WO2022099087A1 - Plate-forme de diagnostic d'agents pathogènes en suspension dans l'air - Google Patents

Plate-forme de diagnostic d'agents pathogènes en suspension dans l'air Download PDF

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Publication number
WO2022099087A1
WO2022099087A1 PCT/US2021/058352 US2021058352W WO2022099087A1 WO 2022099087 A1 WO2022099087 A1 WO 2022099087A1 US 2021058352 W US2021058352 W US 2021058352W WO 2022099087 A1 WO2022099087 A1 WO 2022099087A1
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Prior art keywords
sample collection
carrier fluid
implementations
sample
fluid reservoir
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Ceased
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PCT/US2021/058352
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English (en)
Inventor
Peter Q. NGUYEN
Luis Ruben Martinez SOENKSEN
James J. Collins
Nina M. DONGHIA
Nicolaas M. ANGENENT-MARI
Helena de Puig GUIXE
Ally HUANG
Rose A. LEE
Evan M. ZHAO
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Beth Israel Deaconess Medical Center Inc
Massachusetts Institute of Technology
Harvard University
Original Assignee
Beth Israel Deaconess Medical Center Inc
Massachusetts Institute of Technology
Harvard University
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Publication of WO2022099087A1 publication Critical patent/WO2022099087A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Measuring devices for evaluating the respiratory organs
    • A61B5/097—Devices for facilitating collection of breath or for directing breath into or through measuring devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Measuring devices for evaluating the respiratory organs
    • A61B5/082—Evaluation by breath analysis, e.g. determination of the chemical composition of exhaled breath
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6801—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be attached to or worn on the body surface
    • A61B5/6802—Sensor mounted on worn items
    • A61B5/6803—Head-worn items, e.g. helmets, masks, headphones or goggles

Definitions

  • the present invention relates generally to wearable sensors. More specifically, the present invention relates to a wearable sensing platform based on cell-free synthetic biology reactions added to a face mask.
  • BACKGROUND [0004] Synthetic biology has provided control of biological systems and has led to developments in biotechnology and medicine. Modular biosensors, genetic logic gates, and output effectors are a part of customized biological circuits. In parallel, recent developments in wireless technology, wearable electronics, smart materials, and functional fibers including mechanical, electrical and optical properties have included the development of biosensing systems. Even though genetically-encoded sensors have been incorporated into bench-top diagnostics, examples of wearable devices using these tools are limited.
  • an aqueous solution activated detection device includes a carrier fluid reservoir, a sample collection unit, a sample processing unit including dried synthetic biological components, and a detection unit.
  • a method of detecting an airborne agent includes; contacting air including the agent to the sample collection unit of the aqueous solution activated detection device, wherein the sample unit collects the agent on a surface of the sample unit; causing a carrier fluid from the carrier fluid reservoir to flow to the sample collection unit, the carrier fluid subsequently flowing to the sample processing unit and contacting the dried synthetic biological components; and reading an output from a detection unit, the output indicative of the presence of the airborne agent in the air.
  • a method of making an aqueous solution activated detection device includes: connecting in series a carrier fluid reservoir, a sample collection unit, a sample processing unit, and a detection unit.
  • a diagnostic system includes a biochemical reaction composed of a combination of reverse transcriptase, nucleic acid-modifying enzymes, amplification enzymes, cell-free lysate, Cas13 or Cas12 enzymes, and other required substrates or reagents for their respective reactions and one or more nucleic acids which may contain complete or partial nucleic acid sequences, including the reverse complement and in DNA or RNA, as disclosed herein.
  • a device for capturing an airborne agent includes a sample collection chamber and a covering.
  • the sample collection chamber captures breath from a subject.
  • the airborne agent is contained in the breath from the subject.
  • the covering is coupled to the sample collection chamber.
  • the covering has a sample collection unit with a porous material.
  • a device for processing a sample includes two or more reaction zones coupled in sequence to each other.
  • the device further includes one or more time delay barriers separating each pair of the two or more reaction zones.
  • FIGs.1A-1C illustrate an aqueous solution-activated sensor fabric, according to some implementations of the description.
  • FIG. 1A depicts a top schematic
  • FIG. 1B depicts an isometric view
  • FIG.1C depicts a detailed view.
  • FIG. 2 illustrates an implementation of a fabric based detector, according to some implementations of the description.
  • FIGs. 3A-3C depict an implementation of a fabric based detector including a hydrophobic material, according to some implementations of the description.
  • FIG. 3A is a first view and FIG. 3C is a second view, both illustrating the hydrophobic material on a web structure.
  • FIG.3C depicts a close up schematic view of the hydrophobic material.
  • FIGs. 4A-4B depict an implementation of an aqueous solution-activated sensor, according to some implementations of the description.
  • FIGs. 5A-5B illustrate another implementation of an aqueous solution-activated sensor, according to some implementations of the description.
  • FIG.5A is an exploded layer view and FIG.5B is a front cross-sectional view through one of the chambers.
  • FIGs. 6A-6H illustrate wearable cell-free synthetic biology, according to some implementations of the description.
  • FIG. 6A depicts how freeze-dried cell-free reactions can be embedded in reaction sachets or chambers that are distributed throughout garments for use by soldiers, clinicians, and first responders.
  • FIG. 6B depicts a schematic of the layer-by-layer assembly of the wearable devices.
  • FIG. 6C depicts an array of assembled reaction chambers showing the elasticity (center) and flexibility (right) of the devices.
  • FIG. 6D depicts portals cut into the outermost layer.
  • FIG. 6E-6F depict various types of synthetic biology circuits can be freeze-dried in these wearable devices, including; constitutively expressed outputs (FIG.
  • FIGs. 7A-7C depict assembly layers and sample activation of colorimetric wFDCF reactions with constitutive P T7 ::LacZ module, according to some implementations of the description.
  • FIG. 7B depict the activation of colorimetric wells or reaction chambers with a control rehydration solution.
  • FIG. 7C depict the activation of the reaction chambers with a rehydrating solution including a triggering compound.
  • FIGs.8A-8C depict sample activation of wFDCF colorimetric devices and a bracelet, according to some implementations of the description.
  • FIG. 8A depicts activation of colorimetric Ebola virus DNA toehold wFDCF sensor.
  • FIG. 8B depicts port wicking into reaction chambers containing reaction disks using dd-H 2 O fluid splash.
  • FIG. 8C depicts activation of the wearable colorimetric bracelet with four independent Ebola virus DNA toehold sensors.
  • FIGs.9A-9G depict the design and validation of fluorescent and luminescent freeze- dried cell-free synthetic biology wearables, according to some implementations of the description.
  • FIG.9A details of assembly and activation of fiber-optic based wFDCF module for fluorescence/luminescence output.
  • FIG. 9B top - diagram depicting the layers of the assembled device; bottom - cross-sectional view of the interior of the device.
  • FIG.9C depicts comparison of fluorescent signal after rehydration of wFDCF constitutive sfGFP template as compared to control.
  • FIG. 9D depicts activation of FDCF riboswitch in a wearable device as compared to a control.
  • FIG.9E depicts a demonstration of fluorescent aptamer being activated by a substrate as compared to a control.
  • FIG. 9F illustrates luminescence output detected from an HIV toehold sensor with nanoLuciferase operon.
  • FIG. 9A details of assembly and activation of fiber-optic based wFDCF module for fluorescence/luminescence output.
  • FIG. 9B top - diagram depicting the layers of the assembled device; bottom - cross
  • FIGs. 10A-10D illustrate that concentrating PURE cell-free reactions increases reaction kinetics, according to some implementations of the description.
  • FIG.10A is a schematic of reaction concentration through the lyophilization of PUREXPRESS® (New England Biolabs, Inc., Ipswich, MA) reactions at varying volumes followed by rehydration at a set volume.
  • FIG.10B depicts representative images of PURE reactions with a LacZ output over one hour, at various concentrations.
  • FIG. 10C depicts quantified PUREXPRESS® reactions with a LacZ output.
  • FIG. 10D depicts the half-maximal values from the curve fitting the data shown in FIG. 10D.
  • FIG. 10A is a schematic of reaction concentration through the lyophilization of PUREXPRESS® (New England Biolabs, Inc., Ipswich, MA) reactions at varying volumes followed by rehydration at a set volume.
  • FIG.10B depicts representative images of PURE reactions with a LacZ
  • FIG. 11 depicts Zika DNA Toehold sensor activation in single mercerized cotton thread, according to some implementations of the description.
  • FIG. 12 depicts antibiotic resistance sensors for spa, ermA and mecA genes using in- wearable sensor demonstrate specific orthogonality, according to some implementations of the description.
  • FIG. 13 depicts POF fabric compatibility with lyophilized transcription-only fluorescent aptamer reactions, according to some implementations of the description.
  • FIG. 14 depicts sensor multiplexing using different fluorescent proteins in a single device, according to some implementations of the description.
  • FIGs. 15A-15B depict NanoLuciferase (nLuc) luminescence experiments, according to some implementations of the description.
  • FIG.15A depicts the dynamic response of a wFDCF Lyme disease RNA toehold switch sensor with luminescence output.
  • FIG. 15B depicts the dynamic response of a wFDCF HIV RNA toehold switch sensor with luminescence output in comparison to constitutive PT7::nLuc expression as a positive control.
  • FIGs.16A-16D depict fabrication of polymeric optic fiber (POF) fabric for wFDCF, according to some implementations of the description.
  • FIG. 16A depicts how hydrophilic yarns were weaved along the weft in combination with POFs as warp.
  • FIG. 16B depicts a three-fiber multi-strip design.
  • FIG.16C depicts a roll of the hydrophilic POF fabric after weaving.
  • FIG.16D depicts a cut section of the hydrophilic POF fabric with indications in reaction zone and bundle ends.
  • FIGs.17A-17G depict fabrication of textile-based wFDCF sensor patch, according to some implementations of the description.
  • FIG.17A depicts a cut strip of hydrophilic POF fabric that was laser-etched.
  • FIG. 17B depicts examples of prepared wFDCF fabric-elastomer layers and final assembly into a three-well sensor for garment integration.
  • FIG.17C depicts a schematic of a POF-fabric-elastomer strip for sensing in a single textile layer including two excitation fibers on the sides of an emission fiber.
  • FIG. 17D depicts a schematic of a double POF-fabric- elastomer strip for sensing with dedicated excitation and emission layers.
  • FIG. 17E depicts a schematic of a single excitation or emission POF-fabric-elastomer layer overlaid on an applied elastomer pattern for creating the impermeable reaction wells or chambers.
  • FIG. 17F depicts a finalized three-well sensor wFDCF device with heat shrunk POF covers and Luer connectors for interface with a portable spectrometer device.
  • FIG.17G depicts a top and bottom views of a final three-well sensor wFDCF device. [0033] FIGs.
  • FIG. 18A-18B depict textile substrate compatibility testing using synthetic biology reactions and sample colorimetric reaction, according to some implementations of the description.
  • FIG. 18A depicts samples of eight fabric types selected as part of the textile screening for wFDCF compatibility.
  • FIG. 18B depicts a sample wFDCF colorimetric activation in a cellulose matrix square containing a protein synthesis solution.
  • FIGs. 19A-19B depict textile screening using model constitutive P T7 ::LacZ assay, according to some implementations of the description.
  • FIG. 19A depicts a sample well plate containing BSA blocked and unblocked discs of different textile types after constitutive P T7 ::LacZ expression following a 12-hour run for reactions containing an protein synthesis solution with plasmid or without plasmid as controls.
  • FIG. 19B depicts examples of qualitative traces of colorimetric signals for these different fabric disks using a plate spectrophotometer.
  • FIG. 20 depicts a compilation of normalized functional scoring for colorimetric wF- DCF textile screening, according to some implementations of the description.
  • FIGs. 21A-21F depict fabrication of wearable microcontroller system with LED illumination and spectrometric capabilities, according to some implementations of the description.
  • FIG.21A is an exploded isometric view of wearable POF spectrometer components with case and electronics.
  • FIG.21B is a photograph of an open assembled device.
  • FIG.21C is a photograph of a fully assembled device ready for imaging.
  • FIG. 21D depict details of a camera used in the device.
  • FIG. 21E is a top view of an assembled device to provide detail of compact electronics arrangement.
  • FIG.21F depicts the arrangement of a wearable POF spectrometer with wireless connectivity in-garment for wFDCF reaction testing.
  • FIGs. 22A-22C depict custom mobile application software, according to some implementations of the description.
  • FIG. 22A depicts a main window of the developed wFDCF sensor mobile application where spectrographic measurements are continuously recorded.
  • FIG.22B depicts an environmental window of the mobile application displaying geolocation information as well as environmental information.
  • FIG.22C depicts an excitation window of the application.
  • FIGs. 23A-23J depict validation of CRISPR-based FDCF wearable sensors, according to some implementations of the description.
  • FIG.23A depicts the sensing mechanism of CRISPR-Cas12a system.
  • FIG.23B depicts a wFDCF mecA CRISPR-based sensor exposed to sample containing mecA trigger.
  • FIG.23C depicts wFDCF spa CRISPR-based sensor exposed to spa trigger.
  • FIG. 23D depicts wFDCF ermA CRISPR-based sensor exposed to ermA trigger.
  • FIG.23E depicts experimental detection of mecA CRISPR-based sensor was statistically distinguishable.
  • FIG. 23F is an orthogonality demonstration of mecA / spa / ermA CRISPR- based multi-sensor wearable.
  • FIG. 23G is a plot depicting the orthogonality.
  • FIG. 23H depicts POF end on light up demonstrating the orthogonality.
  • FIG.23I depicts garment-level integration of fabric-based wearable synthetic biology sensors.
  • FIG. 23J depicts connection of fabric-based module to wearable POF spectrometer with wireless connectivity capabilities.
  • FIG. 24 depicts the limit of detection of wFDCF CRISPR-Cas12a based sensor activated in-fabric, according to some implementations of the description. [0040] FIG.
  • FIGs. 26A-26E depict integrated wFDCF sample Lysis, according to some implementations of the description.
  • FIG. 26A depicts detergent combinations for cellular lysis were tested against CRISPR-Cas12a SHERLOCK reactions.
  • FIG. 26B depicts assembly of the wFDCF with lysis.
  • FIG.26C depicts in-wearable wFDCF mecA sensors containing a lyophilized lysis buffer challenged with intact E. coli cells either containing the target mecA gene or a negative control plasmid.
  • FIG.26D depicts some non-ionic surfactants used as freeze-dried lysis reagents: top row left to right Triton X-100, NP-40, and Tween-20; bottom row left to right Brij- 58, Brij-C10, and Brij-S20.
  • FIG. 26E depicts some ionic surfactants used as freeze-dried lysis reagents: left to right; sodium dodecyl sulfate, CHAPS hydrate, and sodium deoxycholate.
  • FIGs.27A-27D depict bioinspired sample-wicking for textile-based wFDCF synthetic biology devices, according to some implementations of the description.
  • FIG.27A is a schematic of the base cover presented for the textile-based wFDCF synthetic biology devices, as well as the underlying biomechanical mechanism of water collection.
  • FIG.27B depicts a modified cover for the textile-based wFDCF synthetic biology devices with wicking ports.
  • FIG.27C depicts a five- second time-lapse of the fluid pinning and port wicking exhibited by the device.
  • FIG. 27D is a photograph of an assembled textile-based wFDCF synthetic biology device including the bioinspired port.
  • FIG. 28A depicts a sensor, according to some implementations.
  • FIG. 28B depicts a wearable mask, according to some implementations.
  • FIG. 28C depicts freeze-dried lysis and detection components, according to some implementations.
  • FIG. 28A depicts a sensor, according to some implementations.
  • FIG. 28B depicts a wearable mask, according to some implementations.
  • FIG. 28C depicts freeze-dried lysis and detection components, according to some implementations
  • FIG. 28D is a plot and FIG. 28E depicts a corresponding Lateral Flow Assay(LFA), according to some implementations.
  • FIG. 28F is a plot and FIG.28G depicts a corresponding Lateral Flow Assay(LFA), according to some other implementations.
  • FIG. 28H depicts a breathing simulator, according to some implementations.
  • FIG. 28I is a plot and FIG. 28J depicts a corresponding Lateral Flow Assay(LFA), according to yet another implementation.
  • FIGs. 29A and 29B depict implementation of Polyvinyl Alcohol (PVA) time delays for multi-stage wFDCF Reactions, according to some implementations.
  • FIG.29A depicts testing of the PVA time delays.
  • PVA Polyvinyl Alcohol
  • FIG.29B shows a representative experiment, from left to right, at 0, 13, 14 and 15 min.
  • FIGs.30A, 30B, 30C1-30C6, 30D-30G show details on the design, performance, and relevant molecular sensor sequences, according to some implementations.
  • FIG. 30A depict sSARS-CoV-2 genomic region targeted by the RT-RPA and SHERLOCK sensor utilized in a face-mask diagnostic of A-version sensors.
  • FIG. 30B depicts a Laser-cut sample collection pad from capillary wicking material.
  • FIG.30C1-C6 depict steps of the ⁇ PAD construction.
  • FIG.30D depicts components of a face-mask sensor before assembly.
  • FIG. 30E depicts a fully assembled sensor.
  • FIGs. 31A-31C depict a face-mask diagnostic B-version sensor design and construction, according to some implementations.
  • FIG.31A depicts a sub-assembly consisting of the sample collection pad, ⁇ PAD (unfolded), and the LFA output strip.
  • FIG. 31B depicts the fully assembled B-version face-mask sensors.
  • FIG. 31C depicts a B-version sensor fully integrated into a face mask.
  • FIGs. 32A-32F depicts a breathing simulator, according to some implementations.
  • FIG. 32A depicts a schematic of the modules used in the breathing simulator.
  • FIG. 32A depicts a schematic of the modules used in the breathing simulator.
  • FIG.32B depicts details of a spontaneous breathing generator shown in FIG.32A.
  • FIG.32C depicts details of the nebulizer and heating assembly shown in FIG. 32A.
  • FIG. 32D depicts the nebulizer reservoir being filled (left) and the nebulized aerosols exiting the tubing (right).
  • FIG. 32E depicts details of a high-fidelity anatomically precise airway manikin shown in FIG. 32A.
  • FIG. 32F depicts details of the air flow path shown in FIG.32A.
  • FIG.33A shows a breathalyzer, according to some implementations.
  • FIG.33B shows a face mask, according to some implementations, for comparison to the breathalyzer shown in FIG.33A.
  • FIG.34 depicts toehold switches, according to some implementations.
  • FIGs. 35A-35U show plots of a LacZ output if screening of a library, according to some implementations (35A control, 35B-35U toehold sensors 1-20 respectively).
  • FIGs.36A-36Q depict a library of gRNAs that were screened to allow Cas13a direct sensing of SARS-CoV-2 vRNA, according to some implementations. Plots of screening data are shown in 36A-36P, a summary plot is shown by 36Q.
  • FIG. 37A depicts a reverse transcriptase amplification reaction, according to some implementations.
  • FIG. 37A depicts a reverse transcriptase amplification reaction, according to some implementations.
  • FIG. 37B depicts the experimental setup for screening a library of 11 gRNAs, according to some implementations.
  • FIG. 37C-37M are plots depicting gRNAs activity for the 11 gRNAs screened according to FIG. 37B.
  • FIG. 37N is a summary plot of the data from FIG. 37C-37M.
  • FIG. 38A is a diagram depicting an RT-RPA reaction, according to some implementations.
  • FIG. 38B depicts the experimental setup for screening of primer pairs, according to some implementations.
  • FIG.38C depicts a primer set that allows for rapid detection of viral RNA fragments and full-length genome, according to some implementation.
  • FIG. 38D depicts the optimization of the primer molar ratio for the primer set of FIG. 38C.
  • FIG. 38A is a diagram depicting an RT-RPA reaction, according to some implementations.
  • FIG. 38B depicts the experimental setup for screening of primer pairs, according to some implementations.
  • FIG.38C depicts a
  • FIG.39A depicts testing of lysis buffer reagents, according to some implementations.
  • FIG. 39B depicts a subset of tested lysis buffer reagents, according to some implementations.
  • FIG.39C depicts compositions of lysis buffer reagents, according to some implementations.
  • Embodiments of various aspects described herein are, at least in part, based on the discovery that synthetic biological reactions can be incorporated into wearable devices and fabrics.
  • the synthetic biological reactions can be selected to function as sensors and expand and complement the scope of use available with live biological sensor systems.
  • the various embodiments enable many applications for synthetic biology, allowing utilization in a wide range of wearable substrates (e.g., functional fibers or fabrics) to assess molecular targets difficult to detect through other technologies.
  • the sensors can be used, for example, by first responders, military personnel, and clinicians at risk to exposure to biological pathogens, viruses and chemical toxins.
  • Cell-free synthetic biology reactions are self-contained abiotic chemical systems with all the biomolecular components required for efficient transcription and translation.
  • Such systems can be freeze-dried into shelf-stable formats using porous substrates, which allow for robust distribution, storage and use without specialized environmental or biocontainment requirements.
  • Genetically engineered circuits, encoded in DNA or RNA can be added to freeze- dried, cell-free (FDCF) reactions for activation by simple rehydration.
  • FDCF genetic circuits are combined with flexible and textile substrates. These can be incorporated and used for the design of practical wearable biosensors.
  • various wearable freeze-dried, cell-free synthetic biology (wFDCF) sensors for small molecule, nucleic acid, and toxin detection have been made.
  • FIGs.1A-1C illustrate an aqueous solution-activated sensor fabric (100), according to some implementations.
  • FIG.1A depicts a top schematic
  • FIG.1B depicts an isometric view
  • FIG.1C depicts a detailed view.
  • the fabric includes an excitation plastic optic fiber (POF) 102, and an emission POF 104 combined with a porous hydrophilic material into a flat web structure 106.
  • the fabric also includes a FDCF synthetic biological component 108 in at least a portion of the web structure.
  • the web structure 100 is a woven structure where the excitation POF 102 and emission POF 104 are woven in the warp direction 112, and the hydrophilic material 110 is woven in the weft direction 114.
  • the excitation POF 102, and the emission POF 104 include an outer cladding 116.
  • FIG.2 illustrates an implementation of a web structure 200.
  • the web structure 200 is a woven structure including a first layer 202 where the excitation POF 102 is woven in a warp direction 112, and the porous hydrophilic material 110 is woven in the weft direction 114.
  • the excitation POF includes a plurality of substantially parallel POFs.
  • the web structure 200 can optionally include a second layer 204 wherein the emission POF 104 is woven in the warp direction 112, and the porous hydrophilic material is woven in the weft direction 114.
  • the excitation POF 102 includes a plurality of substantially parallel excitation POFs 102.
  • the emission POF 104 includes a plurality of parallel emission POFs 104.
  • the FDCF synthetic biological component is spatially contained by being surrounded by patterns of a hydrophobic material.
  • FIG.3A -3C illustrate one possible configuration. A hydrophobic material 302 is shown in FIG 3A, and shown in outline in FIG. 3B. The synthetic biological component 108 is surrounded by the hydrophobic material 302.
  • FIG.3C depicts a depicts a close up schematic view of the hydrophobic material 302.
  • the hydrophobic material 302 forms a chamber 308, shown as a dashed outline.
  • the port 306 provides a fluid connection to the chamber 308 through a conduit 310.
  • the FDCF synthetic biological components 108 are disposed (e.g., deposited or placed) in the chamber 306 (not shown for clarity).
  • An excitation POF 102 and emission POF 104 are shown passing through the hydrophobic material 302, and through the chamber 308. Additional fibers of POFs can be included. For clarity, the porous hydrophilic material 110 is also not shown.
  • a first end 322 of the excitation POF 102 is treated with a reflective coating.
  • a second end 332 of the excitation POF 102 can be connected to an excitation source, such as an LED light.
  • a first end 324 of the emission POF 104 is treated with a reflective coating.
  • a second end 334 of the emission POF 104 can be connected to a detector.
  • the chamber volume is between about 0.1 ⁇ L and about 500 ⁇ L (e.g., between about 1 and 150 ⁇ L).
  • the port 306 is between 0.1 ⁇ m 2 and 50 mm 2 (e.g., between 1 ⁇ m 2 and 10 mm 2 ).
  • FIG. 3A-3C Although illustrated in FIG. 3A-3C as web structure 106, other web structures, such as the web structure 200 (FIG. 2) can also be used.
  • no POFs are used and a top portion 312 of the hydrophobic material 302, all through the hydrophobic material 302 to the chamber 308 (FIG.3C), is transparent.
  • FIG. 4A and 4B depict an implementation of an aqueous solution-activated sensor 400.
  • FIG. 4A is an exploded perspective layer view and FIG. 4B depicts separated layer of the sensor 400 from a top view.
  • a chamber 402 is formed by a bottom layer 404 of a flexible material, a middle layer 406 of a second flexible material, and a top layer 408 of a third flexible material.
  • the first, second and third flexible materials can have the same or different compositions.
  • the bottom layer 404 defines a bottom wall 414 of the chamber, the area of which is shown in encircled by a dashed line (e.g., the boundary) in FIG. 4B.
  • the boundary defining the bottom wall 414 is provided by a cut out in the middle layer 406.
  • the middle layer 406 defines a side wall 416 of the chamber, by the continuous open space or cut out in the middle layer 406.
  • a top layer 408 defines a top wall of the chamber 418, shown by a dashed outline (opposite an exterior surface 412). Synthetic biological components 108 are disposed in the chamber.
  • a port 410 fluidly connects the exterior surface 412 of the third layer 408 of the flexible material to an interior of the chamber 402.
  • the port is defined by a continuous open space or cut out in the top layer 408.
  • the port 410 allows an aqueous solution in contact with the exterior surface 412 to be wicked to the interior of the chamber 402.
  • the FDCF synthetic biological components are hydrated upon exposure to the aqueous solution to form rehydrated synthetic biological components.
  • the rehydrated synthetic biological components are formulated to provide an optical signal transmittable through a light transmitting medium.
  • the optical signal is responsive to the presence or absence of a triggering compound in the aqueous solution wicked to the interior of the chamber.
  • the top layer 404, or a portion thereof is a UV-Vis light transmitting medium and provides an optical connection to the interior of the chamber 402.
  • a dried lysate is disposed in the chamber.
  • the dried lysate is disposed in the chamber between the port 410 and the FDCF biological components 108.
  • the dried lysate is absorbed on or in a porous hydrophilic material.
  • a dissolvable membrane or dissolvable material is disposed between the dried lysate and the FDCF biological components 108.
  • the dissolvable material can provide a time delay allowing the lysate to act on components, such as cells and viruses, in the aqueous solution.
  • the aqueous solution, and lysates in the aqueous solution subsequently contact the biological components 108.
  • a delay is provided by a tortuous path.
  • a barrier is provided that is made of material that is impermeable to the aqueous solution but has a tortuous channel.
  • the tortuous channel fluidly connects the dried lysate and the FDCF biological compounds.
  • tortuous can include a winding path for the channel creating a large distance for the aqueous solution to flow through, and can include constrictions and narrowing restricting.
  • FIG. 5A and 5B illustrate another implementation of an aqueous solution-activated sensor 500.
  • FIG.5A is an exploded perspective view and FIG.5B is a front cross-sectional view through one of the chambers. The cross-section is perpendicular to the direction of the parallel POFs 102, 104.
  • FIG.4A, 4B Some aspects are similar to the implementation 400 (FIG.4A, 4B).
  • sensor 500 features a chamber 402 formed in a flexible material by a bottom layer 404 of the flexible material, a middle layer 406a and 406b of the flexible material (a single middle layer 406 is used in the embodiment shown in FIG.4A), and a top layer 408 of the flexible material.
  • a first UV-Vis light transmitting medium is the emission POF 104
  • the first UV-Vis light transmitting medium in FIG.4A is a portion of the top layer 408.
  • a second UV-Vis light transmitting medium is the excitation POF 102.
  • a port 410 fluidly connects the exterior surface of the third layer 408 of the flexible material to an interior of the chamber 402, similar to the implementation shown in FIG.4A.
  • a portion of an outer cladding of the emission POF 104 is removed or etched, as previously described with reference to FIG. 1C. This provides the first optical connection from the interior of the chamber 402.
  • a portion of an outer cladding of the excitation POF 102 can also be removed or etched to provide the second optical connection to the interior of the chamber 402.
  • One end of the emission POF 104 and excitation POF 102 can be connected to a spectrophotometer.
  • the excitation POF 102 is connected to a light source such as an LED light
  • the emission POF 104 is connected to a detector, such as a CCD detector.
  • the other end of the emission POF 104 and excitation POF 102 can be treated with a reflective compound to provide a reflective surface.
  • light from the reactions enter the POFs through ends that are cut (i.e., transmission through the end of the fiber).
  • Different ways generated light can be absorbed into the POF includes: (a) through the side of the fiber where the cladding has been removed, (b) through the end of the fiber, and/or (c) through some light-focusing material (e.g., some kind of geometric waveguide that can absorb emited photons and route them to the POF).
  • an opaque barrier 504 is inserted between the port 410 and both of the emission POF 104 and excitation POF 104.
  • the opaque barrier is selected to reduce or eliminate light transmission from the port 410 to the emission POF and excitation POF.
  • the optical barrier includes fluid connectivity to the chamber 402, for example shown as a gap 505 in FIG. 5B. Any form of fluid connectivity such as holes and perforations through the optical barrier 504 can be use provided light is eliminated or reduced. For example, in some implementations the light is reduced by at least 80%, at least 90%, at least 95%, or at least 99%, when the optical barrier 504 is used. [0075] Still referring to the implementation depicted by FIG. 5A-5B, the emission POF 104 and the excitation POF 102 are combined with a porous hydrophilic material.
  • the POFs 102, 104 can be interwoven with the porous hydrophilic material providing a woven fabric 110 as previously described and shown in FIG.1A-1C and FIG.2.
  • the emission POF 104 is interwoven with a first portion of porous hydrophilic material providing a first woven fabric (e.g., layer 204 in FIG.2)
  • the excitation POF is interwoven with a second portion of the porous hydrophilic material providing a second woven fabric (e.g., layer 202 FIG. 2).
  • FIG. 5B only shows a single hydrophobic material 110 for clarity, but multiple layers of hydrophobic material and POFs is also contemplated as a possible implementation.
  • the porous hydrophilic material 110 is embedded in the flexible material.
  • the porous hydrophilic material 110 passes from the chamber 402, through region 405 of layer 406a, 406b, and out of the sensor 500 to region 506.
  • the sensor 500 includes a plurality of conical spikes 508 perpendicular to the exterior surface and proximate to the port. The conical spikes aid in collecting and attracting aqueous solutions close to the port 410.
  • Some implementations relate to methods for making an aqueous solution-activated sensor.
  • the method includes providing a layer of a first material.
  • the layer of the first material can include the bottom layer 404, as depicted in FIG 4A and 4B, 5A and 5B.
  • a layer of a second material is provided on the top surface of the first material.
  • the layer of the second material can include the middle layer 406, 406a or 406b.
  • a layer of a third material is provided on a top surface of the second material.
  • the layer of the third material can include the top layer 408.
  • the method further includes adding synthetic biological components into the chamber. [0079] In some implementations, the synthetic biological components are freeze dried after being placed in the chamber 402. In some other implementations, the synthetic biological components are freeze-dried or otherwise dried prior to placement in the chamber 402.
  • the FDCF biological components are absorbed on a porous hydrophilic material.
  • the material can be inserted into the chamber 402 through the port 402, for example, where the top layer 408 is made of an elastomeric material.
  • the method includes addition of lysate, optionally absorbed on a porous hydrophilic material.
  • a time delay barrier such as a dissolvable barrier or a barrier having a tortuous channel there through, is placed between the lysate and the biological components.
  • the method includes curing any one or more of the first material, the second material, and the third material prior to, during, or after providing the first material, second material, or third material as a layer.
  • any one of the materials can comprise a cross linking polymer that cross-links upon heat curing, exposure to oxygen or after adding an initiator or catalyst.
  • the method includes solidifying any one or more of the first material, the second material, and the third material from a molten state prior to, during, or after providing the first material, second material, or third material as a layer.
  • the material can be a thermoplastic which is heated, cast to form one or more layers 404, 405, 406a, 406b, or 408 and then cooled so that it solidifies.
  • the thermoplastic is formed by additive manufacturing such as 3D printed to form the layers.
  • thermoplastic is formed by a subtractive process, such as milling (e.g., CNC machining).
  • one or more of the layers are formed by injection molding.
  • the method includes forming, by a polymerization reaction, any one or more of the first material, the second material, and the third material from monomeric precursors, during, or after providing the first material, second material, or third material as a layer.
  • Hydrophobic materials [0085] According to some implementations, any hydrophobic material can be used. For example, a low molecular polymer or oligomer such as a wax.
  • the hydrophobic material is an elastomeric material such as one or more of EPDM, a silicone, a neoprene rubber, a natural rubber, a nitrile rubber, a butyl rubber, a thermoplastic elastomer, or any hydrophobic elastomer.
  • the elastomeric material is a silicone.
  • Porous hydrophilic materials can include any material that can be wet by an aqueous solution and adsorbs between 10 wt.% and 1000 wt.% water.
  • materials having hydrophilic or hydrogen bonding groups such as hydroxyls, esters, carboxylates, ketones, amines, amides, sulfates and phosphates.
  • the material can be a fiber that can be formed into a flat shape, including fibers that can pressed together into a web structure or mesh structure.
  • the material can also be a fiber that is formed into a yarn and then woven into a web structure or pressed together into a mesh structure.
  • the porous hydrophilic material can include one or more of one or more of a cellulose, starch, maltodextrin, glycerin, sugar, sucralose, dextrose, gum arabic, cotton, wool, silk, rayon, hemp, spandex/lycra/elastane, polyester, polyamide, linen, nylon, or combinations thereof.
  • Chamber for holding FDCF biological components Chambers or reaction chambers, wells or sachets are described herein and refer to a space, for example, where the FDCF biological components are disposed, placed or contained.
  • the chamber volume is between about 0.1 ⁇ L and about 500 ⁇ L, between about 1 and 150 ⁇ L, or between about 1 and 100 ⁇ L.
  • the chambers include a port or small opening (e.g., FIG. 3C port 306, FIG. 4A-4B, FIG. 5A-5B port 410).
  • the port is between 0.1 ⁇ m 2 and 50 mm 2 , such as between 1 ⁇ m 2 and 10 mm 2 ).
  • the port is configured to allow fluid access into the chamber and in some implementations is not self-sealing. The fluid access should be fast, for example within at least five minutes. In some implementations within 1 minute. In some implementations within at least 30 seconds.
  • the port has a cover, for example to seal off the chamber from liquids when the sensor device is not in use, is not usable or when the device may be intentionally exposed to a liquid that is not expected to contain a triggering compound.
  • the user may wish to deactivate the sensor by covering the port before the sensor is immersed in water or when the user is in a wet environment such as in an area with precipitation.
  • the cover can be any form such as a friction fit plug or adhesively attached.
  • the ports are designed for wicking in small volumes, such as from splashes of between with volumes a low as about 1 ⁇ L (e.g., between 10 and about 500 ⁇ L) at relative humidities between about 20-40%.
  • the chamber and port are also configured to reduce the amount of evaporation once an aqueous solution has entered the chamber.
  • the evaporation rate is less than about 1% volume/hr (v/hr).
  • the evaporation rate is less than about 5% v/hr.
  • the evaporation rate is less than about 10% v/hr.
  • the evaporation rate is less than about 15% v/hr.
  • the evaporation rate is less than about 20% v/hr.
  • FDCF Biological Components are used as circuits that are triggered by a triggering compound to provide a detectable signal.
  • the synthetic biological components provide the optical signal when activated with the triggering compound by synthesizing, activating, or suppressing, a colored, fluorescent or luminescent protein.
  • the synthetic biological components include toehold sensor components, transcription-factor sensor components, aptameric sensor components, enzyme sensor components, antibody sensor components, CRISPR DNA sensor components, CRISPR RNA sensor components, ribonucleoprotein sensor components, and combinations thereof.
  • the biological components can be supplied from a commercial source.
  • a commercial source For example, cell-free NEB PUREXPRESS® reaction components (New England Biolabs, Inc., Ipswich, MA).
  • the reaction components such as an A and a B component are combined and diluted with water to a specified concentration according to the manufactures specification for use. It has been found that using a higher concentration than the specified concentration range provides faster kinetics according to some implementations of this disclosure. However, at too high a concentration, the signal kinetics of the reaction are negatively impacted.
  • the rehydrated synthetic biological components have a concentration between 1 and 2.4 times a specified concentration.
  • CRISPR Microbial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems contain programmable endonucleases, such as Cas12a Cpf1 (also referred to as Cpf1) and Cas9.
  • RNA-guided RNases Although both Cas12a and Cas9 and target DNA, single effector RNA-guided RNases also have been recently discovered (Shmakov et al., 2015) and characterized (Abudayyeh et al., 2016; Smargon et al., 2017). These programmable endonucleases and RNases provide a platform for specific nucleic acid (DNA or RNA) sensing. DNA-guided endonucleases, such as Cas 12a and Cas9 can be easily and conveniently reprogrammed using CRISPR guide RNA (gRNAs) to cleave target DNAs.
  • gRNAs CRISPR guide RNA
  • RNA-guided RNases such as C2c2
  • CRISPR RNA CRISPR RNA
  • crRNAs CRISPR RNA
  • target DNA e.g., double-stranded DNA
  • RNA-Cas endonucleases and RNases exhibit promiscuous non-specific DNase or RNase activity.
  • the CRISPR-Cas endonucleases and RNases can lead to “collateral” cleavage of any non-targeted DNAs or RNAs present in proximity.
  • a CRISPR-Cas or CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas12a, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) to guide Cas, such as Cas12a, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • the CRISPR-Cas effector protein can be from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Hel
  • the effector protein can comprise a chimeric effector protein comprising a first fragment from a first effector protein (e.g., a Cpfl) ortholog and a second fragment from a second effector (e.g., a Cpfl) protein ortholog, and wherein the first and second effector protein orthologs are different.
  • a first effector protein e.g., a Cpfl
  • a second effector e.g., a Cpfl
  • At least one of the first and second effector protein (e.g., a Cpfl) orthologs can comprise an effector protein (e.g., a Cpfl) from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibaci
  • sordellii Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can be DNA or RNA.
  • target nucleic acid refers to a polynucleotide being or comprising the target sequence.
  • the target nucleic acid can be a polynucleotide or a part of a polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR effector protein and a gRNA is to be directed.
  • the effector protein can be a DNA targeting CRISPR-Cas protein or an RNA targeting CRISPR-Cas protein.
  • Exemplary CRSIPR-Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • orthologue also referred to as “ortholog” herein
  • homologue also referred to as “homolog” herein
  • a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins can but need not be structurally related, or are only partially structurally related.
  • An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins can but need not be structurally related, or are only partially structurally related.
  • Homologs and orthologs can be identified by homology modelling (see, e.g., Greer, Science vol.228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or “structural BLAST” (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a “structural BLAST”: using structural relationships to infer function. Protein Sci. 2013 Apr;22(4):359-66. doi: 10.1002/pro.2225.). See also Shmakov et al. (2015) for application in the field of CRISPR-Cas loci. Homologous proteins can but need not be structurally related, or are only partially structurally related.
  • the effector protein has a sequence homology or sequence identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, with the wild- type sequence.
  • sequence homology or sequence identity of at least 60%, more particularly at least 70, such as at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, with the wild- type sequence.
  • the CRISPR-Cas effector protein can be from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methyl obacter
  • the effector protein is a promiscuous non-specific DNase or RNase such as Cas 9, Cas12a, Cas13a, or Cas14.
  • the effector protein is Cas12a, also known as Cpf1 or Cas 13a, also known as C2c2.
  • Selection of a promiscuous non-specific DNase or RNase activity Detections is by addition of a short nucleotide sequence that is coupled to a fluorescent reporter and a quencher. Cleavage of the nucleotide allows separation of the quencher from the fluorescent report providing the detectable signal.
  • SHERLOCK refers to “Specific High-sensitivity Enzymatic Reporter un-LOCKing.” SHERLOCK works by amplifying RNA (or DNA with a reverse transcriptase) using recombinase polymerase amplification (RPA) which is an isothermal nucleic acid amplification. SHERLOCK is useful for biosensors, such as wearable biosensors, because isothermal amplification does not require specialized instrumentation, such as PCR, as it uses a single temperature.
  • RPA recombinase polymerase amplification
  • the amplified nucleotides are combined with an effector protein (e.g., Cas 13a), a guide RNA that matches the nucleic acid sequence of interest, and a short nucleotide sequence that is coupled to a fluorescent reporter and a quencher. If the target sequence is present in the pool of amplified nucleotides, the non-specific RNAse activity of effector protein becomes activated and the RNA reporter will be cleaved resulting in activation of the fluorophore. Therefore, the fluorescent signal is used as an indicator to determine whether the target sequence is present in the original pool of nucleotides.
  • an effector protein e.g., Cas 13a
  • a guide RNA that matches the nucleic acid sequence of interest
  • a short nucleotide sequence that is coupled to a fluorescent reporter and a quencher.
  • Light-up aptamers are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. A non-hindered fluorogen can be excited and have its energy dissipated by non-radiative pathway such as molecular vibrations (heat). Once tightly bound by an aptamer, the fluorophore is stabilized and radiative fluorescence decay pathways predominate, leading to a large fluorescence increase.
  • the RNA aptamers can be selected or designed to target specific molecules (trigger compounds) such as small molecules and metabolites.
  • aptamers MFA, BFR, DIT-Apt1, Spinach, Spinach2, Mango, Broccoli, and dimeric Broccoli can be used. Any congnate fluorgen ligand can be used as the trigger compound.
  • the fluorogen is DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone), Malachite green, Hoechst 1C, DIR, DMHBI, DMABI, 2-HBI, 2-HBI, DFHBI, T01-Biotin, T030Biotin, DFHBI-1T, DFHBI-2T, and PFP-DFHBI.
  • Toehold switch sensors are synthetic riboregulators that control the translation of a gene via RNA-RNA interactions. They utilize a designed hairpin structure to block gene translation in cis by sequestration of the ribosome binding site (RBS) and start codon.
  • Transcriptional factor based biosensors consist of a repressor or activator protein regulating the transcriptional activity of a specific promoter.
  • a cis-regulatory DNA sequence (generally called operator or enhancer) adjacent to the promoter is the core DNA element that binds with a TF restricting or enhancing the access of RNA polymerase (RNAP) to the promoter.
  • RNAP RNA polymerase
  • a repressor binds to the operator and prevents RNAP proceeding forward to decrease transcription
  • an activator binds to the enhancer elements and promotes the formation of more stable RNAP-promoter complex to increase transcription.
  • TFs also contain a ligand-binding domain which is the sensor domain that responds to small molecules or environmental stress signal (salt, osmosis, pH, oxygen, redox, light or radiation etc.).
  • Transcriptional activators can be any activators that are coupled to the specific repressor or activator protein used.
  • Riboswitch are RNA-based sensors that utilize chemically induced structural changes in the 5′-untranslated region of mRNA to regulate expression of downstream genes. Riboswitches are quickly synthesized in vitro, flexible in engineering (both aptamers and expression platforms), and can provide a fast response to recognize elements due to the avoidance of complicated protein–protein interactions, even before considering their high specificity and sensitivity.
  • a typical riboswitch construct includes two domains linked to each other, a sensory domain and the regulatory domain.
  • the aptamer binds to the target ligand and causes sufficient conformational changes or stability changes which then trigger the desired readout in the expression platform (switch output) through different mechanisms depending on the choice of expression control at the translation or transcription level.
  • the switch output is described below. [00119]
  • Switch output [00120] In some implementations, the switch output can be expression of any protein providing colorimetric, fluorescent or phosphorescent output.
  • the protein is selected from one or more of GFP, LacZ, Luciferase, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, or T-Sapphire; cyan proteins: ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midorishi-cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKO ⁇ , mKO2, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRu
  • Green Fluorescent Protein is a single polypeptide gene product of 238 amino acids discovered in the jellyfish Aequorea victoria. The protein has a natural green fluorescence. GFP is quite stable and withstands a number of chemical treatments and procedures. GFP requires no biochemical transformation, contrast agent or the use of harmful ionizing radiation in order to be visualized. Enhanced GFP (EGFP) has been engineered to be expressed at higher levels in mammalian cells and to fluoresce more intensely.
  • the lacZ gene encodes beta-galactosidase, which catalyzes the cleavage of lactose to form galactose and glucose. Beta-galactosidase activity can be identified by when incubated with the beta-galactosidase substrate X-gal. Beta-galactosidase cleaves X-gal, a chromogenic substrate, resulting in an insoluble blue dye, thus allowing for the identification of lacZ activity.
  • Luciferase are a class of oxidative enzymes that produce bioluminescence. Luciferase enzymes isolated from different animal species have inherent variability in light emission. For example, Luciferase enzymes are commercially available from the organisms Photinus pyralis, Luciola cruciate, Luciola italic, Luciola lateralis, Luciola mingrelica, Photuris pennsylvanica, Pyrophorus plagiophthalamus, Phrixothrix hirtus, Renilla reniformis, Gaussia princeps, Cypridina noctiluca, Cypridina hilgendorfii, Metridia longa, and Oplophorus gracilorostris.
  • the triggering compounds can be any compound for which the synthetic biology switch is designed or selected.
  • the triggering compound can include natural or synthetic molecules including, but not limited, peptides, oligonucleotides polypeptides, proteins, peptidomimetics, antibodies, antibody fragments (e.g., antigen binding fragments of antibodies), carbohydrate-binding protein, e.g., a lectin, glycoproteins, glycoprotein-binding molecules, amino acids, carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids, steroids, hormones, lipid-binding molecules, cofactors, nucleosides, nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivatives of nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, small molecules, and any combinations thereof.
  • small molecules refers to natural or synthetic molecules including, but not limited to, amino acids, peptides, peptidomimetics, polynucleotides, aptamers, nucleotide analogs, organic or inorganic compounds (i.e., including heterorganic and organometallic compounds), saccharides (e.g., mono, di, tri and polysaccharides), steroids, hormones, pharmaceutically derived drugs (e.g., synthetic or naturally occurring), lipids, derivatives of these (e.g., esters and salts of these), fragments of these, and conjugates of these.
  • the small molecules have a molecular weight less than about 10,000 Da, organic or inorganic compounds having a molecular weight less than about 5,000 Da, organic or inorganic compounds having a molecular weight less than about 1,000 Da, organic or inorganic compounds having a molecular weight less than about 500 Da. In some implementations the small molecule has a molecular weight of less than about 1000 Da.
  • the triggering compound can include aptamers.
  • aptamer means a single-stranded, partially single-stranded, partially double- stranded or double-stranded nucleotide sequence capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation.
  • Aptamers can include, without limitation, defined sequence segments and sequences comprising nucleotides ribonucleotides deoxyribonucleotides nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branchpoints and nonnucleotide residues, groups or bridges.
  • the oligonucleotides including aptamers can be of any length, e.g., from about 1 nucleotide to about 100 nucleotides, from about 5 nucleotides to about 50 nucleotides, or from about 10 nucleotides to about 25 nucleotides.
  • the triggering compound is a component that is extracted or lysed from a microbe.
  • the terms “microbes” and “pathogens” generally refer to microorganisms, including bacteria, fungi, protozoan, archaea, protists, e.g., algae, and a combination thereof.
  • microbes also includes pathogenic microbes, e.g., bacteria causing diseases such as plague, tuberculosis and anthrax; protozoa causing diseases such as malaria, sleeping sickness and toxoplasmosis; fungi causing diseases such as ringworm, candidiasis or histoplasmosis; and bacteria causing diseases such as sepsis.
  • pathogenic microbes e.g., bacteria causing diseases such as plague, tuberculosis and anthrax
  • protozoa causing diseases such as malaria, sleeping sickness and toxoplasmosis
  • fungi causing diseases such as ringworm, candidiasis or histoplasmosis
  • bacteria causing diseases such as sepsis.
  • microbe or microbes can also encompass non-pathogenic microbes, e.g., some microbes used in industrial applications.
  • the term “microbe” or “microbes” also encompasses fragments of microbes, e.g., cell
  • the trigger molecule is a “molecular toxin,” which refers to a compound produced by an organism which causes or initiates the development of a noxious, poisonous or deleterious effect in a host presented with the toxin.
  • Such deleterious conditions may include fever, nausea, diarrhea, weight loss, neurologic disorders, renal disorders, hemorrhage, and the like.
  • Toxins include, but are not limited to, bacterial toxins, such as cholera toxin, heat-liable and heat-stable toxins of E. coli, toxins A and B of Clostridium difficile, aerolysins, and hemolysins; toxins produced by protozoa, such as Giardia; toxins produced by fungi.
  • Molecular toxins can also include exotoxins, i.e., toxins secreted by an organism as an extracellular product, and enterotoxins, i.e., toxins present in the gut of an organism.
  • exotoxins i.e., toxins secreted by an organism as an extracellular product
  • enterotoxins i.e., toxins present in the gut of an organism.
  • a lysate e.g., a prokaryotic or a eukaryotic cell lysate is used.
  • the lysate can be combined with the FDCF biological components prior to contact with an aqueous solution, or the lysate can be first combined with the aqueous solution.
  • the lysate includes one or more of Triton X-100, NP-40, Tween-20, Brij non- ionic surfactants, CHAPS hydrate, lysozyme, and disaccharides or polysaccharides such as sucrose, mannitose, or trehalose.
  • the lysate is freeze-dried.
  • the lysate is dried by another method, such as by evaporating the solvent above the freezing temperature (e.g., under vacuum).
  • the lysate does not include a cationic surfactant.
  • the amount of ionic surfactant by weight of total dry lysate is less than about 20%, less than about 10%, less than about 5%, or less than about 1%.
  • Dissolvable membranes or dissolvable materials [00133] In some implmentations a dissolvable membrane can be integrated into a sensor, for example, in order to allow control of sample flow. The membrane acts as a time-barrier film, by stopping the sample flow until it is dissolved. The control of sample flow in sensor critical areas, such as cell lysing regions, allows increased exposure time for the lysing reagents to act. This helps to ensure higher sensitivity, reactivity and in some cases reduces false-positive signals.
  • Dissolvable membranes contain a water-soluble polymer, sugars such as sucrose, inorganic salts, patterned hydrophobic materials, or other compounds to provide a fluidic delay.
  • the water-soluble polymer is a hydroxylpropyl-methylcellulose, polyvinylpyrrolidone, polyvinyl-alcohol (PVA), carboxymethyl-cellulose, polyethylene-oxide, hydroxylpropyl-cellulose, hydroxylethyl-cellulose, methyl-cellulose, pullulan, gelatin, pectin, sodium alginate, maltodextrin, polymerized rosin, and xanthan.
  • a plasticizer is added, for example, to improve mechanical properties such as brittelness.
  • the plasticizers is glycerol, propylene glycol, poly (ethylene glycol), glycerine, dimethyl phthalate, diacetyl phthalate, dibutyl phthalate, triacetrin, castor oil, citrate ether, and tryethyle citrate.
  • Blocking agents As used herein a “blocking agent” or “molecular blockers” are compounds used to prevent non-specific interactions.
  • the blocking agent can be a coating on a surface, e.g., of the substrate, that prevents non-specific interactions or fouling of the surface when it is contacted with the test sample.
  • a blocking agent includes a compound that either covalently bonds with the material it is blocking or uses non-covalent interactions to block the material with a desired physiochemical characteristic. Blocking agents can be used to treat any surfaces and materials described herein.
  • the interior or exterior surfaces of sensors are treated with blocking agents.
  • the porous or non-porous hydrophilic materials are treated with blocking agents.
  • hydrophobic materials such as elastomers are treated with blocking agents.
  • Non-specific interactions can include any interaction that is not desired between the target molecule (e.g., a triggering compound) and the surface (e.g., a porous hydrophilic material) or between other components in solution.
  • the blocking agent can be a protein, mixture of proteins, fragments of proteins, peptides or other compounds that can passively absorb to the surface in need of blocking.
  • proteins e.g., BSA and Casein
  • poloxamers e.g., pluronics
  • PEG-based polymers and oligomers e.g., diethylene glycol dimethyl ether
  • cationic surfactants e.g., DOTAP, DOPE, DOTMA.
  • Some other examples include commercially available blocking agent or components therein that are available from, for example, Rockland Inc. (Limeric, PA) such as : BBS Fish Gel Concentrate; PBS Fish Gel Concentrate; TBS Fish Gel Concentrate; Blocking Buffer for Fluorescent Western Blotting; BLOTTO; Bovine Serum Albumin (BSA); ELISA Microwell Blocking Buffer; Goat Serum; IPTG (isopropyl beta-D- thiogalactoside) Inducer; Normal Goat Serum (NGS); Normal Rabbit Serum; Normal Rat Serum; Normal Horse Serum; Normal Sheep Serum; Nitrophenyl phosphate buffer (NPP); and RevitablotTM Western Blot Stripping Buffer.
  • BBS Fish Gel Concentrate PBS Fish Gel Concentrate
  • TBS Fish Gel Concentrate TBS Fish Gel Concentrate
  • BSA Bovine Serum Albumin
  • ELISA Microwell Blocking Buffer Goat Serum
  • Goat Serum IPTG (isopropyl beta-D- thioga
  • the blocking agent is BSA.
  • the blocking agent can be a monomer.
  • a monomer (with a single binding site) has no free binding site after binding to the target-binding agent.
  • saccharide-based monomeric blocking agent for example, saccharide-based monomeric blocking agent.
  • the blocking agent can be a monosaccharide or modification thereof, including, e.g., but not limited to, diose, triose, tetrose, pentose, hexose, heptose, linear chain monosaccharides, open chain monosaccharides, cyclic isomers (e.g., furanose form and pyranose of monosaccharides such as hexose), pyranose, fructose, galactose, xylose, ribose, amino sugars (e.g., but not limited to, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, N-acetyl-muramic acid, sulfosugars (e.g., but not limited to sulfoquinovose).
  • diose triose
  • tetrose pentose
  • hexose
  • the aqueous solution can include a biological fluid.
  • biological fluids can include, but are not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, and any mixtures thereof.
  • a biological fluid can include a homogenate of a tissue specimen (e.g., biopsy).
  • an aqueous solution is a suspension obtained from homogenization of a solid sample obtained from a solid organ or a fragment thereof.
  • Optical fibers are used to transmit excitation or emissions.
  • Optical fibers are waveguide fibers designed for transmission of light.
  • Optical fibers typically include a core surrounded by a transparent cladding material with a lower index of refraction. Light is kept in the core by total internal reflection.
  • Optical fibers can include glass (silica, fluorozirconate, fluoroaluminate, and chalcogenide glasses) or plastic optical fibers (POF).
  • Glass optical fiber can be made having a high fidelity and low transmission loss, and are often regarded as the fiber of choice for many optical applications, such as communications and long range transmission. For low speed short data links, POFs can often be implemented. POFs also have the advantage of being more flexible than glass optical fibers. POFs are also more economical and the optical fiber of choice for many consumer products, such as digital home appliance networks, home networks and car networks. Being flexible, POFs are rugged and easy to install without fear of damage. POFs generally have a diameter about 8 times that of glass optical fibers. [00143] In some implmentations the optical fiber is a POF.
  • the POFs have a diameter between about 2000 ⁇ m and 200 ⁇ m, between about 1500 ⁇ m and 500 ⁇ m, or between about 1200 and about 800 ⁇ m.
  • one end of the POF is coated with a reflective coating.
  • the coatings ensure light that would escape from the end that is not connected to an emission source or to the detector is not lost.
  • Any reflective coating can be used that reflects at least about 10% of incident light (e.g., at least 20%, at least 50%, at least 80%).
  • reflective coatings can include a metal coating such as gold and silver.
  • the sensors described herein can be configured as, although not limited to, a wearable item. Without limitation these can include a shirt; a jacket; pants; a skirt; a laboratory coat; a full-body garment; an exterior worn armor; a wrist, arm, head or ankle band; a scarf; gloves; socks; shoes or boots; a necklace; a ring; a hat; a helmet; a brooch; a face mask; a patch; or other wearable garments.
  • an aqueous solution activated detection device includes a carrier fluid reservoir, a sample collection unit, a sample processing unit including dried biological components, and a detection unit.
  • the dried biological components are dried synthetic biological components.
  • the carrier fluid reservoir is connected to the sample collection unit
  • the sample collection unit is connected to the sample processing unit
  • the sample processing unit is connected to the detection unit.
  • the detection device further includes a covering (e.g. a face covering). The carrier fluid reservoir, the sample collection unit, the sample processing unit, and the detection unit are attached to the covering
  • the sample collection unit is attached to an inner (e.g., face contacting or facing) surface of the covering for detection of a subject’s respiratory infection status.
  • the covering is attached to an outside (e.g., external to the covering) for environmental detection of exposure.
  • the covering is a face mask or a part of a face mask, e.g., as shown in FIG. 33B.
  • the covering is implemented in a breathalyzer.
  • the breathalyzer may have a sample collection chamber (e.g., a tube).
  • a breathalyzer including a tube or mouthpiece as shown as 3314 in FIG.33A. In operation, the tube is used to engage a subject’s mouth and the user exhales into the tube.
  • the covering is positioned in the tube to capture breath from the subject.
  • the tube opens into an expansion chamber, and the covering is located within the expansion chamber.
  • the tube can be adaped for attachment to a nose.
  • the detection device can be an air monitor.
  • the breathalyzer can be adapted as an air monitor by attaching a fan to draw ambient air into the the tube of the device.
  • the covering is implemented as a nasal cannula.
  • the covering is implemented as part of a helmet.
  • the carrier fluid reservoir is attached to the outer surface of the face mask.
  • the detection unit is hidden during a sample collection when using the device, for example, to protect the subject’s privacy.
  • the carrier fluid reservoir includes a valve connecting and controlling the flow of an aqueous solution from the fluid reservoir to the sample collection unit.
  • the valve is a pressure valve configured to open when the pressure difference across the pressure valve exceeds a predefined threshold. For example, the valve opens when the pressure difference is at least 0.5 psi. In some implementations, the valve opens when the pressure difference is at least 1 psi. In some implementations, the valve is configured to open under finger pressure.
  • the valve is configured as a two-way valve.
  • the two-way valve can be a manually or otherwise actuated valve.
  • the two-way valve can be actuated by a solenoid.
  • the fluid reservoir is configured as a blister pack.
  • a blister pack is an impermeable enclosure or sachet containing the fluid.
  • the valve is configured as a puncture zone forming a wall of the blister pack, the puncture zone opening under conditions of an applied pressure to the blister pack (e.g. at least 0.5 psi, or at least 1 psi, finger pressure, or mechanically actuated and optionally electrically controlled such as using a solenoid).
  • the device further comprising a button, the button actuating a spike positioned opposite the puncture zone and puncturing the puncture zone when the button is depressed (eg manually or otherwise actuated as previously described).
  • the spike is embedded in a compressible material for protecting the puncture zone prior to depressing the button and actuating the spike.
  • a spike is activated by a sliding mechanism. The spike is positioned over the puncture zone and punchtures the puncture zone when activated.
  • the carrier fluid reservoir is a pierceable sachet that is configured to be mechanically activated by piercing, cutting, pressure-based rupturing, or a combination thereof.
  • the device further includes a barrier between the fluid reservoir and the control of fluid from the reservoir to the sample collection unit is controlled by opening of the barrier including sliding (e.g.
  • the carrier fluid reservoir includes a venting hole configured as a one-way vent.
  • the one-way vent allows ambient air into the carrier fluid reservoir when a pressure differential between an exterior to the carrier fluid reservoir and interior of the pressure reservoir is greater than a threshold value.
  • the interior pressure that allows opening of the one-way vent is at least 0.1 psi, at least 0.5 psi, or at least 1psi. This allows air into reservoir when pressure in the reservoir is at least lower than the atmosphere outside the reservoir.
  • the pressure enters the reservoir due to the elasticity of the reservoir that will tend to restore the reservoir to its original shape if it is compressed.
  • the venting hole material includes a hydrophobic coating that prevents bulk fluid from escaping the reservoir while allowing air to equilibrate between the interior of the reservoir and the external atmosphere.
  • the sample collection zone includes a porous material allowing a carrier fluid to flow (e.g. by capillary action) in a direction from the carrier fluid reservoir to the sample processing unit.
  • the porous material is a hydrophilic material comprises one or more of a cellulose, starch, maltodextrin, glycerine, sugar, sucralose, dextrose, gum Arabic, cotton, wool, silk, rayon, hemp, spandex/lycra/elastane, polypropylene, polycarbonate, polyester, polyamide, linen, nylon, polyurethane, glass, metal, or a blend thereof.
  • the porous material is a high release media such as a polyurethane high release media (e.g. POREX® porous high release media). Table 1 and FIG.20 include materials suitable for the porous material.
  • the porous material includes fibers or threads and the majority of the fibers or threads are aligned in a direction pointing from the carrier fluid reservoir to the sample processing unit.
  • at least 50% (e.g., at 60%, 70%, 80%, 90% by wt.% or alternatively by vol% fibers) of fibers are oriented as in a direction pointing from the carrier fluid reservoir to the sample processing unit, thereby providing a faster aqueous solution flow in this direction.
  • the porous material includes fibers or threads and the majority of the fibers or threads are aligned in a direction orthogonal to the direction pointing from the carrier fluid reservoir to the sample processing unit.
  • At least 50% (e.g., at least 60%, 70%, 80%, 90% by wt.% or alternatively by vol%) of fibers are oriented in a direction orthogonal to the direction pointing from the carrier fluid reservoir to the sample processing unit, thereby providing a slower aqueous solution flow in this direction.
  • the fibers of the porous material can facilitate flow of the carrier fluid in one direction of the porous material compared to orthogonal direction(s).
  • Other implementations are contemplated where the majority of fibers are oriented at any one angle thereby modulating the flow of the solution through the device.
  • the sample collection zone’s porous material can be any porous high-release media substrate (e.g.
  • Porous high-release fiber media can include polyolefin fibers made of a combination 15-85% polypropylene and 15-85% polyethylene as well as fiber additives such as nonionic emulsifiers, antistatic chemicals and mixtures of them.
  • the porous material has electrostatic properties for driving separation of molecules in the carrier fluid during flow.
  • the porous material electrostatically binds and sequesters molecules in the carrier fluid during flow.
  • the porous material is covalently modified with biomolecules that alster or bind to molecules in the carrier fluid during flow.
  • the porous material is fully or partially filled with dissolvable material.
  • the sample processing unit of the device includes subunits. In some implementations, the sample processing unit includes a lysis subunit. In some implementations, the sample processing unit includes an amplification subunit. In some implementations, the sample processing unit includes a reporter activation subunit.
  • the lysis subunit includes dry or freeze dried lysis components;
  • the amplification subunit includes dry or freeze-dried nucleic acid, protein or lipid modifying enzyme components such as reverse transcriptase (RT), RNase H, RNA polymerase, oligonucleotide primers, an RT-RPA, RT-LAMP;
  • the reporter activation subunit includes dry or freeze-dried cell-free lysate, cell-free transcription and translation reactions, Cas13 or Cas12 collateral cleavage enzyme, one or more guide RNA, and a corresponding dry or freeze-dried reporter molecule;
  • the detection unit includes a lateral flow assay (LFA) configured to the detect the reporter molecule and a cleaved product of the reporter molecule.
  • LFA lateral flow assay
  • the lysis subunit includes dry or freeze dried lysis components. In some implementations, the lysis subunit includes freeze dried lysis components. In some implementations, the amplification subunit includes dry or freeze-dried reverse transcriptase (RT), RNase H, RNA polymerase, oligonucleotide primers, an RT-RPA, RT-LAMP. In some implementations, the amplification subunit includes freeze-dried reverse transcriptase (RT), RNase H, RNA polymerase, oligonucleotide primers, an RT-RPA, RT-LAMP. In some implementations, the amplification subunit includes dry or freeze-dried RNase H.
  • the amplification subunit includes dry or freeze-dried RNA. In some implementations, the amplification subunit includes dry or freeze-dried polymerase. In some implementations, the amplification subunit includes dry or freeze-dried oligonucleotide primers. In some implementations, the amplification subunit includes dry or freeze-dried RT-RPA. In some implementations, the amplification subunit includes dry or freeze-dried RT-LAMP. In some implementations, the reporter activation subunit includes dry or freeze-dried cell-free lysate, cell-free transcription and translation reactions, Cas13 or Cas12 collateral cleavage enzyme, one or more guide RNA, and a corresponding dry or freeze-dried reporter molecule.
  • the reporter activation subunit includes freeze-dried cell-free lysate, cell- free transcription and translation reactions, Cas13 or Cas12 collateral cleavage enzyme, one or more guide RNA, and a corresponding dry or freeze-dried reporter molecule.
  • the reporter activation subunit includes dry or freeze-dried cell-free lysate.
  • the reporter activation subunit includes dry or freeze-dried cell-free transcription and translation reactions.
  • the reporter activation subunit includes dry or freeze-dried Cas13 collateral cleavage enzyme, one or more guide RNA, and a corresponding dry or freeze-dried reporter molecule.
  • the reporter activation subunit includes dry or freeze-dried Cas12 collateral cleavage enzyme, one or more guide RNA, and a corresponding dry or freeze-dried reporter molecule.
  • the detection unit includes a lateral flow assay (LFA) configured to the detect the reporter molecule and a cleaved product of the reporter molecule.
  • the dry or freeze dried lysis components include less than 1% non-ionic surfactants (e.g., less than 0.1%).
  • the LFA includes a sample region in contact with the reporter activation subunit, a control line configured to change color upon contact with the intact reporter molecule, one or more detection lines configured to change color upon contact with the cleaved products of the reporter molecules, and an adsorption zone or well at a position distal from the sample region.
  • the reporter activation subunit includes Cas12a and FAM-Biotin probe, and the reporter molecule is a FAM-biotin probe.
  • the lysis subunit includes a lysis chamber
  • the amplification subunit includes an amplification chamber
  • the reporter activation subunit comprise a reporter activation chamber
  • first flow channel connecting the lysis chamber and amplification chamber
  • second flow channel connecting the amplification chamber to the reporter activation chamber
  • third flow channel connecting the reporter activation chamber to the detection unit.
  • one or more of the first flow channel, the second flow channel and the third flow channel includes a time delay barrier.
  • the first flow channel includes a time delay barrier.
  • the second flow channel includes a time delay barrier.
  • the third flow channel includes a time delay barrier.
  • the time delay barrier is a dissolvable membrane or dissolvable material.
  • the dissolvable membrane incudes polyvinyl alcohol (PVA), pullulan, sugars such as sucrose, inorganic salts, patterned hydrophobic solids, or other compounds to provide a fluidic delay based on solubility.
  • the time delay barrier is PVA.
  • the dissolvable material is compatible with the biological functioning of reagents found in the lysis chamber, amplification chamber, reporter activation chamber, or detection unit. The dissolvable material can allow spatial segregation of Cas12, Cas13, or Cas14 enzymes from other reactions.
  • the time delay barrier is integrated into the porous material of the device.
  • the lysis subunit, the amplification subunit and the reporter activation subunit is configured as a layered micro-pad ( ⁇ PAD) or any other geometrically arranged porous material in which the fluid path is controlled to allow flow between different reaction units.
  • the geometrically arranged porous material or ⁇ PAD includes time delays (e.g., PVA barriers).
  • the ⁇ PAD is in a fold over accordion configuration having the chambers and flow channels defined by a hydrophobic material (e.g. wax).
  • one or more of the chamber subunits and one or more of the flow channels includes a blocking agent disposed on an internal surface of the chambers/flow channels (e.g. BSA/Triton X-100).
  • the one or more chamber and one or more flow channels includes a dissolvable material.
  • the device mass is less than 10g (e.g., less than about 8, 5, or 3 g).
  • the device is light enough to wear as a mask or hold in one hand as a device such as a breathalyzer.
  • the lysis subunit, the amplification subunit, and the reporter activation subunit are geometrically layered porous material in which the fluid path is controlled to allow flow between different reaction units.
  • the layered porous material can be in a stack configuration as opposed to a fold over accordion configuration.
  • Each stack of the lysis subunit [00159]
  • the carrier fluid reservoir holds water (e.g. at least 90% water).
  • the carrier fluid reservoir holds nucleases free water.
  • the detection unit provides a visual output, a fluorescent output and/or an electrical output. In some implementations, the detection unit provides a visual output. For example, an LFA showing colored bands.
  • the detection unit provide a fluorescent output.
  • the detection unit provides an electrical output.
  • the unit provides a combination of one or more outputs, such as a visual output an and electrical output; or a visual output and a fluorescent output; or an electrical output and a fluorescent output.
  • the sample collection unit can be included in a breathalyzer for collecting the airborne sample, and the sample collection unit can be removed from the breathalyzer for further processing to characterize the airborne sample.
  • the sample processing unit can be used for analyzing any sample in a lab and does not need to be incorporated in a same device as the other individual modules.
  • Methods of using the Devices implementable as, for example, a face covering, mask or air monitor.
  • Some implementations include methods of detecting an airborne agent. The methods use the devices implementable as, for example, a face covering, mask or air monitor described herein.
  • the methods include: contacting air including the airborne agent to the sample collection unit of the device, wherein the sample unit collects the airborne agent on a surface of the sample unit; causing a carrier fluid from the carrier fluid reservoir to flow to the sample collection unit, the carrier fluid subsequently flowing to the sample processing unit and contacting the dried synthetic biological components; and reading an output from the detection unit, the output indicative of the presence of the airborne agent in the air.
  • the airborne agent is a pathogen, a virus or a toxin.
  • the airborne agent is a virus.
  • the virus is SARS-CoV-2.
  • the airborne agent is nucleic acid (e.g., environmental DNA or RNA derived from lysed bacteria, viruses, or hot cells).
  • the airborne agent is an aerosol (e.g. a colloidal suspesions of the agent suspended in air).
  • the air includes respiratory droplets or other exhalations from a subject.
  • the respiratory droplets or other exhalations comprises the airborne agent.
  • contacting the air to the sample collection unit lasts for at least 10 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at least 20 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at least 30 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at least 60 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at least 120 min prior to causing the carrier fluid to flow to the sample collection unit.
  • contacting the air to the sample collection unit lasts for less than 120 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for less than 60 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at less than 30 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at less than 20 min prior to causing the carrier fluid to flow to the sample collection unit. In some implementations, contacting the air to the sample collection unit lasts for at least 10 min prior to causing the carrier fluid to flow to the sample collection unit.
  • a positive output for the agent is indicated within 12 hours (e.g.9, 6, 3 hours) of contacting the air with the sample collection unit. In some implementations, a positive output for the agent is indicated within 9 hours of contacting the air with the sample collection unit. In some implementations, a positive output for the agent is indicated within 6 hours of contacting the air with the sample collection unit. In some implementations, a positive output for the agent is indicated within 3 hours of contacting the air with the sample collection unit. In some implementations, a positive output for the agent is indicated within 1 hour of contacting the air with the sample collection unit. In some implementations, a positive output for the agent is indicated within 30 min of contacting the air with the sample collection unit.
  • the carrier fluid includes water or buffer and after causing the carrier fluid from the carrier fluid reservoir to flow to the sample collection unit, the fluid flows through the sample collection unit, and the sample processing unit, and the water hydrates at least a portion of dry materials therein when the water reaches the sample collection unit, and the sample processing unit.
  • the fluid after causing the carrier fluid from the carrier fluid reservoir to flow to the sample collection unit, the fluid flows sequentially through each of the sample collection unit, the sample processing unit, and the detection unit substantially by capillary action.
  • flow through to or through one or more of the units is provided by a pump, such as a micro pump.
  • flow through to or through one or more of the units is at least partially driven by gravity.
  • Some implementations include methods of making an aqueous solution activated detection device.
  • the methods include connecting in series a carrier fluid reservoir, a sample collection unit, a sample processing unit, and a detection unit.
  • an outlet from the carrier fluid reservoir is connected to an inlet of the sample collection unit
  • an outlet from the sample collection unit is connected to an inlet of the sample processing unit
  • an outlet of the sample processing unit is connected to an inlet of the detection unit.
  • the sample proceesing unit includes dried synthetic biological components.
  • the method includes adding wet biological components to the processing unit and in situ lyophilization (e.g.
  • the dry biological components are added in a dry state to the processing unit.
  • Some implementations further include positioning a valve at the outlet from the carrier fluid reservoir.
  • Some implementations further include providing a pressure release vent to the carrier fluid reservoir by punching a hole in a wall of the carrier fluid reservoir and covering the hole with a film of breathable material.
  • the breathable material is a material allowing air to pass therethrough while not allowing larger molecules such as water to pass through.
  • the breathable material is a rayon breathable hydrophobic porous film.
  • Some implementations further include providing a one-way air valve that allows air into the carrier fluid reservior to offset building up of vacuum.
  • the sample collection unit is made by cutting a porous sheet into a strip, the strip having a central collection region and two opposite distal regions, where a first distal region forms the inlet of the sample collection unit and a second distal region forms an outlet of the sample collection unit.
  • the central collection region is broader (e.g. elliptical in shape) than the first and second distal regions.
  • the porous sheet comprises fibers wherein a majority (e.g > 50%) of the fibers are aligned in a direction pointing from the first distal region to the second distal region.
  • the porous material includes fibers or threads and the majority (e.g.50%) of the fibers or threads are aligned in a direction orthogonal to the direction pointing from the first distal region to the second distal region.
  • one side of the sheet includes a hydrophobic barrier.
  • the sheet is covered or coated with a hydrophobic, non-porous or non-absorbent material, or layer of a hydrophobic, non-porous or non-absorbant material.
  • the sample processing unit is configured as a ⁇ PAD and the method further includes: (a) printing a hydrophobic ink or material (e.g.
  • a hydrophilic sheet e.g. filter paper
  • the hydrophobic ink diffusing through the hydrophilic sheet and forming an array of bounding features
  • one or more of the fluid outlets includes a time delay barrier.
  • a dissolvable polymer can be coated at or on the fluid outlet to form a dissolvable plug.
  • each of the fluid outlets have a volume that is smaller than a volume of each of the subunits.
  • the method further includes depositing lysate components in the lysis subunit, depositing amplification components in the amplification subunit, and depositing reporter activation components in the reporter activation subunit. In some implementations the method further includes depositing lysate components in the lysis subunit. In some implementations the method further includes depositing amplification components in the amplification subunit.
  • the method further includes depositing reporter activation components in the reporter activation subunit.
  • depositing comprises applying the components in an at least partially dissolved state to the units, and removing the water by, for example, drying or freeze drying.
  • the method further includes further attaching the carrier fluid reservoir, the sample collection unit, the sample processing unit, and the detection unit to a face mask, wherein the sample collection unit is attached to an inner surface of the face mask.
  • the carrier fluid reservoir is positioned on an outside surface of the face mask, and the outlet from the carrier fluid reservoir, inlet of the sample collection unit, or an area proximate to this inlet and outlet is passed from the outer surface of the face mask, to the inner surface of the face mask through a hole.
  • the carrier fluid reservoir is attached to the inside surface of the face mask.
  • the carrier fluid reservoir is integrated into an interior of the face mask.
  • connecting in series includes applying a water proof adhesive to matching surfaces of the outlet of the fluid reservoir and inlet of the collection unit, applying a water proof adhesive to matching surfaces of the outlet from the sample collection unit and inlet of the sample processing unit, and applying a water proof adhesive to matching surfaces of the outlet of the sample processing unit and inlet of the detection unit, wherein the water proof adhesive bridges any gap between the corresponding surfaces.
  • Diagnostic systems include applying a water proof adhesive to matching surfaces of the outlet of the fluid reservoir and inlet of the collection unit, applying a water proof adhesive to matching surfaces of the outlet from the sample collection unit and inlet of the sample processing unit, and applying a water proof adhesive to matching surfaces of the outlet of the sample processing unit and inlet of the detection unit, wherein the water proof adhesive bridges any gap between the corresponding surfaces.
  • a diagnostic system includes a biochemical reaction composed of a combination of reverse transcriptase, nucleic acid-modifying enzymes, amplification enzymes, cell-free lysate, Cas13, Cas12, or Cas14 enzymes, and one or more nucleic acids which may contain complete or partial nucleic acid sequences, including the reverse complement and in DNA or RNA, as disclosed herein (e.g. Table 7).
  • other substrates including ATP, reaction enhancers such as BSA or polyethylene glycol, and freeze drying enhancers such as phosphates and glycine are combined with one or more of the reverse transcriptase, nucleic acid-modifying enzymes, amplification enzymes, cell-free lysate, Cas13, Cas12, or Cas14 enzymes, and one or more nucleic acids.
  • other substrates include ATP.
  • other substrates include reaction enhancers such as BSA.
  • other substrates include freeze drying enhancers such as phosphates, disaccharides such s trhalosse and mannose, and glycine.
  • the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").
  • other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the invention ("consisting essentially of”). This applies equally to steps within a described method as well as compositions and components therein.
  • small molecules refers to natural or synthetic molecules including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms
  • FIGs.6B, 7A These prototypes were assembled layer-by-layer to form reaction chambers fluidly connected to top sample portals (FIGs.6B, 7A).
  • the devices are flexible, elastic, and can rapidly wick in splashed fluids through capillary action (FIGs. 6C,6D). Pinning geometries throughout the device direct sample fluids towards enclosed hydrophilic paper networks allowing for reaction rehydration (FIGs. 6B and 8B).
  • CPRG chlorophenol red- ⁇ -D-galactopyranoside
  • FIG. 6F a toehold switch for Ebola virus RNA detection
  • FIG. 6H a theophylline riboswitch for small-molecule sensing
  • Genetic circuits using transcriptional regulators are among some of the most common elements used in synthetic biology.
  • the wFDCF TetR sensor demonstrates the capacity of the colorimetric platform for facile integration of well-established genetic modules into a wearable format (FIG. 6F).
  • toehold switches have been developed as highly programmable nucleic acid sensors capable of detecting any target RNA. It was shown that a wFDCF Ebola virus RNA toehold sensor in the wearable device is capable of rapid and sensitive detection of biothreats (FIG. 6G).
  • Similar viral or bacterial wearable nucleic acid sensors can be made. Furthermore, a functional theophylline riboswitch wFDCF circuit is functionally validated in these platforms for the environmental detection of small molecules via engineered cis-regulated RNA circuits (FIG. 6H). This specific riboswitch was selected as a model test case, although a plethora of similar riboswitches for various targets have been reported and can be used in a modular fashion. All of the colorimetric wFDCF sensors reported here exhibited visible changes within ⁇ 40-60 min after exposure to the respective trigger molecules or inducer, and were performed at ambient conditions of 30-40% RH and 30oC to simulate the average skin surface temperature.
  • FIGs. 9A-9G presents various demonstrations of a highly sensitive, textile- based system (FIGs. 9A, 9B) capable of containing and monitoring the activation of wFDCF reactions with fluorescent (FIGs.9C-9E, 11-14) or luminescent (FIGs.9F, 15A-15B) outputs.
  • a second wearable platform was made that integrates: (a) hydrophilic threads (85% polyester / 15% polyamide) for cell-free reagent immobilization, (b) patterns of skin-safe hydrophobic silicone elastomers for reaction containment, and (c) inter-weaved polymeric optic fibers (POFs) for signal interrogation (FIGs. 9A, 9B, 16A-16D, 17A-17G).
  • This fabric was chosen as the main immobilization substrate after conducting a compatibility screening of over 100 textiles (eg silks cotton rayon linen hemp bamboo wool polyester polyamide nylon and combination materials) using a lyophilized constitutive lacZ cell-free reaction FIGs. 18A, 18B, 19A, 19B, 20).
  • FIGs. 9B, 21A-21F The analysis of sensor outputs was done using a custom-built wearable POF spectrometer (FIGs. 9B, 21A-21F) that could be monitored with a mobile phone application (FIGs.22A-22C).
  • FIGs.9C-9F A sample activation through fluid splashing can be seen in FIG.9A, where the sample wicks through the entry ports with blackout fabrics to rehydrate the freeze-dried, cell-free synthetic biology reactions immobilized within the hydrophilic textile fibers. These fibers are located within the excitation and emission layers of the device as shown in FIG. 9A, 9B.
  • a third fluorescence demonstration was done via activation of a 49-nucleotide Broccoli aptamer (FIG.9E) with substrate-specificity to (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl- 1H-imidazol-5(4H)-one (DFHBI-1T), evincing functionality of this emerging class of fluorescent sensors in synthetic biology. Furthermore, demonstrations utilizing luminescence outputs were conducted using a nanoLuciferase operon downstream of an HIV RNA toehold switch (FIG. 9F, 15A), as well as a B. burgdorferi RNA toehold switch for the wearable detection of Lyme disease (FIG.15B).
  • the wearable nerve agent sensor was validated using paraoxon-ethyl as a nerve agent simulant at levels that are four orders of magnitude lower than the reported lethal dose (LD 50 ) by dermal absorption in mammals (FIG.9G).
  • LD 50 lethal dose
  • FIG.9G dermal absorption in mammals
  • the light emitted from the activated system is then collected by the second set of emission POFs, which exit the fabric weave and bundle into a connection to the optical sensor (FIG. 9B) of the wearable spectrometer (FIGs.17A-17G, 21A-21F).
  • Signals coming from each of the devices are filtered (FIGs. 17D) and processed to generate temporally and spatially resolved fluorescence images of the POF bundle-ends (510 nm) and averaged pixel intensity traces per channel for quantitative analysis (FIG. 9B).
  • all POFs bundles are treated as signal inputs, without the need for sample illumination.
  • CRISPR programmable clustered regularly interspaced short palindromic repeat
  • Cas CRISPR-associated enzymes
  • the advantages of CRISPR-based systems over existing biosensors include high sensitivity, rapid output, single base-pair resolution, freeze- drying compatibility, and the notable programmability to target any DNA or RNA sequence through interchangeable guide RNAs (gRNAs).
  • Cas13a and Cas12a were used for the detection of RNA and DNA, respectively.
  • Cas12a ortholog from Lachnospiraceae bacterium LbaCas12a was used that displays a non-specific collateral cleavage activity towards single- stranded DNA (ssDNA) after detection of a gRNA-defined double-stranded DNA (dsDNA) target.
  • This Cas12a-based sensor was paired with recombinase polymerase amplification (RPA) and freeze-dried into a one-pot reaction to demonstrate state-of-the-art detection limits for wearable clinical applications.
  • RPA recombinase polymerase amplification
  • isothermally generated RPA amplicons activate Cas12a-gRNA complexes.
  • active Cas12a engages in trans-ssDNase activity and cleaves quenched ssDNA fluorophore probes, resulting in a fluorescence output (FIG. 23A).
  • gRNAs were designed against three common resistance markers in Staphylococcus aureus: specifically, the mecA gene common in methicillin-resistant S. aureus (MRSA), the spa gene which encodes the protein A virulence factor, and the ermA gene conferring macrolide resistance.
  • MRSA methicillin-resistant S. aureus
  • spa spa gene which encodes the protein A virulence factor
  • ermA gene conferring macrolide resistance When tested in wFDCF format, the RPA-Cas12a sensors displayed detectable signals within 56-78 min (P ⁇ 0.05) with femtomolar limits of detection (FIGs.23B-23D).
  • femtomolar limits of detection FIGs.23B-23D
  • each device was splashed with dd-H 2 O containing different targets, each specific to only one Cas12a-gRNA complex.
  • the orthogonal behavior of the CRISPR-based wearable sensors is shown in FIGs. 23G-23H, where higher fluorescence was observed for the cases in which the dsDNA trigger matched the pre-defined Cas12a-gRNA complex at each sensor location.
  • a jacket was designed that contained a distributed arrangement of wFDCF multi-sensor arrays (FIG. 23I).
  • the various optical fibers carrying the output emission signals can be routed into a single bundle for centralized imaging analysis or interrogated as separate modules, which was demonstrate using a wFDCF CRISPR- Cas12a based MRSA-sensing array containing spa, ermA and mecA sensors that was activated in the wearable prototype with a fluid splash containing 100 fM of spa DNA trigger (FIG. 12). Only the well containing the spa sensor generated a fluorescent signal upon activation.
  • the platform is also compatible with transcription-only outputs, such as rehydrated fluorescent aptamer reactions (FIG. 13), where the fluorescence signal is monitored by microscopy over time.
  • the optical sensor allows for facile fluorescent output multiplexing simply by using fluorescent proteins with orthogonal emission profiles (FIG.14).
  • wFDCF reactions for three constitutively expressed fluorescent output proteins eforRed, dTomato, and sfGFP
  • additional fluorescent outputs including orthogonal quenched fluorophore probes for SHERLOCK-based sensors, can be employed to increase the signal multiplexing of this wearable platform.
  • the wFDCF POF system is fully compatible with integrated lyophilized lysis components, allowing for the release and detection of a plasmid-borne mecA gene when challenged with intact bacterial cells (FIGs. 26A-26D).
  • the detector system was integrated with a custom wireless mobile application that enables continuous cloud-based data logging, signal processing, geolocation tracking, and on-the-fly control of various detector components through a smart phone or other networked digital device (FIG.23J). All images and spectral data presented in FIGs. 9A-9G, 23A-23J were collected and processed using wFDCF devices fully integrated with the wearable spectrometer and mobile phone application.
  • FIGs. 21A-21F Further details on the hardware (FIGs. 21A-21F) and software design (FIGs.22A-22C), as well an implementation of a novel Opuntia microdasys bioinspired fluid collection add-on for improved sample harvesting and routing splashes outside of the sensor zones into the wFDCF modules (FIGs.27A-27D).
  • the wearable synthetic biology sensors demonstrated here thus imbue programmable and highly sensitive diagnostic sensing to protective apparel.
  • the wFDCF system are adapted to key wearable gear, face masks, that have been shown to be critical in reducing the transmission of this highly infectious virus.
  • face masks are placed on all incoming patients that are presumptive SARS-CoV-2 carriers, confirmation through burdened laboratory diagnostics may result in delays that could negatively impact rapid triaging or effective contact tracing of patients.
  • Patients suspected of an infectious respiratory disease are fitted with a face mask upon clinical admission as a preventative measure to reduce transmission.
  • Diagnosis is commonly undertaken by nasopharyngeal sampling, which may cause reflexive sneezing and increase exposure risk to clinical workers.
  • Respiratory droplets and aerosols are the transmission routes for respiratory infectious diseases, but their use as a non-invasive diagnostic sample has been underutilized historically.
  • the SARS-CoV-2 face-mask NAT sensor Unlike other current nucleic acid tests (NATs) that require laboratory equipment and trained technicians, the SARS-CoV-2 face-mask NAT sensor describe here requires no power source, operates autonomously without liquid handling, is shelf-stable, functions at near-ambient temperatures, provides a visual output in under 2 hours, and is only ⁇ 3 g in weight. All the user has to do is press a button to activate a reservoir containing nuclease-free water. [00195]
  • the SARS-CoV-2 sensor contains four modular components: a reservoir for hydration, a large surface area collection sample pad, a wax-patterned ⁇ PAD (microfluidic paper-based analytical device), and a lateral flow assay (LFA) strip (FIGs. 28A and 28B).
  • Each module can be oriented on the outside or inside of the face mask, with the exception of the collection pad, which must be positioned on the mask interior facing the mouth and nose of the patient.
  • Capillary action wicks any collected fluid and viral particles from the sample collection pad to the ⁇ PAD, which contains an arrangement of freeze-dried lysis and detection components (FIG. 28C).
  • the use of the ⁇ PAD format allowed us to rapidly prototype and optimize a passively regulated multi-step reaction process.
  • Each reaction zone is separated by polyvinyl alcohol (PVA) time delays that enable tunable incubation times between each reaction, greatly improving the efficiency of the sensor compared to that of a one-pot lyophilized reaction.
  • PVA polyvinyl alcohol
  • the first ⁇ PAD reaction zone contains lyophilized lysis reagents including components known to lyse viral membranes.
  • the second ⁇ PAD reaction zone is an RT-RPA reaction zone containing a customized isothermal amplification reaction developed to target a non-overlapping region of the SARS-CoV-2 S gene.
  • the final ⁇ PAD reaction zone contains a Cas12a SHERLOCK sensor with an optimized gRNA for detection of the amplified dsDNA amplicon. In the presence of SARS- CoV-2 derived amplicons, the activated Cas12a enables trans-cleavage of a co-lyophilized 6- FAM-(TTATTATT)-Biotin ssDNA probe.
  • the first, second, and final ⁇ PAD reaction zones are temporally and spatially separated.
  • the ⁇ PAD is merely used as an example but other material can be used. Although three reaction zones are disclosed, any number of reaction zones may be achieved (e.g., two reaction zones, four reaction zones, ten reaction zones, etc.).
  • the spatial separation is achieved with separate reaction zones on separate pieces of material
  • the temporal separation is achieved with dissolving membranes. The dissolving membranes allow automated sequence of reactions in order. For example, a first reaction in the first reaction zone ends before a second reaction in the second reaction zone starts.
  • the dissolving membrane separating the first reaction zone and the second reaction zone times the start of the second reaction.
  • This methodology of separating reactions into reaction zones allows attaining higher sensitivity compared to a one-pot reaction where the first, second, and third reactions are mixed together. Ordered reaction as described herein preserves efficiency of each reaction when compared to the one-pot reaction. The spatial separation improves efficiency in labs so that a technician is not required to monitor and move materials between reactions.
  • This methodology is compatible with CRISPR.
  • PVA is used as an example of the dissolving material, but other materials can be used or substituted in cases where PVA may not be inert or may affect the sequenced reactions. [00197] From activation of the face-mask sensor to a final readout only takes ⁇ 1.5 hours.
  • the limit of detection observed for the sensors is 500 copies (17 aM) of SARS-CoV-2 in vitro transcribed (IVT) RNA, which matches that of WHO-endorsed standard laboratory-based RT- PCR assay (FIG. 28D-28E).
  • the sensors also do not cross react to RNA from other commonly circulating human coronavirus strains (HCoVs) (FIG. 28F). Most critically, the hands-off diagnostic reaction proceeds to full completion even at room temperature, which is considered sub-optimal for RT, RPA, and Cas12a activities.
  • the SARS-CoV-2 face-mask sensor was also validated using a precision lung simulator attached to a high-fidelity human airway model (FIGs. 28H, FIG 32E).
  • the target RNA was nebulized to replicate lung emissions with aerosol diameters matching those naturally occurring in breath exhalation plumes.
  • the breath temperature was regulated to 35°C and the relative humidity in the mask microclimate was measured to be 100% RH.
  • the face-mask sensor was able to detect SARS-CoV-2 vRNA after a breath sample collection period of 30 minutes, with a calculated accumulation of 106-107 vRNA copies on the sample pad, as determined by RT- qPCR (FIG. 28A-28J). Clinical measurements have previously shown that the SARS-CoV-2 breath emission rate of infected patients could reach an output 103-105 copies/min.
  • SARS-CoV-2 NAT This is the first SARS-CoV-2 NAT that is able to achieve high sensitivity and specificity while operating fully at ambient temperature ranges, thus obviating the need for any heating instruments and allowing for integration into a wearable format.
  • the rapid face-mask-integrated SARS-CoV-2 diagnostic presented here can relieve strained medical systems by combining protection and sensing into a simple and easy-to-deploy wearable system, greatly improving patient outcomes.
  • This face-mask system can be adapted to discriminate between SARS-CoV-2 and other respiratory viruses, as well as different emerging SARS-CoV-2 variants, allowing rapid triaging of patient populations and isolation of specific positive cases to minimize the spread of infection.
  • the wFDCF platform are complementary to cell-based synthetic biology sensors.
  • Such living sensors are capable of self-replication, can operate continuously to provide dynamic sensing, and they can actively draw upon environmental resources for energy.
  • storage and biocontainment concerns limit their use for wearable technologies.
  • cell-free synthetic biology systems can be used to build practical wearable biosensors that are shelf-stable, genetically programmable, and highly sensitive.
  • the wFDCF sensors are responsive to external rehydration events, such as splashes with contaminated fluids, and withstand inhibitory evaporative and dilutive effects in open- environment conditions (30-40% RH and ⁇ 25-30oC). These freeze-dried systems generate measurable colorimetric, fluorescence, or luminescence outputs upon exposure to relevant real- world targets.
  • wFDCF POF sensors continuous monitoring enables rapid alert to an exposure event.
  • the integration of these device designs into garments that are compatible with wireless sensor networks to provide real-time dynamic monitoring of exposure using custom smartphone applications is also demonstrated.
  • laboratory testing may be more sensitive, the wFDCF sensors have the distinct advantages of a wearable format, autonomous functioning, and rapid results.
  • the presented platform is the first wearable technology demonstrated to detect nucleic acids from potential viral or bacterial pathogens in contaminant fluid samples with sensitivities rivaling those of traditional laboratory tests at ambient temperatures.
  • the wFDCF platform evinces a number of distinct advantages over existing POC diagnostics, which similarly attempt to eliminate the need for time-consuming laboratory tests.
  • wFDCF synthetic biology sensors can be networked to provide sensing arrays of lyophilized reactions and lightweight polymer fabrics, thus cloaking the user and continuously generating high-density, real-time outputs without sacrificing comfort or agility in the field.
  • the platform is also designed to operate autonomously, unlike most current POC instruments that require training for use and multiple operations by the user to acquire the final results. This feature removes the need to perform regular exposure checks, freeing those in the field to focus on their core tasks.
  • these modular wearable sensors can detect environmental threats or patient samples through nucleic acid, protein, or small molecule detection.
  • electrochemical sensors have been integrated into a wearable format, they only detect chemicals and an easily programmable wearable form for sensitive nucleic acid detection does not exist to date.
  • the wFDCF components are inexpensive, with cell-free reactions costing only $0.01-0.03 per ⁇ L.
  • a single 10 mm-diameter sensor would currently only cost ⁇ $1 in reagents.
  • the optical fiber textiles are woven from common polymer fibers, and are also inexpensive.
  • the wearables could be utilized as disposable protective garments with advanced sensing technology.
  • the sensors are also highly modular and adapted to various form factors, such as clothing.
  • Field applications that would greatly benefit from these wFDCF synthetic biology platforms include soldiers and first-responders (e.g., Hazmat personnel, Firemen) operating in environments where a specific chemical or biological threat is suspected.
  • the apparel of disposable wFDCF sensors could be used to maintain situational awareness, with continuous spatio-temporal monitoring of exposure and bodily resolution down to centimeters.
  • Another set of potential uses for this platform involves the environmental awareness of clinicians, health workers, and researchers working in high-risk areas.
  • the wearable sensing platforms could enable rapid responses to contagion so that any exposed users could begin decontamination and neutralization procedures immediately.
  • wFDCF-enabled coats and gowns in hospitals could provide alerts to prevent the spread of nosocomial infections to vulnerable populations, such as immune-compromised patients or newborns.
  • An additional promising application is patient-worn sensor-enabled wearables such as the face mask presented here that can provide inexpensive, shelf-stable, and labor saving POC diagnostics to rapidly inform clinicians in outbreak events, such as the current COVID-19 pandemic that has rapidly overwhelmed the resources of worldwide medical infrastructures.
  • any animal such as mammals, can use the wFDCF.
  • a dog associated with a soldiers and first responders can be deployed with or separated with the associated human.
  • the wFDCF can be attached to a robot sent in a hazardous environment.
  • the wFDCF can be taken off (e.g., the human, dog, robot) and left to collect and relay or monitor a specific chemical or biological threat.
  • Fabrication of colorimetric synthetic biology wearable modules [00203] Translucent (FIG. 6B top) and opaque (FIG.
  • 6B middle/bottom layers were made using skin-safe ECOFLEX® silicone elastomer (Smooth-On, Inc, Macungie, PA), precast overnight and laser-cut on a 75W Epilog Legend 36EXT according to the layouts shown in FIG. 6B and 7A.
  • the silicone pieces were placed in a warm wash (45°C) with TERGAZYME® detergent (Alconox, Inc., White Plains, NY) for one hour with agitation, followed by three washes in 18- ⁇ pure water and a final wash in 70% ethanol, before allowing them to air dry.
  • reaction disks saturated with the cell-free reaction components were finally snap-frozen in liquid nitrogen and freeze-dried for 8 - 12 hours in an SP Scientific Freezemobile lyophilizer (SP Industries, Inc., Warminster, PA).
  • SP Scientific Freezemobile lyophilizer SP Industries, Inc., Warminster, PA.
  • Freeze-dried reaction disks were then inserted through the wicking ports of the elastomer chambers for assembly.
  • the silicone elastomer chambers in the colorimetric device exhibit three 3 x 5 mm curved wicking ports in each of the four reaction chambers, which allow routes for fluid entry while delaying evaporation of cell-free reaction (FIG. 7A).
  • the device chamber walls were aligned and bonded using uncured elastomer, to prevent flow or lateral diffusion of the reaction after rehydration.
  • each colorimetric wFDCF reaction used for lyophilization was a 75 ⁇ L cell-free NEB PUREXPRESS® reaction (New England Biolabs, Inc., Ipswich, MA).
  • NEB PUREXPRESS® reaction New England Biolabs, Inc., Ipswich, MA
  • each rehydrated reaction is a 1.5x-concentrated cell-free reaction based on the suggested reaction composition indicated by the manufacturer.
  • Each reaction consisted of: 30 ⁇ L of PUREXPRESS® Component A, 22.5 ⁇ L of PUREXPRESS® Component B, 0.6 mg/mL of chlorophenol red- ⁇ -D-galactopyranoside (CPRG; MilliporeSigma, St.
  • CPRG chlorophenol red- ⁇ -D-galactopyranoside
  • TetR transcriptional regulation circuit FPLC- purified recombinant TetR protein was supplemented in the reaction at a concentration of 120 ⁇ g/mL.
  • the Ebola RNA genome trigger was acquired by an in vitro transcription reaction utilizing the HISCRIBETM T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA), using a DNA template. Each wFDCF reaction was applied to a BSA-blocked cellulose disc inserted into a 2 mL microcentrifuge tube. After the reaction was absorbed into the disc, the tubes were submerged in liquid nitrogen to snap freeze the disc and allowed to lyophilize for 12 hours.
  • Each square was freeze-dried with 100 ⁇ L of a 1x PUREXPRESS® cell-free reaction with CPRG substrate and a constitutive LacZ plasmid.
  • Various temperature (27-32oC) and fluid exposure conditions were investigated in combination with different coverage ratios of the rehydrated test squares to assess evaporation reduction. Suitable activity of the rehydrated reactions was assessed by visual inspection of the conversion of the colorimetric substrate from yellow to purple.
  • the port designs shown in FIG.7A, 8B, 17G were selected empirically due to suitable activation of synthetic biology reactions with reduced evaporation rates ( ⁇ 20% of initial fluid volume in 2 hours) at 30-40% relative humidity. [00210] Kinetic enhancement by freeze-dried concentration of cell-free reaction components.
  • the time to half-maximal output signal for each base or concentrated reaction was calculated by a least square fitting of the acquired data.
  • Screening of textiles for freeze-dried cell-free synthetic biology reactions was tested in 103 different fabrics materials (e.g., silks, cotton, rayon, linen, hemp bamboo, wool, polyester, polyamide, nylon, and combination threads) under activation conditions (FIG. 18A- 18B). A detailed list of the textiles used for this substrate screening can be found in Table 1. This compatibility of these textiles to FDCF synthetic biology reactions was compared to samples using WHATMAN TM No.
  • Cell-free PUREXPRESS® in vitro protein synthesis solution (New England Biolabs, Inc., Ipswich, MA) was combined with a constitutive LacZ template containing 0.6 mg/mL CPRG and spotted (1.8 ⁇ L) on each of the fabric wells.
  • Control wells containing 2 mm disks of Whatman No.4 filter-paper were also filled with 1.8 ⁇ L constitutive LacZ test reactions, whereas 7 ⁇ L were spotted on empty wells as liquid controls.
  • a transparent adhesive PCR cover compatible with freezing was then placed over the plate and pressed with a roller to seal chambers.
  • Such POFs were used for distributed optical interrogation of fluorescent or luminescent synthetic biology reactions within this fabric (three fibers per well).
  • Polymeric optic fibers were weaved into this hydrophilic combination fabric using a standard industrial loom (DREAMLUX, Samsara Srl., Milan, IT), according to the design presented in FIGs 16A 16D
  • DREAMLUX Standard industrial loom
  • Samsara Srl. Milan, IT
  • FIGs 16A 16D Once fabric samples were manufactured three strip arrangements of this hydrophilic POF fabric were cut to fit the device and laser-etched (5 mm) to disrupt the cladding in the POFs sections within the reaction zones (FIGs. 17A-17G). Black elastomer layers (top and bottom in FIG.17B) were precast overnight and laser-cut according to the layout shown in FIGs.17B, 17E.
  • the silicone elastomer chambers in this device exhibit two 3 x 5 mm curved wicking ports that allow for fluid entry while still delaying evaporation within reaction fabric.
  • Uncured black silicone elastomer was stamp-patterned onto the precast layers as well as into the internal POF fabric strips to be aligned and assembled, preventing air bubble formation between device layers and elastomer wicking in reaction zones.
  • Final assembly of the base three- well sensor “patch” can be seen in FIGs. 17B, 17F, 17G. Devices were then placed under vacuum for 15 minutes to remove bubbles and were allowed to cure overnight at 65oC.
  • the fluorescent POF prototypes were thoroughly sprayed with RNase Away Decontaminant (Thermo Fisher Scientific, Waltham, MA) and washed with 70% ethanol twice before being stored in petri dishes. Once the assembled device was fully cured, POF fibers were separated into excitation and emission bundles and then covered with blackout adhesive fabric as well as black heat shrink tubing (6 mm) to prevent environmental light leakage. Blackout fabric disks (10 mm) made of black polyester knit Item#: 322323 (MoodFabrics Inc. New York, NY) were soaked in RNase Away Decontaminant for 5 minutes, washed thoroughly with 70% ethanol followed by water.
  • the washed blackout fabric was incubated in 0.1% Triton X-100 for 5 minutes (as a wetting agent to enhance the ability of the textile to absorb water) and then excess solution was removed and the fabric pieces allowed to air-dry.
  • the final blackout fabric discs were placed inside the reaction chamber with tweezers to aid in environmental light-blocking over sensing fibers.
  • quick-turn stainless steel coupling sockets #5194K42 (McMaster-Carr Co., Elmhurst, II) were added to the ends of the sensor device bundles for connection with the wearable spectrometer.
  • the finalized wFDCF sensor device can be seen in FIGs.17F, 17G.
  • FIG. 21A Hardware / software implementation of wearable POF spectrometer
  • FIG. 21A A custom-made wearable spectrometer with internal processing and wireless connectivity modules was fabricated to provide unsupervised sensing of on-body synthetic biology reactions.
  • the device electronics were based on a Raspberry Pi Zero W Version 1.3 architecture (Raspberry Pi Foundation, Cambridge, UK) with connection to a custom shield for battery power, an environmental sensing module, an LED illumination module, and a flexible camera for imaging (FIG. 21A).
  • the Raspberry Pi Zero W was selected as microprocessing for this application, due to its low cost ( ⁇ $15.00), small profile/weight (65 x 30 x 5 mm / 12 g) high performance (1 GHz single core ARM1176JZF S CPU 512 MB RAM VideoCore IV GPU) and on-board wireless connectivity (802.11 b/g/n LAN, Bluetooth(R) 4.1, Bluetooth Low Energy -BLE). Regulated battery power was achieved using a PiZ-UpTime module, which is an uninterruptible power supply shield for Raspberry Pi Zero (Alchemy Power Inc., Santa Clara, CA), which uses rechargeable a Lithium-Ion 14500 battery (Battery & Power management in FIG.
  • the wearable spectrometer was covered by a two-part case fabricated using black photoreactive resin and a stereolithography 3D printing method using a Form 2 printer (Formlabs Inc., Summerville, MA) as seen in FIG.21A.
  • FIG.21B A view of the open device is shown in FIG.21B, while a closed view is shown in FIG.21C.
  • This case included geometrical features to fit and align the camera/lens arrangement and the removable 3 mm diameter amber acrylic filter for fluorescence readings (slot arrangement in FIG. 21D). Also, the case features a slot for the 4-LED arrangement, a vent for the environmental sensors (FID.21D), as well as female Luer connection (FIG. 21A) to fit quick-turn stainless steel coupling sockets #5194K42 (McMaster-Carr Co., Elmhurst, II). A top view of the assembled wearable POF spectrometer is shown in FIG. 21E, while the integration of this device within a wearable garment with wFDCF sensors is shown in FIG. 21F.
  • the final volume of the wearable spectrometer device was approximately 235 cm 3 with a total weight of around 173.8 grams (6.13 ounces), with a total cost of material and consumable supplies under $100 USD.
  • Base data-collection software (test version) implemented in python for control of the Raspberry Pi Zero W within the wearable POF spectrometer was also provided. [00218] Preparation of optimized fluorescence wearable synthetic biology reactions.
  • Constitutive sfGFP expression reactions for wFDCF testing were prepared by combining 50 ⁇ L of 1x NEB cell-free PUREXPRESS® in vitro protein synthesis solution with 0.5% Roche Protector RNase Inhibitor and 10 ng/ ⁇ L constitutive PT7-sfGFP plasmid (+) or without as controls (-). Prepared reactions were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device. Activation of sensors was achieved by rehydration with a fluid splash of dd-H 2 O.
  • Theophylline riboswitch sensor reactions for wFDCF testing were prepared using 1x NEB cell-free PUREXPRESS® with 10 ng/ ⁇ L
  • Theophylline riboswitch sensor E mRNA in dd-H 2 O prepared sensor reactions (50 ⁇ L per well) were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device.
  • Activation of sensors was achieved by rehydration with a fluid splash of dd-H2O spiked with 1 mM theophylline for the positive samples, while 0 mM theophylline was used for controls.
  • Dimeric Broccoli fluorescent aptamer sensor reactions for wFDCF testing (FIG. 9E) were prepared using 1.5x NEB cell-free PUREXPRESS® with 25 ng/ ⁇ L of pJL1-F30-2xd- Broccoli aptamer DNA in dd-H 2 O. Prepared sensor reactions (50 ⁇ L per well) were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device.
  • Activation of sensors was achieved by rehydration with a fluid splash of dd-H2O spiked with 50 ⁇ M of the substrate (5Z)-5-((3,5-Difluoro-4-hydroxyphenyl)methylene)-3,5-dihydro-2-methyl-3 -(2,2,2-trifluoroethyl)-4H-imidazol-4-one (DFHBI-1T; Tocris Bioscience, Minneapolis, MN) substrate for the positive samples, while 0 ⁇ M DFHBI-1T substrate was used for controls.
  • DFHBI-1T Tocris Bioscience, Minneapolis, MN
  • the fluorescent wearable device for the nerve agent was altered for the detection of near-infrared fluorescence by replacing the optical components with excitation using a 627 nm red quad-LED array module (Quadica Developments Inc. - Luxeon, Alberta, Canada). Additionally, the emission camera was substituted with a NoIR Zero Spy Camera without infrared filter, on top of which was positioned three gel transmission filters No. 381, 382 and 383 (Rosco Laboratories Inc., Stamford, CT) to form a dedicated emission filtering stack with ⁇ 1% cutoff at 660nm and peak transmittance at 740nm.
  • HIV RNA toehold switch sensor reactions for luminescence wFDCF testing were prepared in 50 ⁇ L batches using 20 ⁇ L of NEB cell-free PUREXPRESS® Component A, 15 ⁇ L NEB Component B, 2.5 ⁇ L murine RNase inhibitor (New England Biolabs, Inc., Ipswich, MA), 6 ng/ ⁇ L HIV toehold sensor template with a nano luciferase (nLuc) output, 0.5 ⁇ L luciferin substrate (Promega Corp., Madison, WI) in dd-H 2 O.
  • 23B-23F were prepared using 100 nM Cas12a (New England Biolabs, Ipswich, MA) and 100 nM gRNA, 1x NEB buffer 2.1, 0.45 mM dNTPs, 500 nM of each RPA primer, 1x RPA liquid basic mix (TwistDx Limited, UK), 14 mM MgCl2, and 5 ⁇ M FAM-IOWA BLACK® FQ quenched ssDNA fluorescent reporter (Integrated DNA Technologies, Coralville, IA) in dd-H2O. Prepared sensor reactions (50 ⁇ L per well) were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device.
  • Base neoprene fabric used for this jacket was of 3mm thickness and treated with a superhydrophobic coating to prevent fluid absorption in places other than the reaction zones.
  • Fabricated wFDCF jacket prototype was specified to fit a medium-sized male torso 36"(chest) by 31"(waist). In- garment sensors were tested on a mannequin at room temperature. [00237] Sensor and reporter sequences Tables S2 and S3 contain the DNA and RNA sequences of sensors and reporters used in this study. The plasmid construct used for the Zika 27B toehold sensor has been previously described elsewhere.
  • the Lyme disease and HIV toehold sensors with a nanoluciferase output were cloned into the pBW121 plasmid backbone (Addgene plasmid #68779). All other plasmid constructs utilized the pJL1 backbone that has been previously described2, 3 .
  • the F30 dimeric Broccoli fluorescent aptamer was subcloned into pJL1 from pET28c-F30-2xdBroccoli which was a gift from Samie Jaffrey (Addgene plasmid #66843; www.n2t.net/addgene:66843; RRID:Addgene_66843).
  • FIGs. 6A-6H depict wearable cell-free synthetic biology.
  • FIG. 6A depicts freeze- dried cell-free reactions can be embedded in reaction sachets or chambers that are distributed throughout garments for use by soldiers, clinicians, and first responders. Upon exposure to an external splash, the reactions are rehydrated, activating dormant synthetic gene circuits that detect pathogens, metabolites, and toxins.
  • FIG. 6B depicts a schematic of the layer-by-layer assembly of the wearable devices.
  • FIG.6C depicts an array of assembled reaction chambers showing the elasticity (center) and flexibility (right) of the devices.
  • FIG. 6D depicts portals cut into the outermost layer allow sample access, which is rapidly drawn into the reaction chambers through capillary action. The hydrophobic chamber walls prevent inhibitory dilution through lateral diffusion.
  • FIGs. 7A-7C depict assembly layers and sample activation of colorimetric wFDCF reactions with constitutive P T7 ::LacZ module.
  • FIG. 7A depicts the layout of elastomer layers in the colorimetric wFDCF device.
  • FIG. 7B depicts activation of colorimetric prototype reaction chambers using 40 ng/ ⁇ L constitutive LacZ-T7 plasmid in a 50 ⁇ L rehydration splash as compared to FIG. 7C which depicts rehydration with no plasmid.
  • PURExpress reactions were conducted at 1.5x concentration. All the reactions were allowed to incubate at 30 ⁇ C, exposed to the ambient environment, and images were taken every 5 minutes. Color change in one replicate was visible in under 20 min. Each row depicts a representative single-well reaction.
  • FIGs. 8A-8C depict sample activation of wFDCF colorimetric devices and bracelet for detection of Ebola virus RNA.
  • FIG. 8A-8C depict sample activation of wFDCF colorimetric devices and bracelet for detection of Ebola virus RNA.
  • FIGs.9A-9G depict design and validation of fluorescent and luminescent freeze-dried cell-free synthetic biology wearables.
  • FIG. 9A depicts details of assembly and activation of fiber-optic based wFDCF module for fluorescence/luminescence output, with a schematic of module layers and components of embedded cell-free reactions. Fiber-optic embedded textiles allow excitation of the samples and detection by sensing emission light. A single layer of blackout cover made of polyester fabric is used to prevent the entry of environmental light into the reaction well. Bottom: An example rehydration event over the device shows the aqueous sample being wicked through the portals and blackout fabric and into internal reaction chambers.
  • FIG. 9B top depicts a diagram showing the layers of the assembled device.
  • FIG.9B bottom depicts a cross-sectional view of the interior of the device, where two layers of hydrophobically patterned fabric inter-woven with polymeric optic fibers are placed in a coplanar arrangement to allow for rehydration of freeze-dried cell-free reaction components as well as to provide light input/output for excitation and emission signals.
  • Excitation POFs are illuminated with a 447-470 nm LED arrangement, and emission fibers are bundled and aligned with an optical sensor containing an amber filter (for fluorescence readings only) and a collimating lens for magnification. The amber filter can be removed from the device in luminescence mode.
  • FIG.9C depicts a rapid fluorescent signal after rehydration of wFDCF constitutive sfGFP template as compared to control. Fluorescent signal in-device is statistically distinguishable from the control after 11 min (P ⁇ 0.05).
  • FIG. 9D depicts activation of FDCF riboswitch with 1 mM theophylline in a wearable device as compared to 0 mM theophylline control. Fluorescent signal in-device is statistically distinguishable from the control after 19.5 min (P ⁇ 0.05).
  • FIG.9E depicts a wearable demonstration of fluorescent aptamer being activated by the presence of 50 ⁇ M DFHBI-1T substrate as compared to 0 ⁇ M DFHBI-1T control.
  • FIG. 9F depicts luminescence output detected from an HIV toehold sensor with nanoLuciferase operon. HIV RNA trigger was added at 10 ⁇ M and was statistically distinguishable from the control after 6 min (P ⁇ 0.05) post- rehydration.
  • FIG. 9G depicts a wearable detection of organophosphate nerve agents using a lyophilized HRP-coupled enzyme sensor rehydrated with 50 mM acetylcholine with and without 3.7 mg/mL paraoxon-ethyl (acetylcholinesterase inhibitor).
  • FIGs. 10A-10D depict concentrating PURE cell-free reactions increases reaction kinetics.
  • FIG. 10A-10D depict concentrating PURE cell-free reactions increases reaction kinetics.
  • FIG. 10A depicts a schematic of reaction concentration through the lyophilization of PURExpress reactions at varying volumes followed by rehydration at a set volume. Using this method, synthetic biology reactions can be concentrated to enhance kinetics through molecular crowding effects or greater density of cell-free components per volume.
  • FIG. 10B depicts representative images of PURE reactions with a LacZ output over one hour, at various concentrations.
  • FIG. 10C depicts quantified PURExpress reactions with a LacZ output in triplicate; the error bars denote standard deviation.
  • FIG. 10D depicts the half-maximal values from curve fitting the data shown in FIG. 10D and indicate that the 1.5x concentrated PURE reaction accelerates the signal output by more than 10 minutes. Error bars are smaller than the data points. [00243] FIG.
  • FIG. 11 depicts Zika DNA Toehold sensor activation in single mercerized cotton thread.
  • sfGFP Zika DNA toehold sensor
  • FIG. 12 depicts antibiotic resistance sensors for spa, ermA and mecA genes using in- wearable sensor demonstrate specific orthogonality. Only reaction chambers with a Cas12a sensor targeting the S. aureus virulence factor-encoding spa-gene generates a detectable signal within 30 min.
  • FIG. 13 depicts POF fabric compatibility with lyophilized transcription-only fluorescent aptamer reactions. The left panel shows a picture of the fabric; the right panel shows a detail magnified view in.
  • FIG. 14 depicts sensor multiplexing using different fluorescent proteins can be detected in a single device.
  • the top row depicts cell-free reactions demonstrating different fluorescent protein outputs generated after 30 min at 30 ⁇ C. All tubes were photographed with illumination using an Invitrogen Safe Imager 2.0 G6600 Blue Light Transilluminator (Carlsbad, CA).
  • the bottom row depicts sensor images of fiber topic bundles in (1) brightfield (intense light is placed over the sensor regions to spatially locate each fiber), (2) image when the sensor is dry, (3) image when wFDCF reaction is hydrated but without plasmid (30 min incubation at 30 ⁇ C), and (4) image when wFDCF reaction is hydrated but with FP plasmids (30 min incubation at 30 ⁇ C).
  • FIGs. 15A-15B depict additional Nanoluciferase (nLuc) luminescence experiments.
  • FIG.15A depicts dynamic response of a wFDCF Lyme disease RNA toehold switch sensor with luminescence output.
  • 50 ⁇ L reactions consisting of 20 ⁇ L of NEB cell-free PUREXPRESS® Component A, 15 ⁇ L NEB Component B, 2.5 ⁇ L NEB murine RNase inhibitor, 19 ⁇ L Lyme disease toehold sensor DNA with nLuc reporter (6 ng/ ⁇ L), 0.5 ⁇ L luciferin substrate (Promega Corp., Madison, WI) and 19 ⁇ L dd-H2O.
  • Prepared sensor reactions (50 ⁇ L per well) were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device.
  • FIG.15B depicts dynamic response of a wFDCF HIV RNA toehold switch sensor with luminescence output in comparison to constitutive P T7 ::nLuc expression as a positive control (+), which was statistically distinguishable from the negative condition after 8 minutes (P ⁇ 0.05).
  • the HIV toehold reaction was prepared in 50 ⁇ L batches using 20 ⁇ L of NEB cell-free PUREXPRESS® Component A, 15 ⁇ L NEB Component B, 2.5 ⁇ L NEB murine RNase inhibitor, 19 ⁇ L HIV toehold sensor DNA template with a nanoLuciferase reporter (6 ng/ ⁇ L), 0.5 ⁇ L luciferin substrate (Promega Corp., Madison, WI) and 19 ⁇ L dd-H2O. Prepared sensor reactions (50 ⁇ L per well) were quickly deposited in-fabric to be snap-frozen and then lyophilized for 4-8 hours within the device.
  • FIG. 16A depicts clean 0.2 mm hydrophilic yarns made of 85% polyester and 15% polyamide were weaved in VELO style along the weft in combination with 0.25 mm un-etched poly(methyl methacrylate) POFs as warp using a standard industrial loom via Dreamlux's process (Samsara S.R.L., Milan, IT).
  • FIG. 16B depicts a three-fiber multi-strip design was achieved with a POF pitch of ⁇ 1 mm and intermediate POFs at 5 mm from the strip center for easy cutting.
  • the reaction zone was cut to be ⁇ 30 mm in length.
  • the width of the fabric roll was arbitrary, usually above 1 m depending on the used loom. Free POFs can then be detached from the un-weaved side to be bundled together.
  • 16C A roll of the hydrophilic POF fabric after weaving.
  • FIGs.17A-17G depicts a fabrication of textile-based wFDCF sensor patch.
  • FIG.17A depicts a cut strip of hydrophilic POF fabric was laser-etched (5 mm) to disrupt the POF outer cladding in the POFs sections closest to the reaction zone.
  • FIG. 17B depicts examples of prepared wFDCF fabric-elastomer layers and final assembly into a three-well sensor for garment integration. POFs in these devices were covered with black heat shrink tubing (6 mm).
  • Top elastomer cover features two 5.19 x 1.85 mm curved sample ports instead of three as in the colorimetric prototypes to reduce direct light leakage on top of the POFs that may cause background light detection.
  • FIG.17C is a schematic of a POF-fabric-elastomer strip for sensing in a single textile layer including two excitation fibers on the sides of an emission fiber.
  • FIG. 17D is a schematic of a double POF-fabric-elastomer strip for sensing with dedicated excitation and emission layers. This design was the one selected for further experiments due to higher hydrophilic fiber content and capacity to immobilize fluid for lyophilization.
  • FIG. 17C is a schematic of a POF-fabric-elastomer strip for sensing in a single textile layer including two excitation fibers on the sides of an emission fiber.
  • FIG. 17D is a schematic of a double POF-fabric-elastomer strip for sensing with dedicated excitation and emission layers. This design was the one selected
  • FIG. 17E is a schematic of a single excitation or emission POF-fabric-elastomer layer overlaid on an applied elastomer pattern for creating the impermeable reaction chambers.
  • FIG. 17F depicts a finalized three-well sensor wFDCF device with heat shrunk POF covers and Luer connectors for interface with a portable spectrometer device.
  • FIG.17G depicts top and bottom views of a final three-well sensor wFDCF device. The blackout fabric can be seen through the sample wicking ports and serve to prevent environmental light penetration into reaction chambers.
  • FIGs. 18A-18B depict textile substrate compatibility testing using synthetic biology reactions and sample colorimetric reaction.
  • FIG. 18A-18B depict textile substrate compatibility testing using synthetic biology reactions and sample colorimetric reaction.
  • FIG. 18A depicts samples of eight fabric types selected as part of the textile screening for wFDCF compatibility. Bottom icons indicate the environmental and hydration conditions that were monitored over time for analysis.
  • FIG. 18B depicts a sample wFDCF colorimetric activation in a 1 x 1 cm cellulose matrix square containing 75 ⁇ L of NEB cell-free PUREXPRESS® in vitro protein synthesis solution (New England Biolabs, Inc., Ipswich, MA) with 40 ng/ ⁇ L constitutive pJL1-LacZ plasmid.
  • FIGs. 19A-19B depict textile screening using model constitutive PT7::LacZ assay.
  • FIG.19A depicts a sample 384-well plate containing triplicates of BSA blocked and unblocked 2 mm discs of 30 different textile types after constitutive P T7 ::LacZ expression following a 12-hour run for reactions containing 1.8 ⁇ L of NEB cell-free PURExpress® in vitro protein synthesis solution (New England Biolabs, Inc., Ipswich, MA) with 40 ng/ ⁇ L constitutive pJL1-pLacZ plasmid (+) or without plasmid as controls (-).
  • FIG.19B depicts examples of qualitative traces of colorimetric signals for these different fabric disks using a plate spectrophotometer (420 nm absorbance).
  • FIG. 20 depicts a compilation of normalized functional scoring for colorimetric wF- DCF textile screening.
  • a normalized functionality score was calculated for each of the 103 evaluated fabrics tested for compatibility with freeze-dried PURExpress reaction generating a LacZ output. This score was generated by measuring six key parameters: peak absorbance intensity at 420 nm, reaction rate, time to maximum signal, lag-time, fabric fiber density and in- fabric autofluorescence, and then multiplying normalized scores for each of these measurements, penalizing longer times to maximum signal, long lag-times and high autofluorescence.
  • FIGs. 21A-21F depict fabrication of wearable microcontroller system with LED illumination and spectrometric capabilities.
  • FIG. 21A depicts an exploded isometric view of wearable POF spectrometer components with case and electronics.
  • the device electronics are based on a Raspberry Pi Zero W Version 1.3 (Raspberry Pi Foundation, Cambridge, UK), assembled with a PiZ-UpTime battery power board (Alchemy Power Inc., Santa Clara, CA), an environmental sensing module, an LED illumination module, and a flexible camera for imaging.
  • FIG.21B depicts a photograph of an open assembled device.
  • FIG.21C depicts a photograph of a fully assembled device ready for imaging.
  • FIG.21D depicts details of camera used in the device as well as the amber fluorescence emission filter and lens for magnification. Slots at the front of the bottom case fit the camera end, the LED arrangement and a vent for the environmental sensors.
  • FIG. 21E depicts a top view of an assembled device to provide detail of compact electronics arrangement.
  • FIG. 21F depicts an arrangement of wearable POF spectrometer with wireless connectivity in-garment for wFDCF reaction testing.
  • FIGs. 22A-22C depict custom mobile application software.
  • FIG. 22A depicts a main window of the developed wFDCD sensor mobile application "Biofabrics" where spectrographic measurements are continuously recorded.
  • Display graphs show independent color channels and bottom icons alert features such as Twitter, email, or messaging as a method of alarm in case of sensor activation.
  • FIG. 22B depicts an environmental window of the mobile application depicts geolocation information as well as recorded measurements of temperature (oC), humidity (%) and CO2 (PPM).
  • FIG. 22C depicts excitation window of the application allows on-the-fly user adjustment of the LED illumination parameters of the four Luxeon Star LEDs installed in the wFDCF device using a Saber Z4 Color Mixing Array (Quadica Developments Inc., Lethbridge, Alberta). LEDs included in the current device were: 447nm, 470nm, 505nm, and 6500K white.
  • FIGs.23A-23J depict validation of CRISPR-based FDCF wearable sensors.
  • FIG.23A depicts the sensing mechanism of CRISPR-Cas12a system is based on catalytic trans-cleavage of fluorophore-quencher ssDNA probes after activation by an RPA-amplified dsDNA trigger.
  • FIG. 23B depicts wFDCF mecA CRISPR-based sensor exposed to sample containing 100 fM mecA trigger.
  • FIG.23C depicts wFDCF spa CRISPR-based sensor exposed to 100 fM spa trigger.
  • FIG. 23D depicts wFDCF ermA CRISPR-based sensor exposed to 100 fM ermA trigger.
  • Statistically distinguishable signals (P ⁇ 0.05) were observed after 72, 56 and 78 min for mecA, spa and ermA sensors respectively.
  • FIG.23E depicts experimental detection of mecA CRISPR-based sensor at 2.7 fM trigger was statistically distinguishable after 75 min (P ⁇ 0.05), corresponding to 10,000 dsDNA-copies per ⁇ L.
  • Each experiment is from three independent wells, each having three fiber optic sensors, for a total of 9 fiber optic outputs. Any fibers that were 1 S.D.
  • FIG. 23F depicts an orthogonality demonstration of mecA / spa / ermA CRISPR-based multi-sensor wearable.
  • FIG. 23G-23H depict rehydration only yielded activation of sensors when the Cas12a-gRNA sensor was in the presence of its programmed trigger dsDNA. Scale bars are 250 ⁇ m.
  • FIG. 23I depict garment- level integration of fabric-based wearable synthetic biology sensors. Distributed continuous sensing of garment activity can be achieved through multi-bundle imaging.
  • FIG. 23J depict Connection of fabric-based module to wearable POF spectrometer with wireless connectivity capabilities.
  • the spectrometer electronics consist of a Raspberry Pi Zero W with a camera module (Raspberry Pi Foundation, Cambridge, UK), as well as LED illumination, environmental sensing, and custom-fabricated shields for battery power.
  • Smartphone application for visualization and alarm of wFDCF sensor activation was based on the blynk.io platform (Blynk Inc., New York, NY) which provides support for Raspberry Pi communication. This application allows for wireless recording of experiments, control of device parameters, as well as environmental and geolocation information.
  • FIG. 24 depict limit of detection of wFDCF CRISPR-Cas12a based sensor activated in-fabric.
  • a statistically significant difference between the negative control and trigger presence was observed at 90 min only for concentrations equal and above that of 2.7 fM of trigger (P ⁇ 0.05), which can be considered a limit of detection for this specific trigger, device configuration and evaluation timepoint.
  • FIG. 25 depict comparison of Cas13a-based SHERLOCK MRSA RNA-sensing in wFDCF in-fabric prototype against signal in a standardized plate reader.
  • a CRISPR-Cas13a based MRSA SHERLOCK RNA sensor was prepared and freeze-dried over a wearable textile device for testing. This reaction contained Cas13a for ssRNA detection instead of Cas12a for dsDNA detection as reported for the other CRISPR-based sensors. Cell-free reactions were freeze-dried in the wearable devices for 4-8 hours and also freeze-dried in a 384-well plate for comparison in 4 ⁇ L reaction aliquots.
  • All reactions contained RNaseAlert substrate, a quenched fluorophore probe that is cleaved by activated Cas13a (Integrated DNA Technologies, Coralville, IA).
  • the wearable sensor was activated with a fluid splash of dd-H2O containing 20 nM mecA RNA trigger, while the plate samples were rehydrated with the same trigger concentrations to the originally deposited reaction volume (4 ⁇ L). Reactions were monitored at 30oC for 30 minutes using the wearable optical device or and a BioTek NEO HTS plate reader (BioTek Instruments, Inc., Winooski, VT) in fluorescence mode (Ex. 470 nm / Em. 510 nm).
  • FIGs. 26A-26D depict integrated wFDCF sample lysis.
  • FIG. 26A depicts detergent combinations for cellular lysis were tested against CRISPR-Cas12a SHERLOCK reactions. Shown are reactions for the SARS-CoV-2 SHERLOCK sensor tested in various detergent dilutions. Based on these results, the 2x dilution was chosen as the optimal lysis buffer. For bacterial samples, the lysis buffer was supplemented with 100 ⁇ g/mL of lysozyme for dissolving peptidoglycan and 5% sucrose to create a hyperosmotic environment.
  • FIG.26B depicts assembly of the wFDCF with lysis: top to bottom; Blackout fabric layer, Disc containing free-dried lysis reagents and lysozyme, dissolvable PVA time delay bridge (edges sealed with elastomer), freeze- dried RPA/SHERLOCK reactions in layer containing POF emission and POF excitation 26C,
  • In- wearable wFDCF mecA sensors containing a lyophilized lysis buffer were challenged with intact E. coli cells either containing the target mecA gene (+, top images) or a negative control plasmid ( -, bottom images).
  • FIG. 26D depicts effectiveness of freeze-dried non-ionic surfactants.
  • the surfactants tested in the top row left to right are Triton X-100, NP-40, and Tween-20.
  • the surfactants tested in the bottom row left to right re Brij-58, Brij-C10, and Brij-S20. All the ionic surfactants show little or no effect on the RFU values.
  • FIG. 26E depicts some ionic surfactants used as freeze-dried lysis reagents. From left to right these are sodium dodecyl sulfate, CHAPS hydrate, and sodium deoxycholate. Only CHAPs Hydrate shows modest decrease in RFU, Sodium dodecyl sulfate and sodium deoxycholate show immediate impact on RFU.
  • FIG.27A-27D depict bioinspired sample-wicking for textile-based wFDCF synthetic biology devices.
  • FIG. 27A depicts a schematic of the base cover presented for the textile-based wFDCF synthetic biology devices, as well as the underlying biomechanical mechanism of water collection at the areoles of the bunny ears cactus, Opuntia microdasys. The high aspect ratio and agglomeration of spikes in these areoles, known as glochids, provide a high wettability gradient, which pins fluid for rapid absorption.
  • FIG. 27B depicts modified cover for the textile-based wFDCF synthetic biology devices with Opuntia-inspired wicking ports.
  • the cover features 3D- printed conical spikes (1 mm base diameter) with an aspect ratio of 1:5 arranged concentrically with 1 mm spacing.
  • the cover was fabricated using an elastic photoreactive resin and a stereolithography 3D-printing method using a Form 2 printer (Formlabs Inc., Sommerville, MA), coated with NEVERWET ⁇ superhydrophobic coating (NeverWet LLC., Lancaster, PA). Contact angle measurements to confirm hydrophobicity of cover surfaces is also shown. 27C, Five-second time-lapse of the fluid pinning and port wicking exhibited by the device.
  • FIG.27D is a photograph of an assembled textile-based wFDCF synthetic biology device including the bioinspired port. Images before and after fluid splash are also shown to evince behavior.
  • FIGs. 29A and 29B depict implementation of Polyvinyl Alcohol (PVA) time delays for optimized multi-stage wFDCF Reactions. PVA fluidic time delays allow for wearable multi- stage reactions to occur rather than one-pot lyophilized reactions. As depicted in FIG.
  • PVA Polyvinyl Alcohol
  • FIG. 29A shows a representative experiment using a 50 ⁇ L dried time delay consisting of ⁇ 67,000 MW PVA (Millipore-Sigma, St. Louis, MO) that allows for a time delay of ⁇ 15 minutes.
  • 20% (w/v) PVA was used for the A-version sensors.
  • FIGs. 30A to 30G show details on the design, performance, and relevant molecular sensor sequences.
  • FIG. 30A depict SARS-CoV-2 genomic region targeted by the RT-RPA and SHERLOCK sensor utilized in the face-mask diagnostic of the A-version sensors, used for the experiments shown in FIG. 28D-28G.
  • the Cas12a gRNA sensor targets a region (highlighted in green in the multiple sequence alignment) in the Spike protein gene between 22-23k of the SARS-CoV-2 region.
  • RNA portion of the SARS-CoV-2 genome corresponding to 22,772:23,083 was generated from a synthetized DNA fragment and used in testing.
  • the multiple sequence alignment shows the aligned homologous regions from SARS- CoV-2 and the three circulating human coronavirus strains (OC43, HKU1, NL83, and 229E).
  • the sequence alignment was generated using Clustal Omega (EMBL-EBI) and BoxShade (SIB Swiss Institute of Bioinformatics). The shown region corresponds to the amplicon generated from RT-RPA with the F4/R4/R3 primer mix.
  • FIG. 30B depicts a Laser-cut sample collection pad from capillary wicking material.
  • the sample includes a polymeric wicking material was laser-cut with a large sample collection area (55 x 20 mm) that will be positioned inside of the mask to collect respiratory droplets and aerosols for virus detection.
  • FIGs. 30C1-C6 depict images of the ⁇ PAD construction.
  • Steps for construction the ⁇ PAD device portion of the sensor are: 30C1, a solid wax printer is used to print an array of ⁇ PADs on filter paper; 30C2, the printed wax pattern is refluxed by application of a hot press, to fully allow the wax to penetrate the filter paper; 30C3, the individual ⁇ PADs are cut from each sheet; 30C4, polyvinyl alcohol is added to the time delay zones and allowed to dry at room temperature overnight; 30C5, fresh lysis reagents, RT-RPA reactions, and Cas12a SHERLOCK reactions are applied to their respective reaction zones–the ⁇ PAD is lyophilized for a minimum of 4 hours; 30C6, after lyophilization, the ⁇ PAD is folded using RNase-free tweezers to overlap the reaction zones.
  • FIG.30D depicts components of the face-mask sensor before assembly. From left to right, is depicted a water reservoir with adjustable orientation, a sample collection area with fixed orientation of an absorbent side facing the patient, a ⁇ PAD with adjustable orientation, and a LFA output with adjustable orientation.
  • the water blister water reservoir
  • the sample collection area absorbs viral particles from the patient.
  • the ⁇ PAD contains the freeze-dried nucleic acid test sensor reactions, separated by PVA time delays.
  • a lateral flow assay generates a visual output based on Cas12a-based cleavage of a FAM-Biotin probe.
  • FIG.30E depicts a fully assembled sensor.
  • FIG.30F depicts a demonstration of sample flow through face- mask sensor. Bromophenol blue dye was spotted at random locations throughout the sample collection zone. Upon hydration from the reservoir, a sample front can be clearly seen sweeping across the sample zone and into the ⁇ PAD.
  • FIG.30G depicts the arrangement that allows for the preservation of patient confidentiality, where the LFA strip for A-version sensors are oriented with the LFA indicator surface facing the mask to hide the output from external view. The clinician must pull the strip back to observe / record the results.
  • FIGs. 31A-31C depict SARS-CoV-2 face-mask diagnostic B-version sensor design and construction. The B-version of the SARS-CoV-2 face-mask sensors contain a number of modifications over the A version sensors that optimize robustness and consistency.
  • FIG.28A-28J FIG.31A sub- assembly consisting of the sample collection pad, ⁇ PAD (unfolded), and the LFA output strip, highlighting key differences between the B-version and A-version sensor components are: (1) the reservoir connection area is enlarged to ensure adequate flow to collection zones; (2) borers of the sample collation pad is rastered to fuse the porex HRM fiber media with the PET backing material, eliminating delamination; (3) porex HRM fiber media is cut with fibers parallel to the longitudinal axis of the sample collection pad to increase flow speed; (4) lysis buffer composition is altered from the A-version–triton X100 is eliminated and lowering of NP-40 percentage prevents erosion of ⁇ PAD wax layers–CHAPS is increased; (5) PVA time delays were adjusted to 18% (w/v), decreasing viscosity for more consistent application; and (6) border areas not containing wax are blocked using hydrophobic ink to prevent fluidic short-circuits during operation.
  • FIG. 31B depicts the fully assembled B-version face-mask sensors.
  • the indicated changes from A-version sensors are (7) venting holes are punched into the water blister reservoir to eliminate buildup of a vacuum–the holes are overlaid with breathable water-repelling adhesive covers and (8) ⁇ PAD area interfacing with the sample collection pat is tightly sealed to prevent unwanted fluidic short circuits.
  • FIG.31C depicts a B-version sensor fully integrated into a face mask.
  • the water blister reservoir is positioned as a flap on the outside of the mask, to prevent potential crushing of the blister while the mask is being worn.
  • FIGs. 32A-32F depicts a breathing simulator for exhaled emission testing of the SARS-CoV-2 face-mask wearable diagnostic.
  • FIG.32A depicts a schematic of the key modules used in the breathing simulator. Dotted lines indicate connecting airflow through the different modules via ventilation tubing connectors. Generally, from left to right is depicted: (1) a spontaneous breathing generator; (2) an in-line aerosol producing device (e.g. nebulizer); (3) a heat sleeve, and; (4) an anatomically precise airway manikin.
  • FIG. 32B depicts the spontaneous breathing generator.
  • the TESTCHEST® Organis GmbH, Switzerland
  • FIG.32A depicts a schematic of the key modules used in the breathing simulator. Dotted lines indicate connecting airflow through the different modules via ventilation tubing connectors. Generally, from left to right is depicted: (1) a spontaneous breathing generator; (2) an in-line aerosol producing device (e.g
  • 32C depicts the nebulizer and heating assembly.
  • the AEROGEN® Solo nebulizer system (Aerogen, Inc., Ireland) was used to generate aerosols in the simulated breath stream.
  • the AEROGEN® platform is a medical-grade inhalation medicine device that uses vibrating mesh technology.
  • the AEROGEN®-produced aerosols have a measured size distribution (0.4 - 4.4 microns) that matches the size range of naturally occurring lung aerosols and droplets.
  • a self-regulating thermal pad sleeve was used to heat the simulated breath to maintain a face mask microclimate of 35°C.
  • FIG. 32D depicts photos of the nebulizer reservoir being filled (left) and the nebulized aerosols exiting the tubing (right).
  • FIG. 32E depicts a high-fidelity anatomically precise airway manikin (7-SIGMA Simulation Systems, Minneapolis, MN), which can simulate exhaled breath as it would exit physiologic airway structures and provides realistic fitment of the mask on a patient’s face.
  • FIG. 32F depicts a photograph showing the full air flow path from the TESTCHEST® to the AEROGEN® Solo nebulizer, through the heating sleeve, and connecting to the 7-SIGMA manikin. [00264] Table 2.
  • This supplemental description encompasses two main bodies of work: 1) an engineered platform for breath-based sensing of nucleic acids for POC diagnostics for the detection of pathogens in a patient’s respiratory droplets / aerosols.
  • This platform can be integrated with the second component: 2) freeze-dried compatible programmable nucleic acid test (NAT) reactions that are shelf-stable and can run isothermally at ambient (25 – 35 ⁇ C) temperatures in an autonomous manner with minimal user intervention.
  • NAT freeze-dried compatible programmable nucleic acid test
  • Breath-based detection of infection and disease is a convenient, non-invasive, and information rich sample source that is underutilized. Breath samples can be used to detect the infection status of a patient for respiratory infections such as SARS-CoV-2 using a customized inexpensive isothermal nucleic acid test (NAT) based sensor. These sensors can be used in a stand-alone device or integrated into a facemask for a combined sensing and protection device.
  • Face masks are effective wearable devices for preventing the spread of infectious respiratory diseases. They work by trapping potentially pathogen-laden droplet and aerosol particles generated by coughing, sneezing, tussis, talking, or breathing.
  • Some embodiments described herein integrate an on-board breath-sensing pathogen diagnostic that can be integrated into a face mask, thus combining both a surveillance as well as a protective function into one system.
  • the inside of the mask collects droplet and aerosol particles as the patient wears the mask.
  • the clinician activates the mask, allowing water from a reservoir to sweep through the sample collection zone and into freeze-dried cell-free reactions embedded in the face mask.
  • the output is visible on the outside of the mask and can be hidden from public view to preserve patient confidentiality.
  • an alternative embodiment is a breath-based diagnostic similar to chemical “breathalyzers” that have a mechanism for breath capture.
  • FIG.33A shows a breathalyzer 3302 –based diagnostic.
  • Components such as a carrier fluid reservoir (e.g., a water reservoir or sachet) 3304, a sample collection unit 3306, a sample processing unit including synthetic biological components 3308 (e.g., a ⁇ PAD) and a detection unit 3310 (e.g. an LFA) can be encased in an enclosure 3312. Except for the detection unit 3310 (or at least a visible output of the detection unit), only the general area of the fluid reservoir 3304, sample collection unit 3306, and sample processing unit 3308 is indicated since these are enclosed in enclosure 3312.
  • the breathalyzer includes a mouth piece 3314 which the user blows or breaths into.
  • the sample collection unit 3306 is disposed in case 3302 positioned to direct breath to the sample collection unit 3306, for example at the base of the mouth piece proximate to where the mouth piece 3314 contacts the enclosure 3312.
  • FIG. 33B depicts a face mask 3322 diagnostic for comparison. Similar components are indicated as the fluid reservoir 3324, the sample collection unit 3326, a sample processing unit 3328 and a detection unit 3310. The components of the face mask have been previously described. [00273]
  • the diagnostic face mask serves to provide a rapid ( ⁇ 1 hour) diagnostic result without any need for hands-on manipulation aside from the activation step. This point-of-care smart personal protective equipment (PPE) ensures that patients can be quickly triaged for proper medical care, while simultaneously protecting healthcare workers and other patients from infectious droplet transmission.
  • PPE point-of-care smart personal protective equipment
  • This autonomous diagnostic smart-PPE will fill an urgent need for a quick presumptive test (and as a mask preventing viral transmission) to supplement laboratory-based diagnostics without additional burden to hospital staff.
  • the face mask NAT sensor are freeze-dried, shelf-stable, and fully autonomous point-of-care (POC) devices.
  • POC point-of-care
  • the face mask system could be further developed to discriminate between SARS-CoV-2 and other respiratory viruses, such as influenza, or for monitoring emerging SARS-CoV-2 mutant strains.
  • SARS-CoV-2 and other respiratory viruses such as influenza
  • Other respiratory viruses such as influenza
  • SARS-CoV-2 mutant strains include various nucleic-acid detection platforms, including transcriptional toeholds, Cas13a, or Cas12a-based switches that work at ambient temperatures.
  • In vitro data is then presented demonstrating the functional performance of the freeze-dried isothermal NAT sensors for detecting SARS-CoV-2 nucleic acids.
  • From initial activation of the face mask sensor to a final readout only takes ⁇ 40-90 min.
  • All of the sensors presented in this invention disclosure target regions of the SARS-CoV-2 Spike gene (FIG. 30A), which encodes for the primary receptor of the virus that is essential for infectivity. Moreover, this region shows high sequence divergence from other circulating human coronaviruses (HCoVs), allowing for specific discrimination for SARS-CoV-2 and variant strains harboring mutations in the spike gene.
  • HoVs circulating human coronaviruses
  • These sensor inserts contain the following modules: a source of water, followed by a wicking area that absorbs droplets and aerosols generated by the patient’s breath, porous materials containing freeze-dried lysis, isothermal amplification, and toehold or CRISPR-based NAT sensors.
  • Each of the four modules are described below. These, and other details, have been previously described, for example with reference to FIG.30B-30G, 31A-31C, and 33B. [00279] 1.
  • Water source This would be an encapsulated sachet containing water or aqueous buffer which can be mechanically or electronically actuated to release the fluid to flow into the remainder of the sensor.
  • Breath sample collection area This area consists of a capillary wicking material which is positioned on the interior of the mask in front of the patient’s nose and mouth, for the absorption and collection of viral or bacterial-laden aerosol or droplets generated from breathing, coughing, sneezing, or speaking.
  • the material could be a polymeric, natural fiber, metal, of nonmetal porous material that would support capillary wicking.
  • This sample collection area is positioned and connected downstream of the activatable water source and upstream of the freeze- dried NAT sensors.
  • NAT sensors will consist of any combination of sample lysis reactions, isothermal or thermal amplification reactions, and nucleic acid sensing reactions such as toehold switches, Cas13a-SHERLOCK and Cas12a- SHERLOCK reactions. All of these reactions are freeze-dried into a substrate which may or may not be porous. They may also be encapsulated in a dissolvable polymeric matrix. They can be mixed into a single “one-pot” reaction or spatially divided into a series of sequential reactions.
  • fluidic time delays may be positioned between each reaction zone. These time delays consist of dissolvable polymeric, sugar, or inorganic freeze-dried barriers that separate one reaction from another, thus providing a tunable time delay allowing for sufficient completion of one reaction before the next reaction is encountered.
  • the output from the combined NAT may be a visual or fluorescence signal which may require electrochemical or electronic optical detection.
  • a simple lateral flow assay device connected to the NAT reactions can convert a positive signal to a visual signal detectable by the eye. [00282] 4.
  • LFA Lateral Flow Assay
  • Custom LFAs can be used to generate a colorimetric or fluorescent response by using spatially immobilized affinity molecules, such as antibodies, other proteins, or nucleic acids and a molecular signal generated from the NAT.
  • affinity molecules such as antibodies, other proteins, or nucleic acids
  • NAT molecular signal generated from the NAT.
  • nucleic acid probes present in Cas12a-SHERLOCK reactions are cleaved when the Cas12a ribonucleoprotein complex attains trans-cleavage DNase activity upon cis-recognition of the target nucleic acid.
  • the cleaved nucleic probes bind to different regions of the LFA, creating a detectable spatial pattern.
  • LFA output is used here as an example, but other output methods can be used as well, whether visual or nonvisual.
  • Toehold switch sensors were previously described (e.g. FIG. 6A). These are programmable synthetic riboregulators that allow protein expression only when a specific trigger RNA is present. These sensors consist of an mRNA molecule designed to include a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding site and start codon. Hybridization to a complementary trigger RNA results in secondary structure rearrangement, facilitating ribosomal translation of an output gene (FIG.6A and FIG.34A).
  • the toehold circuit can be encoded as DNA with a transcriptional promoter with a cell-free system (either a cell lysate or reconstituted system) and the entire reaction can be freeze-dried for shelf-stable storage and transport.
  • a library of toehold sequences was designed and screened to detect regions in the S gene of the SARS-CoV- 2 virus (FIG.
  • FIG.34 depicts toehold switches which are RNA transcripts locked through designed secondary structure.
  • FIG. 35A-35U depict plots screening of a library targeting regions in the SARS-CoV-2 S-gene. All output was LacZ (beta- galactosidase enzyme), which generates a colorimetric response using the substrate chlorophenyl red- ⁇ -D-galactopyranoside (CPRG). Negative control (EBOV) is an unrelated toehold designed against the Ebola virus. Positive control is LacZ with no toehold. Plots show trigger (blue) and no trigger (orange).
  • Cas13a-SHERLOCK sensors leverage the ability of Cas13a ribonucleoprotein (RNP) complexes for sensitive detection of RNA nucleic acids.
  • RNP ribonucleoprotein
  • the Cas13a enzyme can be easily programmed to detect different sequences.
  • the key aspect of these enzymes that allow for their use in NAT diagnostics is their unique ability as an activatable nonspecific nuclease.
  • the enzyme undergoes an irreversible conformational change which activates two HEPN domains, allowing it to cleave non-specific RNA molecules in the environment (known as trans-cleavage or collateral cleavage).
  • a reporter molecule is designed in which a fluorescent moiety is rendered non-fluorescent through physical coupling to a quenching molecule through an RNA bridge.
  • the cis-activated Cas13a begins to degrade the probe via trans- cleavage and thus generates fluorescence.
  • FIGs. 36A-36Q depict a library of gRNAs that were screened to allow Cas13a direct sensing of SARS-CoV-2 vRNA.
  • the Cas13a sensors with the best switching activity (high signal and low background) for the detection of SARS-CoV-2 were gRNAs 7, 8, 9, and 11 as shown by the summary plot FIG. 36Q.
  • These experiments utilized the Cas13a ortholog from Leptotrichia wadei, although other Cas13 enzymes could be used with the same spacer sequences presented here that target the vRNA.
  • the cis-activated Cas12a-gRNA complex Upon recognition of the dsDNA amplicon, the cis-activated Cas12a-gRNA complex gains indiscriminate ssDNA nuclease activity, allowing for detection through cleavage of a quenched ssDNA fluorophore probe (FIG. 37A).
  • PAM protospacer adjacent motif
  • each Cas12a-gRNA complex was challenged with water only (negative control) or water spiked with a dsDNA fragment encoding for the entire spike gene region of SARS-CoV-2 and monitored the reactions for fluorescence, indicative of activated Cas12a trans-cleavage of the fluorescent probe (FIG.37B).
  • FIG. 37A depicts that when coupled with a reverse transcriptase amplification reaction, Cas12a SHERLOCK sensors can detect dsDNA amplicons and cleave ssDNA reporters.
  • FIG. 37B depicts the experimental setup for screening a library of 11 gRNAs targeting a dsDNA fragment of the SARS-CoV-2 S- gene for activity.
  • FIG.37C-37N depict that all gRNAs showed switching activity in the presence of amplicon (green data points), with low signal when no amplicons are present (gray data points). The data is summarized in the plot shown in FIG.37N.
  • the Cas12a sensors were selected as the final sensors for integration into the face mask device due to the following reasons: robust performance, relative stability of the ssDNA probes, and the minimal components needed for operation.
  • the amplicon generation requires a step in which reverse transcriptase (RTase) is paired with an amplification reaction such as PCR, NASBA, LAMP, or RPA.
  • RTase reverse transcriptase
  • This critical step generates a dsDNA amplicon from the viral RNA that can be detected by the Cas12a RNP (FIG. 38A).
  • the amplification also serves to amplify the nucleic acid target of interest to enhance sensitivity.
  • a key requirement here is the identification of conditions for reverse-transcriptase and isothermal amplification that is capable of sufficiently amplifying the SARS-CoV-2 vRNA at ambient temperatures (25-35°C) that do not require external heating.
  • RNA secondary structure is problematic for efficient probe hybridization for RTase reactions11,12
  • Viral genomes, especially coronaviruses such as SARS-CoV-2 contain an uncommonly high degree of secondary structure motifs that make probe hybridization very difficult without using a heating step13-15
  • all commercially available RTases have optimal activities at 42–48°C.
  • Several primer combinations were screened under the desired conditions for the detection of SARS-CoV-2 RNA, using an RNA fragment (named F5R11) corresponding to a region of the spike gene for screening.
  • the amplification method chosen was a one-pot RTase (PrimeScript from Takara Bio) and RNase H (from ThermoFisher) reaction paired with RPA (Recombinase polymerase amplification, from TwistDx).
  • the cDNA is generated from the RTase + RNaseH activities and is then amplified by the RPA reaction.
  • RPA was chosen over other isothermal DNA amplification methods due to its capacity for lyophilization, ability for amplification under ambient temperatures, and compatibility with Cas12a-SHERLOCK sensors.
  • the majority of the RT-RPA primers screened performed poorly, which is likely due to the aforementioned RNA secondary structure complexity of the SARS-CoV-2 genome.
  • RT-RPA primers that were able to generate an extremely fast sensor response in a two-pot RT-RPA > Cas12a SHERLOCK reaction (using gRNA-06) were identified. The reaction was able to reach a signal plateau within only 10 minutes, when challenged with the F5R11 RNA fragment.
  • the primer set consists of one forward primer and two reverse primers (F4 / R4 / R3), can successfully amplify full-length SARS-CoV-2 vRNA, and has a determined optimal concentration ratio of (10:10:20 pmols). See FIG.38B-38E.
  • FIG.38A is a diagram of the RT-RPA reaction which isothermally generates DNA amplicons from a viral RNA target.
  • FIG. 38B depicts the experimental setup for screening of primer pairs for successful RT-RPA amplification.
  • FIG. 38C depicts the F4-R4-R3 primer set allows for rapid detection of SARS- CoV-2 RNA fragments and full-length genome.
  • FIG.38D depicts the optimization of the primer molar ratio for the F4-R4-R3 set.
  • FIG.38E depicts the signal over background at 30 min.
  • 30A-30G, 31A-31C 1) a water sachet or reservoir that can be activated by the clinician to drive the reactions.2) downstream of the water reservoir is a large sheet of porous material that is the sample collection zone. This module is positioned over the nasal and oral passageways of the patient to collect aerosolized breath samples, such as SARS-CoV-2, that have been shown to be present in exhaled breath. 3) downstream of the sample collection zone is an arrangement of the freeze-dried NAT reaction components -- lysis reagents, RT-RPA for isothermal amplification, and toeholds/Cas12a/Cas13a for nucleic acid detection. These can be arranged as separate reactions or as one-pot reactions.
  • ⁇ PADs Microfluidic Paper-based Assay Devices
  • PVA polyvinyl alcohol
  • the clinician would press a button on the mask that activates the water reservoir by puncturing the sachet. This event would mark the beginning of the sensor activation and is the only hand-on time required from the clinician.
  • the water or buffer would begin to flow by capillary wicking action through the sample collection material, sweeping along any virions along the sample front.5) the sample would then enter the lysis reaction zone in the ⁇ PAD. The rehydrated detergents and osmotic additives would release the nucleic acid inside the viral particles.
  • the sample after dissolving the first PVA time delay, the sample would then move into and rehydrate the RT-RPA zone, allowing for isothermal amplification of the liberated viral genome, converting the vRNA into a dsDNA signal. 7) after dissolving the second PVA time delay, the sample moves into the final reaction zone containing precomplexed Cas12a-gRNA and a nucleic acid probe.
  • the type of probe may be variable, but here was used a ssDNA probe which has a FAM molecule on one end and a Biotin molecule on the other (FB probe).
  • the Cas12a-gRNA will cis-cleave the target and enter a trans-DNase state allowing it to repeatedly cleave the FB probe.8) after dissolving the third and last PVA time delay, the sample enters into a LFA in which freely floating gold nanoparticles conjugated with anti-FAM antibodies are freeze-dried. Also on the LFA are areas in which affinity molecules have been immobilized: The “C” band consists of a biotin-binding protein and the “T” band consists of an antibody that binds to the anti-FAM antibody that is conjugated to the gold nanoparticles.
  • the FB probe In the event of a negative SARS-CoV-2 result, the FB probe is not cleaved, resulting in the gold nanoparticles being tethered to the “C” line due to the FB probe. In the event of a positive result, the FB probe is cleaved and the gold nanoparticles flow past the “C” band and are bound at the “T” line. In the tests of the face mask sensors, it was found that negative results using the FB probe often results in the appearance of “C” and “T” lines, likely due to gold nanoparticles that are able to escape binding at the “C” line.
  • the “C/T” ratio was determined based on the relative intensities at these bands to ascertain a negative test result from a positive test result.
  • the SARS-CoV-2 in-mask breath diagnostic consists of the sensor assembly containing the lyophilized reactions which was then inserted in an N95-equivalent face mask.
  • the laser-cut wicking material is then adhered to a white PET double-adhesive backing material (3M Microfluidic Diagnostic Tape, #9965).
  • a sterile sealed blister pack containing nuclease-free water.
  • the ⁇ PAD device is created by wax printing hydrophobic patterns onto WHATMAN® Grade 1 chromatographic filter paper (Thermo Fisher Scientific, Waltham, MA) using a Xerox Phaser 8560 solid ink printer.
  • the printed ⁇ PAD sheets were then wax reflowed by hot pressing for 15 sec at 125°C using a Cricut EASYPRESSTM (Cricuit Inc., Fork, UT), and then left untouched to cool at room temperature. After wax reflow, the reaction zones have an aperture diameter of 5 mm, while the intervening PVA time delays have an aperture diameter of 3 mm.
  • the PVA time delays were placed onto the time delays zones first, by pipetting 4 ⁇ L of 10% ⁇ 67,000 MW PVA (Millipore-Sigma, St.
  • the lysis reaction added to each sensor lysis zone can consist of 0-100 mM Tris-HCl (pH 7.5), 0-5% Triton X-100, 0-5% NP-40, 0-10% CHAPS, 0-500 ⁇ g/mL lysozyme, and/or 0- 15% sucrose.
  • the RT-RPA reaction added to the isothermal amplification zone was 15 ⁇ L of a single lyophilized TWISTAMP® lyophilized RPA pellet (TwistDx Limited, UK) that was rehydrated to 50 ⁇ L using a rehydration reaction of 29.6 ⁇ L Twist Rehydration Buffer, 9.6 ⁇ L of a primer mix (see Table S2; RT-RPA-F4, RT-RPA-R4, RT-RPA-R3 primer in the mix are at a ratio of 10 ⁇ M : 10 ⁇ M : 20 ⁇ M). Other primer concentrations and other reaction volumes may be used.
  • the output end of the laser-cut Porex sample collection section was carefully inserted on top of the lysis zone, while the input end of a Milenia HybriDetect-1 Universal Lateral Flow Assay (TwistDx Limited, UK) was inserted on top of the last PVA time delay.
  • the entire ⁇ PAD section was carefully sandwiched and taped together to compress all of the layers.
  • the entire blister-pack water reservoir – Porex sample collection area – ⁇ PAD – LFA test strip is secured using the double-sided backing to the inside of an N95 equivalent mask, positioning the sample collection area in the region directly in front of the mouth and nose.
  • the LFA test strip is routed to the outside of the mask through a small slit in the mask and the indicator can be oriented to hide the results from external viewing, to ensure patient confidentiality. To access the results in this configuration, the test strip must be bent over to view the results.
  • a button was affixed to the outside of the mask directly over the water blister reservoir. The button contains a small spike embedded in a compressible foam double-sided adhesive material. When pressed down, the button pierces the foil on the blister, allowing the nuclease-free water to flow through the same collection zone, the ⁇ PAD reaction zones and time delays, and finally into the LFA indicator strip.
  • the sample collection pads for the B-version sensors were laser-cut from sheets of Porex high release media #36776 with the dominant fiber direction along the long axis of the pad to allow faster flow of the hydration front.
  • the pad geometry was adjusted to enhance water flow by moving the reservoir puncture point to the distal end of the water blister, increasing the pad area in contact with the water reservoir, and reducing the sample collection region.
  • Approximately 2 mm of the outer border of the sample pad was rastered during laser- cutting to heat seal the Porex material to the PET backing material, preventing delamination. Before assembly, approximately 1 cm of the backing material was peeled away and cut off from the end of the sample pad region that is to be in contact with the reservoir.
  • each reaction zone area was blocked with 5 mL of 1% BSA + 0.02% Triton X-100 and allowed to air dry for 12 hours to prevent nonspecific adsorption of the biochemical reaction components to the filter paper matrix.
  • PVA at a concentration of 18% (w/v) at a volume of ⁇ 5 ⁇ L was applied to each time delay zone and allowed to air dry for 24 hours.
  • the lysis buffer for the B-version sensors was reformulated to 10 mM Tris-HCl (pH 7.5), 5% Sucrose, 0.02% NP-40, and 2% CHAPS.
  • the amount of non- ionic surfactants in the lysis buffer were reduced to prevent observed degradation of the wax barrier, an observation made during design and testing of the A-version ⁇ PADs.
  • the CHAPS concentration was increased as it was not found to degrade the wax and this zwitterionic detergent has previously been shown to be effective in lysing coronavirus particles.
  • a volume of 10 ⁇ L of this lysis buffer was added to the ⁇ PAD lysis zone.
  • the RT-RPA and Cas12a SHERLOCK reaction compositions, volumes, and lyophilization parameters were unchanged.
  • both the sample pad:: ⁇ PAD and the ⁇ PAD::LFA contact regions were fully sealed using precut sterile aluminum PCR foil seals (#60941-076, VWR Intl., Radnor, PA) to improve contact transfer and prevent any fluidic short-circuiting that may occur from undesired droplet contact to the folded ⁇ PAD edges.
  • venting holes were introduced into the water-containing blister mold to prevent vacuum buildup inside the blister during flow. Three venting holes were punched into the blister surface using an 18-gauge needle and then sealed with a 6-mm adhesive disc of a single-sided rayon breathable hydrophobic porous film (#60941-086 VWR Intl., PA).
  • the water reservoir module was positioned on the exterior of the mask to minimize unwanted contact pressure on the blister pack during wearing of the mask.
  • the sensor activation mechanism is the same as the A-version sensors.
  • 1 cm slits were cut into KN-95 masks through which the sensor ends were threaded and subsequently sealed using adhesive.
  • regions of the paper-based ⁇ PAD can be inserted which are designed to act as time delays.
  • time delay devices that can be integrated into the paper matrix are useful for functioning of the sensor.
  • Many of the enzymatic reactions of the sensor require a sequential progression through the different reactions in a multi-step process. Much of this is due to different buffer conditions required for each enzymatic reaction. It was found that the most significant incompatibility occurs when mixing RPA and Cas12a SHERLOCK reactions in a single “one-pot” reaction, which results in poor product yields for both reactions.
  • a programmable time delay that would regulate the spatial and temporal dynamics of each reaction in the ⁇ PAD was designed using a dissolvable polymeric barrier made of polyvinyl alcohol (PVA) to improve the sensor performance.
  • PVA time delays are created by applying molten PVA solutions to the paper substrate of the ⁇ PAD and allowing it to completely air dry.
  • the dried PVA barrier acts as a plug to prevent the rehydration front from progressing immediately to the next reaction zone.
  • the timing of the PVA dissolution can be tuned simply by altering the percent solution of the PVA, where there is a positive correlation between the % of PVA and the amount of time delay.
  • ⁇ PAD it was empirically determined that a 15-20% percentage solution of PVA with an average molecular weight of ⁇ 67,000 and 86.7-88.7 mol% hydrolysis (Millipore-Sigma, 81383) was optimal to create the needed time barriers in the SARS-CoV-2 ⁇ PAD modules.
  • each data point consisted of a face mask sensor in which a defined amount of synthetic SARS-CoV-2 RNA fragment containing the specific gRNA targeting region of the SARS-CoV-2 spike gene was generated by in vitro transcription using the HISCRIBETM T7 Quick High Yield RNA Synthesis Kit (NEB, Ipswitch, MA) using synthetic DNA templates with a T7 promoter (Integrated DNA Technologies, Inc., Coralville, Iowa, and Twist Bioscience, San Francisco, CA).
  • Corresponding homologous regions to the spike gene for the commonly circulating human coronavirus strains 229E, HKU1, NL63, and OC43 were determined by sequence homology alignment of the respective spike genes and the RNA targets were generated using the same method described above. The indicated amount of target RNA was spotted onto the breath collection zone to simulate viral deposition. All SARS-CoV-2 face mask sensors were tested at room temperature at ambient humidity. After activation and LFA output formation ( ⁇ 20-30 min), the LFA strips were digitized using the scanner function on a Ricoh MP C3504 on default contrast settings. This ensured equal brightness and contrast across all strips in comparison to photography. Each “T” and “C” output line from each strip was quantified in ImageJ from the raw scanned images without any adjustments to brightness or contrast.
  • the limit of detection observed for these sensors is 500 copies (17 aM) of SARS-CoV-2 in vitro transcribed (IVT) RNA, which matches the sensitivities of current WHO-endorsed standard laboratory-based RT-PCR assays (FIG.28F and 28G).
  • the limit of detection threshold, +3 S.D. of the no-template control (NTC) is shown as a red dotted line.
  • the sensors also do not cross react to RNA from other commonly circulating human coronavirus strains (HCoVs) as shown in FIG. 28I and 28J).
  • the simulator consisted of four modules that performed the following functions: spontaneous breath generation, aerosol production, heating control, and physiologic airway and head simulation.
  • the TESTCHEST® lung simulator (Organis GmbH, Switzerland), a highly accurate artificial lung that uses an actuated dual bellows design to replicate lung mechanics such as lung vial capacity and tidal volume, was employed.
  • the TESTCHEST® was connected through ventilator tubing to all other downstream modules for simulated spontaneous breathing. Directly downstream of the TESTCHEST®, was placed an in-line AEROGEN® Solo nebulizer (Aerogen, Inc., Ireland).
  • the AEROGEN® Solo is a medical-grade vibrating-mesh nebulizer for the administration of lung inhalation therapeutics. Previous studies have demonstrated that the nebulizer generates aerosol droplets that are similar in diameter to those that occur naturally from human lung emissions. Furthermore, previous work has used the AEROGEN® system to deliver therapeutic RNA in an animal model, showing that it can be used to produce transmissible RNA-laden aerosols.
  • the tubing is next wrapped in a temperature-regulated heat pad (Zoo Med Laboratories, Inc., San Luis Obispo, CA) that maintains the output temperature at 35 ⁇ C.
  • the tubing is connected to a lung input tube in a high-fidelity airway manikin (7-SIGMA Simulation Systems, Minneapolis, MN) that faithfully replicates pulmonary and nasopharyngeal structures as well as head movement ranges.
  • the other simulated lung and the simulated esophagus are clamped shut to direct breath output only through the oral cavity.
  • B-version face masks were assembled with the blister pack reservoir and LFAs on the outside of the mask.
  • the face mask tests on the breathing simulator used a spontaneous breathing rate of 12 breaths per minute and a temperature was set to maintain an outflow temperature of 35 ⁇ C.
  • a 5 mL solution of SARS-CoV-2 F5R11 vRNA IVT target was then nebulized using the AEROGEN® Solo unit.
  • the simulated breath was allowed to collect in the face mask and on the sensor collection pad for a period of 30 minutes, then the sensor was activated on the manikin for processing while maintaining the breathing and heating.
  • the LFA outputs for all sensors were scanned using a Ricoh MP C3504 printer system using default settings.
  • the total amount of aerosolized vRNA collected after 30 minutes on each mask sensor for a given concentration of vRNA IVT target solution was estimated by RT-qPCR analysis of a 6 mm filter paper disc affixed to the sample pad area.
  • the diagnostic system comprising a biochemical reaction including a combination of reverse transcriptase, nucleic acid-modifying enzymes, amplification enzymes, cell-free lysate, Cas13 or Cas12 enzymes, and one or more nucleic acids comprising complete or partial nucleic acid sequences or the reverse complement thereof, wherein the nucleic acids are DNA or RNA corresponding to: a) Toehold Sequences selected from the group consisting of: SARS2-TH-01 (CGAUCCCGCGAAAUUAAUACGACUCACUAUAGGGAUAAUUACCACCAACC UUAGAAUCAAGAUUGUUAGAGGACUUUAGAACAGAGGAGAUAAAGAUGUCUAAC AAUCUUACCAUGAUUACGGAUUCACUGGCCGUC), SARS2-TH-02 (CGAUCCCGCGAAAUUAAUACGACUCACUAUAGGGGAUUGUUAGAAUUCCA AGCUAUAACGCAGCCUGUAAGGACUUUAGAACAGAGGAGAUAAAGAUGUUACAG GC
  • the diagnostic system according to the first alternative embodiment, wherein the system includes one or more SARS-CoV-2 S-gene amplification primers comprising a fragment of the Seq1 (Tcaaacttctaactttagagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccacc agatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaag tgtatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatg
  • the diagnostic system according to the first or second alternative embodiments, wherein the amplification primers are from about 1 nucleotide to about 100 nucleotides, from about 5 nucleotids to about 50 nucleotides, or from about 10 nucleotides to about 25 nucleotides.
  • the diagnostic system according to any one of the first through third alternative embodiments, wherein the biochemical reactions are freeze-dried components.
  • SEQUENCE LISTING Table 5 DNA and RNA sensor sequences used in this study. The sequences presented in this table are SEQ ID NO: 1 to SEQ ID NO: 36 in their order of appearance.
  • Table 6 Reporter sequences used in this study. The sequences presented in this table are SEQ ID NO: 37 to SEQ ID NO: 42 in their order of appearance.
  • Table 7 Sequences for SARS-CoV-2 Molecular Sensing and Isothermal RT-RPA Amplification. The sequences presented in this table are SEQ ID NO: 43 to SEQ ID NO: 96 in their order of appearance.

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Abstract

Sont décrits dans la description, des modes de réalisation de divers aspects relatifs à un dispositif de détection activé par une solution aqueuse. Le dispositif comprend un réservoir de fluide porteur, une unité de collecte d'échantillon, une unité de traitement d'échantillon comprenant des constituants biologiques synthétiques séchés et une unité de détection. Le dispositif peut être intégré dans un masque facial destiné à la détection d'agents pathogènes, de virus et de toxines en suspension dans l'air.
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