WO2019028162A1 - Détermination de la viabilité de bactéries par mesure de la production transitoire d'amines biogènes - Google Patents

Détermination de la viabilité de bactéries par mesure de la production transitoire d'amines biogènes Download PDF

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WO2019028162A1
WO2019028162A1 PCT/US2018/044850 US2018044850W WO2019028162A1 WO 2019028162 A1 WO2019028162 A1 WO 2019028162A1 US 2018044850 W US2018044850 W US 2018044850W WO 2019028162 A1 WO2019028162 A1 WO 2019028162A1
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biosensor
capture probe
glycoenzyme
viability
microorganism
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Eric Scott MCLAMORE
Ishika Islam KHONDAKER
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the presently disclosed subject matter relates to generally to a method, system, and products for determining bacteria viability.
  • the presently disclosed subject matter provides a product that comprises a biosensor having a sensor surface, a glycoenzyme, and a capture probe.
  • the glycoenzyme and the capture probe are adsorbed to the sensor surface.
  • the capture probe comprises a binding material that is capable of binding to a microorganism.
  • the presently disclosed subject matter provides a kit that comprises a glycoenzyme, a capture probe, and an exogenous amino acid (EAA) cocktail comprising an amino acid.
  • the glycoenzyme and the capture probe are in a formula to be adsorbed to a sensor surface of a biosensor.
  • the capture probe comprises a binding material that is capable of binding to a microorganism.
  • the EAA cocktail is placed separately from the glycoenzyme and the capture probe.
  • the presently disclosed subject matter provides a method for determining the viability of a microorganism.
  • the method comprises: capturing a microorganism in a sample with a capture probe adsorbed to a sensor surface of a biosensor, wherein a glycoenzyme is adsorbed to the sensor surface; adding an EAA cocktail to the sensor surface to form a mixture comprising the glycoenzyme, the EAA cocktail, and the microorganism; and measuring the change in pH or hydrogen peroxide of the mixture, thereby determining the microorganism viability.
  • a quantifiable change in pH or hydrogen peroxide of the mixture is associated with the number of viable microorganisms.
  • FIG. 1 is a schematic diagram illustrating a general scheme for viability detection combined with a generic capture step according to one embodiment of the presently disclosed subject matter.
  • FIG. 2 is a schematic illustration showing an example of reaction cascade for a two-step analysis of bacterial capture/detection (step 1) and viability testing (step 2) according to one embodiment of the presently disclosed subject matter.
  • FIG. 3 is a schematic illustration showing stimulus-response LBL protein nanobrush on reduced graphene oxide-nanoplatinum (rGO-nPt) electrodes according to one embodiment of the presently disclosed subject matter.
  • FIG. 4 is a graph showing representative Nyquist plots for LBL assembly of lectin- glycoenzyme nanobrush according to one embodiment of the presently disclosed subject matter.
  • FIG. 5 illustrates graphs showing chronoamperometric detection results of biogenic amine production using the LBL protein nanobrush (5 protein layers) according to one embodiment of the presently disclosed subject matter.
  • FIG. 7 illustrates graphs showing proof of concept demonstration of capture and viability cascade according to one embodiment of the presently disclosed subject matter.
  • a value or property is "based" on a particular value, property, the satisfaction of a condition, or other factor, if that value is derived by performing a mathematical calculation or logical decision using that value, property or other factor.
  • biosensor refers to a device that is used for detecting or monitoring organisms, such as microorganisms, cells, bacteria, pathogens, etc., based on the specific molecular (or macromolecular) interaction between a target organism and a sensor coating. The interaction results in an intermediate complex that leads to transduction of signal to an acquisition system.
  • a biosensor can be an analytical device which converts a biological response into an electrical signal, optical signal, or other transduction scheme.
  • exogenous amino acid (EAA) cocktail refers to a solution including one or more exogenous amino acids.
  • EAA exogenous amino acid
  • Exogenous refers to amino acids that are not endogenous to the specific sample (for example a food product, water sample, surface, etc. that contains natural amino acids).
  • the term “cocktail” refers to a mixture of amino acids and micronutrients that is suitable for a particular type of bacteria and a particular sample.
  • the term “enhance” refers to increasing or prolonging either in potency or duration of a desired effect.
  • free amino acid refers to amino acids that are available to a microorganism for metabolic activity within a 24 hour time frame, and excludes amino acids that may be available to bacteria over long periods of time (such as amino acids that result from the decay of natural material).
  • micronutrients refers to nutrients that an organism needs for healthy growth and development.
  • a non- limiting list of examples of micronutrients includes carbon, hydrogen, nitrogen, oxygen, phosphorus, potassium, sodium, calcium and magnesium, as well as trace elements such as iron, sulfur, chlorine, manganese, zinc, nickel, molybdenum, copper, iodine, selenium and cobalt.
  • target refers to an organism or a biological molecule to which some other entity, like a molecule, is directed and/or binds.
  • room temperature refers to a temperature of from about 20 °C to about 25 °C.
  • real time refers to a transaction that is processed fast enough for the result to come back and be acted on as transaction events are generated.
  • a real time measurement refers to the measurement of reactants or end-products during a chemical or other dynamic process.
  • Embodiments disclosed herein provide an approach for rapid determination of organism (e.g. bacteria) viability by measuring transient biogenic amine production (TBAP) after adding an EAA cocktail.
  • organism e.g. bacteria
  • TBAP transient biogenic amine production
  • Biosensors are devices that are used for monitoring pathogens based on the specific molecular (or macromolecular) interaction between the target organism and a sensor coating. The interaction results in an intermediate complex that leads to transduction of signal to an acquisition system.
  • many rapid biosensors have been developed for measuring pathogens based on interactions of the target cell with coatings composed of nucleotides, viruses, proteins, polymers, or peptides. 5 ' 6 ' 7 ' 8 ' 9 ' 10
  • nearly all of these sensors lack the ability of discerning pathogen viability after capture, which persists as one of the most difficult challenges in sensing.
  • cell membrane permeability analytical techniques include cell live/dead labeling assays, 14 vPCR, 15 16 and molecular viability testing, or MVT. 13 ' 17 ' 18
  • Cell labeling uses a combination of membrane-impermeable stain (e.g., propidium iodide) and membrane-permeable stain (e.g., SYT09) for analysis by fluorescence microscopy and/or flow cytometry.
  • membrane-impermeable stain e.g., propidium iodide
  • membrane-permeable stain e.g., SYT09
  • live/dead stains are known to have major issues with false positives.
  • the vPCR also uses the live/dead tagging concept, but the detection mechanism is based on inhibition of PCR amplification by a cell impermeant, photoactivated reagent (e.g., propidium monoazide).
  • MVT is also a PCR based method but uses RT-qPCR to detect production of a species specific macromolecule in response to exogenous nutrients.
  • BA There are a wide range of analytical techniques for detecting BA, including standard analytical tools such as spectrometry, chromatography, 27 PCR, 28 and biosensors. 29 ' 30 ' 31 ' 32 While high levels of BA are indeed a qualitative indicator of microbial spoilage, measurement of BA alone does not correlate with cell concentration or viability. Rather, the measurement of BA alone is used as a rapid qualitative screening tool for food samples as BA (such as histamine) can be directly toxic to humans upon ingestion. Amino acid content of various foods has been studied in detail. 33 ' 34 In such work, BA are used as a marker of amino acid metabolism by bacteria that are present in a sample.
  • Disclosed embodiments of the presently disclosed subject matters provide a novel, simple, low cost method for measuring bacteria viability without the need for expensive equipment or reagents.
  • a single enzyme system is used to monitor or test the transient production of BA using a reaction that is similar to a glucometer.
  • the testing period for monitoring the transient production can be within about 60 minutes at room temperature.
  • a system for measuring microorganism viability without the need for expensive equipment or reagents.
  • the system comprises a biosensor having a sensor surface, a diamine oxidase adsorbed to the sensor surface, and a capture probe adsorbed to the biosensor surface.
  • the biosensor can be any suitable sensor, such as a colorimetric paper test strip, a commercial metal electrode, a flexible electrochemical biosensor, etc.
  • the capture probe comprises a binding material that can bind to a microorganism.
  • the capture probe can be a mannose binding lectin, a DNA aptamer, or any other kind of binding material that can bind to a microorganism such as a bacterium or a pathogen.
  • the system can be used to determine microorganism viability, e.g., bacteria viability or pathogen viability, by measuring transient biogenic amine production produced by the microorganism after the addition of an exogenous amino acid.
  • the system disclosed herein further comprises an EAA cocktail comprising an exogenous amino acid.
  • An exogenous amino acid can be tyramine, histamine, agmatine, etc.
  • the EAA cocktail can be a specific cocktail that is prepared to represent the free amino acid and micronutrient content of a particular sample.
  • the EAA cocktail is a solution comprising one or more amino acids that can be adsorbed to a sensor surface of a biosensor.
  • the EAA is to be added to a sensor assay to rapidly assess microorganism viability based on the BA production produced by a microorganism after the addition of an exogenous amino acid.
  • the EAA cocktail is used for measuring transient biogenic amine production of a sample based on the chemical reaction between BA and a glycoenzyme used in the test solution.
  • the EAA cocktail is placed separately from the glycoenzyme, the capture probe, and the biosensor.
  • a lectin-based capture of target pathogens can be combined with a viability assay using a glycoenzyme such as diamine oxidase (DAOx).
  • a system for microorganism viability such as bacterial viability, can comprise a biosensor fabricated by developing a layer by layer (LBL) protein nanobrush on graphene- nanoplatinum electrodes.
  • the protein nanobrush comprises two distinct features: 1) the ability to selectively capture microorganism, e.g., a pathogen, a bacterium, based on stimulus- response capture (measured with impedance spectroscopy), and 2) determination of viability of the microorganism based on the decarboxylation of exogenous amino acids to biogenic amines based on enzymatic activity of DAOx (measured with chronoamperometry). If a viable organism is present, decarboxylation of the amino acids by the viable organism produces biogenic amines that can be measured in real time due to enzymatic production of peroxide by diamine oxidase.
  • microorganism e.g., a pathogen, a bacterium
  • stimulus- response capture measured with impedance spectroscopy
  • determination of viability of the microorganism based on the decarboxylation of exogenous amino acids to biogenic amines based on enzymatic activity of DAOx (measured with chronoamp
  • the protein nanobrush can be a multilayer nanobrush comprising stimulus-response materials, such as, but not limited to, a capture probe (e.g. mannose binding lectin such as concanavalin A,Con A) and a glycoenzyme diamine oxidase (DAOx), assembled layer-by-layer on a surface of a g-nanoplatinum electrode.
  • a capture probe e.g. mannose binding lectin such as concanavalin A,Con A
  • DAOx glycoenzyme diamine oxidase
  • the nanobrush can be created by drop-casting a solution of Con A and a solution of DAOx on an electrode surface and rinsing the electrode surface in distilled water.
  • the concentration of the solution of Con A can be about 0.1 mg/ml.
  • the concentration of the solution of DAOx can be about 0.1 mg/ml.
  • the lectin and enzyme concentration for creating the nanobrush layers can be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with a time of about 20 min at room temperature using PBS as a binding buffer.
  • a multilayer nanobrush can comprise 3, 5, or 7 layers of the stimulus-response materials.
  • a multilayer nanobrush can also contain layers of the stimulus-response materials with other numbers.
  • the outermost layer of the stimulus-response material can be terminated with a 64mer DNA aptamer specific to a microorganism (e.g. Listeria monocytogenes ) for targeted capture based on impedance.
  • a microorganism e.g. Listeria monocytogenes
  • the outermost layer of the stimulus-response material can be terminated with a generic capture probe such as Con A for detecting gram negative bacteria, for example.
  • Lectin-mediated organism capture is interrogated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry.
  • EIS electrochemical impedance spectroscopy
  • the protein nanobrush further contains a glycoenzyme (e.g., diamine oxidase) that is used to monitor the production of biogenic amine by viable cells of microorganism in real time using DC potential amperometry.
  • a kit for rapid determination of microorganism viability.
  • the kit comprises a glycoenzyme, a capture probe, and an EAA cocktail comprising an amino acid and, optionally, one or more micronutrients.
  • micronutrients includes carbon, hydrogen, nitrogen, oxygen, phosphorus, potassium, sodium, calcium and magnesium, as well as trace elements such as iron, sulfur, chlorine, manganese, zinc, nickel, molybdenum, copper, iodine, selenium and cobalt.
  • the micronutrients included in the EAA cocktail and amounts thereof will be adjusted depending on the nature of the sample tested.
  • the EAA cocktail is placed separately from the glycoenzyme and the capture probe before use.
  • the capture probe and the glycoenzyme are prepared in a formula, respectively, for being adsorbed to a sensor surface of a biosensor.
  • the glycoenzyme comprises diamine oxidase.
  • the capture probe can be, for example, a mannose binding lectin, a DNA aptamer, or a binding material that is suitable or capable of binding to a microorganism such as a microorganism (e.g. bacterium or a pathogen).
  • the capture probe and the glycoenzyme can be prepared in solutions respectively and be drop-cast to a sensor surface of a biosensor to form a layer of the capture probe and the glycoenzyme on the sensor surface.
  • a glycoenzyme and a capture probe can be assembled in a layer-by-layer (LBL) approach on a sensor surface.
  • LBL layer-by-layer
  • a mannose binding lectin and a diamine oxidase can be assembled in a layer-by-layer approach on a sensor surface of an electrode to form a LBL protein nanobrush.
  • Disclosed embodiments of the presently disclosed subject matter further provide a method for rapid determination or detection of microorganism viability.
  • This method includes capturing microorganisms such as bacteria from a sample and measuring in real time the transient biogenic amine production (TBAP) produced by viable microorganisms after adding an EAA cocktail. More specifically, in one embodiment, a microorganism in a sample is first captured with a capture probe adsorbed to a sensor surface of a biosensor. Then an EAA cocktail is added to the sensor surface to form a mixture comprising the EAA cocktail, the microorganism, and the glycoenzyme. After the addition of the EAA cocktail, the change in pH of the mixture or the change in hydrogen peroxide contained in the mixture is measured. A quantifiable change in pH or hydrogen peroxide is associated with the number of viable microorganisms. Thereby, the microorganism viability is detected or determined.
  • TBAP transient biogenic amine production
  • the glycoenzyme is diamine oxidase (DAOx).
  • DAOx diamine oxidase
  • the capture probe used to capture a microorganism in a sample can be a mannose binding lectin, a DNA aptamer, or any other kind of binding material that can bind to a microorganism such as a bacterium or a pathogen.
  • the EAA cocktail can comprises be tyramine, histamine, agmatine, and other amino acids.
  • the EAA cocktail can be a specific cocktail that is prepared to represent the free amino acid and micronutrient content of a particular sample. This method can be used for rapid determination of bacteria viability in food, water, or other sample.
  • This method can be applied to any pathogen biosensor, with a specific EAA solution tailored to match the endogenous amino acid and micronutrient concentration of a particular sample (e.g., a specific food product such as lettuce).
  • a particular sample e.g., a specific food product such as lettuce.
  • the method can be incorporated into any electrochemical sensor directly, and can also be modified for use as a colorimetric assay in optical biosensors.
  • FIG. 1 is a scheme illustrating the general scheme for viability detection according to one embodiment of the presently disclosed subject matter.
  • Panel a) of FIG. 1 is a scheme for analysis of bacteria viability based on a cascade reaction involving metabolism of an EAA cocktail according to one embodiment of the presently disclosed subject matter.
  • a glycoenzyme diamine oxidase, DAOx
  • a secondary capture probe 120 such as a mannose binding lectin or a DNA aptamer, is also adsorbed to the biosensor surface 110 of the biosensor 106.
  • Step 1 is a capture step to capture a microorganism such as a bacterium using the biosensor 110.
  • a viability test is conducted.
  • the viability test includes adding an EAA cocktail to the sensor surface 110 and monitoring the transient byproduct of EAA metabolism (biogenic amines).
  • the microorganism can be a bacterium or pathogen.
  • the term "bacterium" is in one sense broader than pathogen as it may include non-pathogenic bacteria. Pathogens may be bacteria or other microorganisms that cause disease.
  • Panel b) of FIG. 1 shows a detailed schematic of the reaction cascade in data analysis.
  • the YES state represents a presence of a viable population of microorganism such as a bacterium or pathogen.
  • the EAA is metabolized by a viable microorganis in the sample within the testing period, leading to a quantifiable change in pH and a concomitant increase in hydrogen peroxide due to the oxidation of B A by diamine oxidase (DAOx), where the pH is measured in real time during the viability test with a pH electrode.
  • DAOx diamine oxidase
  • oxidation of BA on the sensor surface is monitored using chronoamperometry, which produces a second signal in the YES (viable) state.
  • Changes in pH or hydrogen peroxide can also be measured using a variety of other transduction schemes such as optical techniques.
  • a quantifiable change in pH and hydrogen peroxide that is measured is linked to the number of cells detected in the capture step.
  • the NO state represents to a state that a non-viable microorganism such as a bacterium or a pathogen is present.
  • the cascade In the NO state (non-viable), no significant metabolism of EAA occurs, the cascade is not activated and no significant change in pH and/or BA occurs within the testing period; no significant change in pH and/or hydrogen peroxide is measured.
  • the quantitative cell numbers detected in the capture step can be attributed to dead cells and/or cell fragments. In either case, the concentration of cells is established in the first step.
  • Embodiments of the presently disclosed subject matter further provide a stimulus- response biosensor for determining bacteria viability using lectin-glycoenzyme nanobrushes.
  • FIG. 2 illustrated a detailed exemplary scheme of the cascade using a nanobrush biosensor. The scheme includes two step analysis of bacteria capture/detection (step 1) and viability testing (step 2).
  • a mannose binding lectin Concavilin A (Con A) 210 is used as the capture probe.
  • a layer-by-layer protein nanobrush is prepared with alternating layers of Con A 210 and the glycoprotein diamine oxidase (DAOx) 212.
  • DAOx glycoprotein diamine oxidase
  • lectin-mediate capture facilitates binding of gram negative cells from a food sample.
  • Lectin-mediated cell capture can be measured using an electrochemical impedance spectroscopy (EIS) or cyclic voltammetry.
  • EIS electrochemical impedance spectroscopy
  • cyclic voltammetry After lectin-mediated capture, an EAA cocktail comprising exogenous amino acids is added.
  • Viable cells captured on the sensor surface decarboxylate the amino acids to produce the biomarker of viability (BA).
  • transient BA production is monitored in real time by measuring the enzymatic production of hydrogen peroxide by DAOx.
  • a DC potential amperometry can be used to detect microbe-produced BA.
  • the total assay time for capture/viability can be about 40 minutes: about 10 min for cell capture and about 30 min for viability.
  • This new sensor approach can be expanded to target specific foodborne pathogens by altering the outermost capture probe of the nanobrush assembly (e.g., use of aptamer- decorated polymer nanobrushes), enabling rapid determination of food pathogen presence and viability without addition of exogenous reagents.
  • the method disclosed herein can be applied with colorimetric paper test strips, commercial metal electrodes, and flexible electrochemical biosensors.
  • the method can be validated using a variety of standard approaches, including: a secondary antibody for validating detection (fluorescein-labeled IgG), 47 an ATP assay for determining live cell metabolic state based on our stratified bienzyme sensor, 35 a commercial live/dead stain (BacLight), the paddle tester kit by Hach ⁇ , the dead cell stain (efluor 660), etc.
  • the product, system, and method disclosed herein can be used to ensure food safety and monitor water quality.
  • a multilayer pH-sensitive nanobrush described herein can be used for measuring pathogen viability in food.
  • the multilayer pH- sensitive nanobrush comprises a mannose binding lectin (concanavalin A, Con A) and a glycoenzyme diamine oxidase (DAOx) assembled in a layer-by-layer approach.
  • Con A mannose binding lectin
  • DAOx glycoenzyme diamine oxidase
  • the outermost layer of the brush was terminated with Con A or a 64mer aptamer to facilitate capture of Escherichia coli 0157:H7 (E.coli).
  • Lectin-mediated cell capture is interrogated using electrochemical impedance spectroscopy and cyclic voltammetry.
  • the optimal lectin and enzyme concentrations for creating the nanobrush layers are determined to be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with an optimum time of lectin/enzyme adsorption of about 20 min at room temperature using PBS as a binding buffer.
  • This new sensor approach can be expanded to target specific foodborne pathogens by altering the outermost capture probe of the nanobrush assembly (e.g., use of aptamer- decorated polymer nanobrushes), enabling rapid determination of food pathogen presence and viability without addition of expensive reagents.
  • the amino acid concentration/type can be further optimized for a particular food.
  • Clean drinking water is a mission-critical asset for deployed defense personnel.
  • the presence of pathogenic bacteria in water systems can cause widespread illness and even death, and thereby significantly hinder warfighter readiness and effectiveness.
  • Army, Air Force and Navy preventive medicine personnel all share similar potable water monitoring requirements, which includes monitoring indicator organisms that are linked to fecal contamination such as generic Escherichia coli (E. coli.) and coliform bacteria.
  • E. coli. generic Escherichia coli
  • coliform bacteria i.e., non spore-forming Gram negative bacteria
  • the total concentration of bacterial coliforms i.e., non spore-forming Gram negative bacteria
  • generic E. coli presence is a hallmark indicator for fecal contamination and potential waterborne pathogens.
  • Culture methods are extremely accurate and can discriminate viable from non- viable cells, but the test requires long incubation times and highly skilled personnel.
  • Biochemical methods such as testing cytosolic biomarkers, are faster than culture methods ( ⁇ 10 min) but cannot trace the results back to specific detection of indicator organisms and also produce false negatives due to incomplete cell lysis.
  • Enzyme-linked immunosorbent assay is a highly specific quantitative method that permits species or serotype level confirmation, but is expensive, low throughput, demands highly skilled personnel, and also requires long-time frame to obtain results (usually at least 24 hours).
  • Labeling techniques using flow cytometry, laser scanning, luminometry, or epifluorescence are rapid (typically ⁇ 1 hour), but fluorescent labels are expensive, susceptible to photobleaching, and some are cytotoxic. In addition, these acquisition systems are cost-prohibitive for large scale or high throughput field monitoring campaigns.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase PCR
  • NASBA nucleic acid sequence -based amplification
  • This water analysis system can detect presence and viability of fecal indicator organisms with a sensitivity of about 1 CFU/100 mL in total test time less than about 4 nr.
  • the technology disclosed herein combines: a rapid concentration/purification of target bacteria from raw water samples, viability discrimination using fluorescent labels, and subsequent analysis using simple imaging via a portable microscope or smartphone.
  • the disclosed technology improves water-testing protocols by reducing test time, cost, and logistical burden.
  • the technology further provides advancements that not only fill a critical technology gap unmet by any other competing technology for defense environments, but also potentially improve sanitation and food safety in other public and private domains (farms, municipal water systems, hospitals/clinics, etc.).
  • the disclosed water analysis system is used to rapidly test for presence and viability of coliform bacteria and E. coli in field water samples.
  • the disclosed water monitoring system is portable, battery-powered, reusable, easy to use, and selective to the specific indicator organism.
  • the disclosed water monitoring system leverages two emergent technologies that have independently developed for biosensing applications.
  • the first emergent technology involves a magnetic pre-concentration step using micrometer- sized magnetic microdiscs coated with capture probes (e.g., aptamers, proteins, etc.) that selectively bind to target bacteria and enable rapid concentration of those targets from about 100 mL samples in a matter of just seconds.
  • the second emergent technology involves the use of carbon quantum dots to enable live/dead viability assay on the magnetically concentrated discs via optical inspection using a smartphone based microscope.
  • the combination of highly selective capture-probe-functionalized microdiscs, magnetic pre- concentration, viability assay, and a portable microscopy system improves measurement time, sensitivity, and viability discrimination compared to other competing technologies.
  • the disclosed technique described herein is applied by using a layer by layer protein nanobrush impedimetric sensor for detection of E. coli 0157:H7.
  • the nanobrush is terminated with a generic capture probe (Concavilin A, a mannose-binding lectin) for detecting gram negative bacteria.
  • the nanobrush also contains the glycoenzyme diamine oxidase, which is used to monitor the production of BA by viable cells in real time using DC potential amperometry.
  • the total assay time for capture and viability test is about 42 minutes: about 10 min for cell capture, about 2 min for adding EAA, and about 30 min for detection of viability.
  • the technique is incorporated with three different sensors, namely a commercial Pt/Ir electrode, a laser inscribed graphene flexible electrode, and a nanomaterial colorimetric test strip.
  • FIG. 3 is a schematic illustrating a stimulus-response LBL protein nanobrush on reduced grapheme oxide-nanoplatnum (rGO-nPt) electrodes according to one embodiment of the presently disclosed subject matter.
  • a mannose binding lectin (Con A) and diamine oxidase (DAOx) are assembled in a LBL approach for developing a multilayer nanobrush.
  • Con A mannose binding lectin
  • DAOx diamine oxidase
  • Yao et al, 40 first developed a LBL stimulus response nanobrush for peroxide sensing using Con A and the glycoenzyme glucose oxidase (GOx), and later extended this to include glucose sensing. 41 Using the methods developed by Yao, 40 ' 41 the LBL protein nanobrush as shown in FIG.
  • the outermost layer of the nanobrush is terminated with a capture probe (e.g. Con A to facilitate capture of Escherichia coli 0157:H7, or other bacteria known to bind to Con A).
  • a capture probe e.g. Con A to facilitate capture of Escherichia coli 0157:H7, or other bacteria known to bind to Con A.
  • the sample is collected in a sample tube, and drop cast onto the sensor surface, or in other applications with high levels of bacteria the sensor may be immersed into a liquid sample that is agitated with a magnetic stir bar or other similar mixing equipment. After a contact time of 20 min, the sensor surface is washed with distilled water or buffer at least three times using a spray bottle prior to testing. Lectin-mediated cell capture is interrogated using EIS. After cell capture, exogenous amino acids are added and metabolism by viable microbes (e.g. E. coli) produce biogenic amines (BA). DC potential amperometry is used to detect microbe-produced BA at +
  • FIG. 4 shows representative Nyquist plots obtained during LBL assembly on rGO- nPt electrodes according to one embodiment of the presently disclosed subject matter.
  • the charge transfer resistance increases significantly with each addition of protein, indicating that the alternating charge interactions lead to formation of a chained nanobrush.
  • electrostaitc interaction with the metal surface caused a significant increase in the charge transfer resistance (Ret); the mannose binding site on Con A is highly negative and adsorbs well to platinum as shown by many others.
  • Ret charge transfer resistance
  • the mannose binding site on Con A is highly negative and adsorbs well to platinum as shown by many others.
  • 40 ' 41 ' 48 Subsequent addition of the glycoenzyme (DAOx) further increases Ret, and this trend continues for up to 7 layers of protein (alternating between Con A 210 and DAOx 212 as shown in FIG 2).
  • Assembly of the nanobrush is based on repeating adsorption of proteins in solution with alternating charges on the surface of the rGO-nPt electrode.
  • 48 lectin-glycoenzyme nanobrushes have stimulus responsive properties, switching between on/off states with changes in temperature, pH, or different chemical environments.
  • the optimal lectin and enzyme concentrations for creating the nanobrush layers are determined to be about 0.8 mg/mL and about 1.0 mg/mL, respectively; with an optimum time of about 20 min at room temperature using PBS as a binding buffer.
  • a LBL nanobrush with 5 layers is prepared and the sensitivity toward biogenic amines (no cell capture) is measured using DCPA at +350 mV.
  • FIG. 5 illustrates graphs showing the results of chronoamperometric detection of biogenic amines using the LBL protein nanobrush (5 protein layers).
  • Panel a) of FIG. 5 illustrates the calibration response of a biosensor toward tyramine, histamine, or agmatine during initial testing (no bacteria).
  • LBL nanobrush sensors are prepared using 5 layers or 7 layers, and EIS is used to test capture efficiency of E. coli 0157:H7 at a cell concentration of 10 3 CFU/mL based on R ct .
  • FIG. 6 shows the average impedance (cutoff frequency - 1 Hz) for rGO-nPt nanobrush electrodes after capture of 10 3 CFU/mL E. coli 0157:H7.
  • Panel A of FIG. 6 is a plot illustrating the charge transfer resistance measured after addition of layers in the synthesis of LBL.
  • Panel B of FIG. 6 are graphs illustrating the testing of 10 3 CFU/mL bacteria capture using a 5 layer system and a 7 layer system. As shown in Panel B of FIG.
  • the net impedance (cutoff frequency of lHz from Bode plot) increased significantly after cell capture, but with 7 layers the nanobrush is unstable and the impedance (-Z") decreases to levels that are similar to a bare rGO-nPt electrode.
  • the LBL capture system cannot be too long or the bacteria will cause the structure to collapse after capture.
  • cell capture is highest for a 3 or 5 protein LBL nanobrush.
  • the LBL is not less than 2 layers or more than 6 layers.
  • FIG. 7 illustrates the proof of concept demonstration of capture and viability cascade.
  • oxidative current is a marker of viable cells captured by the nanobrush.
  • the inset plot shows the differential current versus a control electrode with no cells.
  • E. coli 0157:H7 is captured with a 5 layer nanobrush (EIS plots not shown for brevity).
  • exogenous histidine 0.5mM
  • the noted increase in oxidative current (at +350mV) in FIG. 7 is due to the production of biogenic amine (histamine) by viable cells.
  • this histamine is then oxidized by DAOx, producing hydrogen peroxide.
  • FIG. 7 is strong evidence that the concept for viability has merit, lectins contain one or more carbohydrate recognition domain, and the structure of the CRD determines the overall specificity.
  • Con A is a mannose binding lectin that has affinity for a wide range of carbohydrate patterns on bacteria, viruses, protozoa and fungi. 49 ' 50 Thus, Con A is fairly non-specific and is used as a proof of concept in this work.
  • the lectin can be replaced with a strain-specific aptamer to enhance capture efficiency to specific organisms or foodborn pathogens. As a result, the kinetics of biogenic amine production by the specific spoilage organisms or foodborn pathogens can be examined.
  • This example includes side-by-side comparisons of the described methods versus established EPA methods using a variety of water samples based on established quality assurance and quality control (QA/QC) protocol.
  • the objective of this example is to detect about 1 CFU/100 mL with test time less than about 8 hr using a laboratory bench-top demonstration.
  • Prior to sample analysis all source water is characterized for general water quality parameters, including: temperature, pH, turbidity, total organic carbon, free and total disinfection residual, and heterotrophic plate count.
  • the Alternate Test Procedure undergoes a side-by-side comparison to the USEPA approved reference method for total coliforms (number SM9221B) and E. coli (number SM9221F).
  • the pathogen analysis follows comprehensive QA/QC guidelines form EPA/USDA/CDC.
  • the method development can include appropriate QA/QC according to USEPA approved standards, including replicate spiked reagent water, positive/negative spike controls, duplicate samples, method blanks, and media sterility checks. Characterization of method performance includes data on: precision/bias, specificity, detection limit, recovery, precision, and false positive/negative rates. Viability methodology
  • a test system is demonstrated to meet the USEPA-Alternate Test Procedure (ATP).
  • ATP USEPA-Alternate Test Procedure
  • a disposable biosensor on cellulose paper, 44 ' 45 an electrochemical (amperometric) viability assay is implemented based on direct measurement of metabolism using a cascade reaction with the bacteria captured on microdiscs. The technique is based on electrochemical detection of amino acid metabolism as described herein.
  • the sensor may utilize enzymatic activity of diamine oxidase (DAOx), which oxidizes biogenic amines produced by viable cells, resulting in oxidative current.
  • DAOx diamine oxidase
  • the working electrode for the disposable sensor is composed of graphene coated cellulose paper, 44 or laser inscribed graphene on plastic films.
  • bacterial cells are positioned on a conductive surface for the secondary confirmation step, which requires about 20 min and produces quantitative output regarding cell metabolism within about 30 min.

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Abstract

L'invention concerne, selon certains modes de réalisation, un procédé et un produit pour la détermination rapide de la viabilité d'un microorganisme. Le procédé consiste à mesurer la production transitoire d'amines biogènes par un microorganisme après l'ajout d'un cocktail d'acides aminés exogènes. Le produit comprend un biocapteur possédant une surface de biocapteur sur laquelle est adsorbée une glycoenzyme et une sonde de capture, la sonde de capture comprenant une substance de liaison qui est capable de se lier à un microorganisme.
PCT/US2018/044850 2017-08-01 2018-08-01 Détermination de la viabilité de bactéries par mesure de la production transitoire d'amines biogènes Ceased WO2019028162A1 (fr)

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CN110373165A (zh) * 2019-06-28 2019-10-25 中国石油集团川庆钻探工程有限公司钻井液技术服务公司 氨基酸改性的氧化石墨烯在水基钻井液中作为包被剂的应用
CN112098384A (zh) * 2020-09-22 2020-12-18 华东交通大学 一种快速预测水质是否生物稳定的简捷方法
WO2021237974A1 (fr) * 2020-05-26 2021-12-02 江苏大学 Dispositif à base de capteur organoleptique en voltampère et procédé de test rapide d'indicateurs physiques et chimiques de la sauce soja
CN114402195A (zh) * 2019-05-28 2022-04-26 拉莫特特拉维夫大学有限公司 用于侦测一病原体生物的数个系统及数个方法

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Publication number Priority date Publication date Assignee Title
CN114402195A (zh) * 2019-05-28 2022-04-26 拉莫特特拉维夫大学有限公司 用于侦测一病原体生物的数个系统及数个方法
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CN110373165B (zh) * 2019-06-28 2021-08-27 中国石油集团川庆钻探工程有限公司钻井液技术服务公司 氨基酸改性的氧化石墨烯在水基钻井液中作为包被剂的应用
WO2021237974A1 (fr) * 2020-05-26 2021-12-02 江苏大学 Dispositif à base de capteur organoleptique en voltampère et procédé de test rapide d'indicateurs physiques et chimiques de la sauce soja
CN112098384A (zh) * 2020-09-22 2020-12-18 华东交通大学 一种快速预测水质是否生物稳定的简捷方法
CN112098384B (zh) * 2020-09-22 2023-09-01 华东交通大学 一种快速预测水质是否生物稳定的简捷方法

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