WO2018031497A1 - Détection sans réactif à une seule étape par biocapteurs électrochimiques à base de protéines utilisant l'interférence stérique - Google Patents

Détection sans réactif à une seule étape par biocapteurs électrochimiques à base de protéines utilisant l'interférence stérique Download PDF

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WO2018031497A1
WO2018031497A1 PCT/US2017/045810 US2017045810W WO2018031497A1 WO 2018031497 A1 WO2018031497 A1 WO 2018031497A1 US 2017045810 W US2017045810 W US 2017045810W WO 2018031497 A1 WO2018031497 A1 WO 2018031497A1
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electrode
sensing element
recognition
redox
target species
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Kevin Plaxco
Di KANG
Shen SUN
Martin KURNIK
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • Electrochemical DNA-based (EDNA) sensors are capable of reagentless, real-time detection of a wide range of molecular targets. These sensors comprise a conformation-changing nucleic acid "probe,” such as an aptamer, that is covalently attached via one monomer to an electrode substrate and modified on a second (or more) with a redox reporter. Upon binding to its target molecule, the probe undergoes a conformational rearrangement or displacement of the redox reporter that generates an electrochemical signal in the form of a modulated redox current. Since this the signal generation mechanism is reagentless (e.g., label free), and the signal-generating change is rapid and reversible, the EDNA platform supports continuous, highly-time- resolved molecular detection.
  • a conformation-changing nucleic acid "probe” such as an aptamer
  • EDNA sensors can provide continuous, real-time measurement of target molecules in complex samples such as serum or whole blood, and have been
  • the EDNA concept potentially provides the art with a sensing platform that can be used in many important biomedical applications.
  • the EDNA concept has not been widely implemented.
  • the major factor limiting the use of EDNA sensors is the availability of nucleic acids binding targets of interest with sufficient affinity and specificity.
  • the ability to generate an aptamer to any selected target, for example, is limited.
  • a complementary aptamer is not available, is not readily generated, or may be impossible to generate due to constraints in nucleic acid chemistry.
  • proteins can selectively bind a range of target species.
  • conformation change upon target binding is typical and expected.
  • conformational change is not necessary, or indeed even typical, greatly reducing the number of potential targets that can be sensed using binding-induced conformational change. Accordingly, to the knowledge of the inventors of the present disclosure, the use of proteins in EDNA-like sensors has not been reported.
  • the inventors of the present disclosure have advantageously developed novel electrochemical sensors using proteins as probes.
  • the sensors of the invention do not require a binding-induced conformational change in the probe protein to generate a detectable signal. Instead, in the sensors of the invention, steric interference caused by target binding alters the efficiency of electron transfer between the reporter and electrode, providing a measurable signal of target binding.
  • the scope of the invention encompasses novel protein- based electrochemical sensors comprising redox reporter-modified proteins capable of selectively binding a target species.
  • a plurality (e.g., thousands to billions) of these functionalized proteins are bound to an electrode substrate.
  • the close association of the target moiety to the protein probe interferes with charge transfer between the redox reporter and the electrode substrate. The result is the dampening or ablation of the signal observed in voltammetry or chronoamperometry, providing a concentration-dependent, measurable signal of target binding.
  • the sensors of the invention may be used for the detection of a wide array of target species, including proteins, lipids, carbohydrates, small molecules, pathogens, drugs, pollutants, and other targets.
  • target species including proteins, lipids, carbohydrates, small molecules, pathogens, drugs, pollutants, and other targets.
  • the scope of the invention encompasses novel methods of detecting such targets using steric-interference based protein sensors.
  • Fig. 1 A is a diagram depicting a sensing element of the invention comprising an electrode substrate (101 ) coated with a thiol monolayer (104).
  • a recognition element polypeptide (102) comprising the bacterial chemotaxis protein CheY, is attached to the electrode by an anchoring moiety (103).
  • the recognition element polypeptide is labeled with a redox reporter (105).
  • charge transfer (106) between the redox reporter and the electrode is unimpeded.
  • Fig. 1 B depicts the recognition element with target species (107), CheY's naturally occurring binding partner CheA-P2, bound. Binding of the target species impedes charge transfer between the redox reporter (105) and the electrode (101 ).
  • Fig. 2A, 2B, and 2C depict cyclic voltammetry measurements for CheY-functionalized sensing element with and without Che-P2 present.
  • Fig 2B depicts cyclic voltammetry measurements for CheY-functionalized sensing element with and without FliM.
  • Fig. 2A and 2B show a decrease in peak faradaic current caused by steric interference.
  • Fig. 2C depicts time response of peak faradic current when a CheY-functionalized sensing element is exposed to 100 ⁇ of the target CheA-P2, with a time constant of 2.8 ⁇ 0.7 min "1 .
  • the error bars reflect standard deviation of measurements performed using multiple independently fabricated sensors and largely reflects sensor-to-sensor fabrication variation.
  • Fig. 3A and 3B depict signal change response to varying concentrations of CheY-P2 target for a for both phosphorylated CheY and non- phosphorylated CheY recognition elements (reporter at position 97).
  • Fig. 3B depicts signal change response to varying concentrations of FliM target for a for both
  • Fig. 4 depicts a CheY recognition element (401 ) and the spatial location of various redox reporter cysteine attachment sites generated by site-directed mutagenesis in different variants of the recognition element: A80C (402) , T71 C (403) , E37C (404), M17C (405), and E117C (407).
  • Bound target CheA-p2 (409) creates steric interference between redox reporter and the electrode (electrode surface attachment region denoted with arrow). Signal gain is dependent on the placement of the redox reporter relative to the target-binding and surface attachment sites with the greatest signal gain generally being observed when the reporter is positioned adjacent to the binding site.
  • Fig. 5A, 5B, 5C, and 5D depict signal response for sensors comprising redox-reporter-modified Green Fluorescent Protein (GFP) or CheY as the recognition element when exposed to anti-GFP antibodies at 10 ng/ml or control anti- FLAG antibodies.
  • Fig. 5B depicts the monotonic response to anti-GFP antibodies at various concentrations obtained when the recognition element is GFP modified with a methylene blue on random lysine residues.
  • Fig. 5A depicts signal response for sensors comprising redox-reporter-modified Green Fluorescent Protein (GFP) or CheY as the recognition element when exposed to anti-GFP antibodies at 10 ng/ml or control anti- FLAG antibodies.
  • Fig. 5B depicts the monotonic response to anti-GFP antibodies at various concentrations obtained when the recognition element is GFP modified with a methylene blue on random lysine residues.
  • FIG. 5C depicts signal change in response to anti-hepatitis B surface antigen antibodies at varying concentrations for sensors comprising hepatitis B surface antigen (HBsAg) recognition element modified with methylene blue on random cysteines, compared to a control sensor comprising a CheY recognition element, when the sample is buffer.
  • Fig. 5D depicts the same sensors as in Fig. 5C, when sample is 20% blood serum.
  • current changes were measured using square wave voltammetry.
  • the scope of the invention encompasses electrochemical sensors comprising redox-reporter-modified polypeptides as a probes which selectively bind a target molecule.
  • the invention encompasses a sensing element, comprising an electrode; and a plurality of recognition element polypeptides attached to the electrode; wherein each recognition element polypeptide is capable of selectively
  • each recognition element polypeptide is functionalized with one or more redox reporters, wherein charge transfer (e.g., faradic current) occurs between the one or more redox reporters and the electrode; and wherein binding of the target species to the recognition element causes a detectable change in the charge transfer between the the one or more redox reporters and the electrode.
  • charge transfer e.g., faradic current
  • sensing elements of the invention may be incorporated into sensor assemblies or sensor systems for the detection of target species by various means
  • the sensing elements of the invention are directed to the detection of a target species.
  • the target species may comprise any inorganic or organic molecule, for example: a small molecule drug, a metabolite, a hormone, a peptide, a protein, a carbohydrate, a nucleic acid, a lipid, or any other composition of matter.
  • the target species may comprise a drug.
  • the target species may comprise a chemical entity.
  • the target species may comprise a naturally occurring factor, for example a hormone, metabolite, growth factor, neurotransmitter, nutrients, and pollutants, pathogen-induced or pathogen-derived factors, etc.
  • the target species may comprise large and complex targets such as pathogenic cells or immune system elements such as antibodies or immune cells.
  • Target size may affect the magnitude of the signal change detected upon binding to the recognition element polypeptide. Typically, larger target species will have a greater steric hindrance on the redox- electrode interactions.
  • Electrodes The sensors of the invention will comprise one or more working electrodes to which recognition elements functionalized with redox reporters are bound.
  • the one or more electrodes may comprise various materials and
  • the electrode may comprise any suitable electrode material for electrochemical sensing, including, for example: gold or any gold-coated metal or material, titanium, tungsten, platinum, carbon, aluminum, copper, palladium, mercury films, silver, oxide-coated metals, semiconductors, graphite, carbon nanotubes, and any other conductive material upon which biomolecules can be conjugated.
  • suitable electrode material for electrochemical sensing including, for example: gold or any gold-coated metal or material, titanium, tungsten, platinum, carbon, aluminum, copper, palladium, mercury films, silver, oxide-coated metals, semiconductors, graphite, carbon nanotubes, and any other conductive material upon which biomolecules can be conjugated.
  • the electrode may be configured in any desired shape or size, including discs, strips, paddle-shaped electrodes, rectangular electrodes, electrode arrays, screen-printed electrodes, and other configurations.
  • a thin wire configuration is advantageous, as the low-profile wire may be inserted into cells, veins, arteries, tissue or organs and will not impede blood flow in blood vessels or cause substantial damage in tissues, for example, a wire having a diameter of 1 to 500 ⁇ .
  • Recognition element polypeptides The novel electrochemical sensors of the invention depart from the prior art EDNA design in the use of polypeptides as the recognition probe, i.e., as the unit that selectively binds the target.
  • the recognition probe comprises a polypeptide wherein a portion of the polypeptide will selectively bind to one or more target species.
  • the recognition probe polypeptide may comprise a polypeptide, i.e., a chain of two or more amino acids.
  • the polypeptide may comprise any combination of amino acids, including the natural amino acids, modified amino acids, non-natural amino acids or amino acid analogs.
  • the polypeptide may comprise bound co-factors, carbohydrate moieties (e.g., the product of glycosylation processes), and other non- amino acid components.
  • the polypeptide may comprise a polymeric protein, e.g., a protein having multiple subunits.
  • the polypeptide may comprise a natural protein isolated from biological materials or cells, or may comprise a chemically synthesized or recombinant polypeptide.
  • the size of the recognition probe may vary, from a few amino acids to thousands of amino acids.
  • the recognition element polypeptide comprises a whole protein, i.e. the entire native sequence of a protein as translated.
  • the recognition probe comprises a subsequence of a protein, i.e., a truncated form or fragment of a complete native protein sequence.
  • the polypeptide may also comprise a mutant or engineered form of a protein or protein domain.
  • the recognition probe polypeptide may comprise any amino acid sequence having a binding affinity for one or more target species, with sufficient specificity to prevent non-specific interactions.
  • Binding affinity means, for example, polypeptides having a picomolar to millimolar dissociation constant for the target ligand may be used.
  • the binding of the target species will occur at a binding site within the recognition probe.
  • the binding site as used herein, will refer to any amino acid residues of a polypeptide that facilitate or are necessary for the binding of the target species to the recognition probe.
  • the polypeptide comprises a receptor. In one embodiment, the polypeptide comprises an extracellular domain. In one embodiment, the polypeptide comprises an epitope. In one embodiment, the polypeptide selectively binds a biomarker of a condition, biological process, or disease state.
  • the polypeptide comprises an antibody or fragment thereof.
  • antibodies can be generated against a broad array of target species, resulting in a variable region having high affinity and specificity for the target species.
  • the recognition probe comprises a whole antibody.
  • the recognition probe comprises an antibody fragment comprising one or more antigen-binding regions derived from an antibody.
  • the recognition element polypeptide does not substantially change conformation when target is bound, i.e., there is no measurable change in faradic current attributable to the conformation change.
  • there is a conformational change upon target binding which, independent of steric effects, increases or decreases the interactions between the redox reporter and the electrode by changing the position of the redox reporter with respect to the electrode.
  • this conformation effect is smaller than the change in signal caused by steric interference, for example accounting for 1 -20% of signal change.
  • this conformation effect causes a major change in signal reduction that is augmented by steric effects on faradic current.
  • upon target binding there is a conformational change that enhances faradic current, which such effect is cancelled or obscured by a greater reduction in faradic current caused by steric interferences.
  • the recognition elements are bound to the surface of the electrode.
  • the recognition element may be conjugated to or otherwise associated with the electrode surface by any appropriate chemistry, for example by covalent bonding to the electrode or to a monolayer on the electrode, or via
  • the polypeptide comprising the recognition element may be modified at one terminal end (i.e., C-terminus or N-terminus) with an anchoring moiety.
  • the anchoring moiety may comprise a species that is capable of directly conjugating to the electrode surface.
  • the anchoring species may be capable of conjugation to a complementary functional group with which the electrode surface has been modified or decorated.
  • Anchoring moieties may comprise elements that form self-assembled monolayers on the electrode surface.
  • polypeptides may be thioiated or activated on their carboxy or amino terminal ends for bonding to the electrode surface, using chemistries known in the art. Amide linkages to lysine residues and thio-ether bond formation to cysteine residues may also be used, in one embodiment, the electrode surface (e.g. gold) is functionalized with nickel-nitrilotriacetic acid. These moieties can bind to histidines from His-tag functionalized proteins using copper or zinc or nickel, forming stable complexes.
  • the recognition element comprises a carboxy- terminal or amino-terminal hexahistidine tag.
  • the electrode for example, a gold electrode
  • NTA copper chelating nitrilotriacetic acid
  • the electrode for example, a metal oxide electrode
  • the electrode is coated with a self- assembled silane monolayer to which polypeptides may be attached by chemistries known in the art.
  • the anchoring moiety may comprise a click chemistry group, as known in the art, which is capable of forming bonds with complementary click chemistry groups conjugated to the electrode surface.
  • a click chemistry group as known in the art, which is capable of forming bonds with complementary click chemistry groups conjugated to the electrode surface.
  • non-natural amino acids are incorporated at specific positions in the protein to provide sites for surface tethering, for example, amino-acid analogs with azide side chains that enable surface attachment through click-chemistry.
  • the anchoring moiety may be an activated silane, as known in the art, which is capable of forming bonds to many oxide surfaces.
  • the anchoring moiety may contain a ligand, which can bind to the surface via coordination bond.
  • the recognition element polypeptides may be deposited on the electrode surface at any desired density, for example, in the range of 1 x10 9 to 1 x10 12
  • Linkers In some implementations, a short linker is present between the electrode surface and the recognition element polypeptide. In one embodiment, the linker comprises a nucleic acid sequence, for example a DNA or RNA sequence.
  • Previously described electrochemical sensors for example as in White et al., 201 1 , “Wash-free, Electrochemical Platform for the Quantitative, Multiplexed Detection of Specific Antibodies," Anal. Chem. 2012, 84, 1098-1 103 ) have used polypeptide recognition elements tethered to double stranded nucleic acids, for example 10-30 nucleotide sequences of DNA, PNA, or RNA, wherein one strand is functionalized with a redox reporter. In such sensors, binding of the target to the peptide moiety changes the conformational flexibility of the reporter-labeled nucleic acid linker, resulting in a signal change. Unlike such previous sensors, the sensors of the present invention do not require the additional nucleic acid elements and do not rely on conformational change of the reporter element to generate signal.
  • Each recognition element polypeptide is functionalized with one or more redox reporters.
  • the redox reporter is any composition of matter that interacts with the selected electrode material creating a faradic current.
  • Exemplary redox species include methylene blue, ferrocene, viologen, anthraquinone or any other quinones, daunomycin, organo-metallic redox reporters, for example porphyrin complexes or crown ether cycles or linear ethers, ruthenium, bis-pyridine, tris-pyridine, bis-imidizole, cytochrome c, plastocyanin, and ethylenetetracetic acid.
  • the redox reporter may be attached to the recognition element by any appropriate chemistry.
  • a particular conjugation chemistry is selected for conjugating the redox reporter to the polypeptide at a "compatible residue," i.e. a residue suitable for conjugation of a redox reporter using a selected linkage chemistry.
  • Any system comprising appropriate chemistry and compatible residue may be utilized for attachment of the redox reporter to the recognition element polypeptide.
  • the redox reporter is conjugated to a cysteine residue within the recognition element polypeptide.
  • the redox reporter is functionalized with maleimide and is conjugated to a cysteine residue in the recognition element polypeptide by thio-maliemide linkage.
  • the redox reporter is a maleimide-functionalized methylene blue.
  • the redox reporter is conjugated to cysteines using iodacetamides, alkynes, or other conjugation reagents known in the art.
  • the redox reporter may be conjugated to the polypeptide at a lysine residue, using linkage chemistries such as N- hydroxysuccinimidyl ester, isocyanate, or benzoyl fluoride conjugation, as known in the art.
  • conjugation of the redox reporter to the polypeptide is achieved by incorporation of one or more non-natural amino acids in the recognition element, which such non-natural amino acids are suitable for conjugation chemistries, e.g., click chemistry.
  • non-natural amino acids are suitable for conjugation chemistries, e.g., click chemistry.
  • azidohomoalanine or propargyl-derivatized lysine are incorporated into the polypeptide at selected sites for conjugation to the redox reporter using copper-catalyzed azide-alkyne cycloaddition,
  • Redox Reporter Placement The detection capability of the sensors of the invention is based on steric interference, such that binding of the target species will impede electron transfer between the redox reporter and the electrode. Within the limits of the sensor design, the detected change in signal will be monotonically related to the concentration of the target species in the sample.
  • the steric-interference based sensors of the invention are highly sensitive, and often the binding of target to the recognition element creates sufficient steric interference to create a measurable signal change regardless of the placement of the one or more redox reporters on the recognition element polypeptide.
  • random residues of the recognition element polypeptide may be modified with redox reporter.
  • redox reporter e.g., lysine or cysteine
  • these may be functionalized with redox reporter in a reaction that randomly distributes one or more redox reporter moieties among the compatible residues.
  • random lysines throughout the polypeptide are modified.
  • epsilon amine group of random lysines may be modified methlyene blue NHS-ester.
  • redox reporters are placed in proximity to the target-binding site.
  • a modifiable residue in proximity to the binding site is selected for modification with the redox reporter and an appropriate chemistry for conjugation is utilized.
  • site-directed mutagenesis is used to create a compatible residue in proximity to the binding site.
  • the reporter moiety may be placed in proximity to the binding site, for example, being within 1 -30 angstroms from the binding site.
  • the sensors of the invention may be fabricated based on methods of fabricating EDNA sensors, as known in the art, with appropriate modification for the attachment of recognition element polypeptides to the electrode.
  • sensors may be prepared by analogy to previously described sensors, for example as described in: Xiao, Y., Rowe, A. A., and Plaxco, K. W. (2007)
  • the electrochemical sensing elements of the invention may be configured in various assemblies to make fully functional sensing systems.
  • a sensor assembly will comprise a collection of elements that may operate together to perform various operations of the sensing process.
  • the invention comprises a sensor assembly comprising a sensing element and a reference electrode, for example an Ag/AgCI electrode, or other reference electrode known in the art.
  • the sensing assemblies of the invention may further comprise an auxiliary or counter electrode, for example, a platinum auxiliary electrode.
  • the sensor assemblies of the invention may be configured in a three- electrode cell system.
  • the three-electrode cell system will comprise one or more sensing, reference, and auxiliary electrodes, appropriately configured for performing electrochemical interrogation measurements.
  • the three-electrode cell system may comprise a mixing chamber or other vessel wherein the electrodes are present and are contacted with the sample.
  • the sensor assemblies of the invention may further comprise or be in connection with appropriate electronic components for performing electrochemical measurements.
  • the electronic components may comprise two or more devices in electrical and/or network connection with one another, or may comprise a single integrated device.
  • Electronic components may include potientiostats or other voltage sources and voltage controllers.
  • the system may further comprise appropriate circuitry for reading sensor outputs, and storing such outputs or routing the outputs to other devices.
  • the systems of the invention may further comprise data processing means, for example, a general-purpose computer or other data processor capable of carrying out the various calculations utilized in the methods of the invention.
  • the scope of the invention further encompasses non-transitory computer-readable recording media having stored thereon an encoding program that causes a computer to execute a process, the process comprising one or more data processing calculations for readout and interpretation of signals from a sensing element.
  • the sensor assembly comprises a tabletop lab apparatus. In other embodiments, the sensor assembly comprises a hand-held device. In other embodiments, the sensor assembly comprises a microfluidic biochip.
  • the sensor assembly of the invention is configured as an in vivo sensor.
  • An "in vivo" sensor means a sensor configured to sample fluids within the body of a living organism.
  • the sensing element is inserted, implanted, or otherwise placed within the body of a living organism such that the sensing element is exposed to in-vivo fluids, e.g., blood.
  • a sensing assembly comprising a thin wire configuration is
  • the low-profile wire may be inserted into veins, arteries, tissue or organs and will minimally impede blood flow in blood vessels or will cause minimal damage in the sampled area.
  • a wire having a diameter of 1 -500 ⁇ , for example, 100 ⁇ may be used.
  • the sensing assemblies are housed in a needle, catheter, or cannula that may be inserted into a vein, blood vessel, organ, tissue, or interstitial space in order to place the sensor in the target environment.
  • the needle, catheter, or cannula may be porous, comprising a plurality of holes or channels distal to the tip in order to allow the flow of blood over the sensor assembly.
  • the sensing element comprises an electrode functionalized with a plurality of redox-label modified recognition element polypeptides capable of selectively binding a target species, such that binding of the target
  • sensing element is in connection with and/or proximity to
  • the sample may be any sample.
  • the sample may comprise blood, serum, saliva, urine, interstitial fluid, spinal fluid, cerebral fluid, tissue exudates, macerated tissue samples, cell solutions, intracellular compartments, water, food, groundwater, or other biological and environmental samples.
  • the sample may be flowing whole blood, interstitial fluid, or other biological material.
  • the sample comprises the surrounding environment in which the sensing element is deployed, e.g., in water, in soil, in vivo, etc. Samples may be unaltered or may be pretreated prior to analysis, for example being filtered, diluted, concentrated, buffered, or otherwise treated.
  • the sensors of the invention are reagentless and have the capacity to operate in complex samples such as whole blood, greatly simplifying the sample preparation process.
  • the interrogation of faradic current may be accomplished by any means known in the art, for example by cyclic voltammetry, differential pulse voltammetry, alternating current voltammetry, square wave voltammetry, potentiometry or
  • chronoamperometry In one embodiment, the use of kinetic differential measurement techniques, as known in the art can be employed to improve signal to noise ratio.
  • Determination of target concentration may be accomplished by
  • the sensors of the invention may be operated for extended periods of time, e.g., days, weeks, or months, for the continuous measurement of target species, for example, in the body of an organism (e.g., a human or test animal) or in the environment.
  • the methods of the invention comprises the steps of withdrawing a sample from a living organism, exposing a sensor of the invention that is directed to detection of a target species to the sample, and measuring the concentration of the target species in the sample.
  • the sample fluid is withdrawn continuously from the living organism and target species concentration is measured on a prolonged basis.
  • a single sample is analyzed.
  • the sample is blood.
  • the sensor is housed in a wearable or otherwise portable device.
  • the sensors of the invention are employed in point of care testing methods.
  • a sample is withdrawn from an animal, subject, or patient and the concentration of a target species is measured using a sensor of the invention.
  • the sample is a blood sample, for example, a pin-prick or finger-prick blood sample, for example, a self-withdrawn pinprick or finger-prick blood sample, or a urine or saliva sample.
  • the sensors of the invention advantageously enable the immediate testing of small samples, obviating the need for processing blood or other samples prior to analysis.
  • the sensors of the invention may be utilized for various biological or environmental measurements.
  • the recognition element polypeptide selectively binds a drug.
  • the sensors of the invention may be utilized to determine the concentration of a target drug in the body, for example in an in-vivo real time monitoring system.
  • the methods of the invention are performed for the detection of a drug.
  • the target species may comprise a drug having significant side effects, such as a chemotherapeutic drug, or a drug having a narrow therapeutic index, wherein accurate measurement of blood levels would allow for safe dosing with minimal side effects.
  • the method of the invention is performed in the operation of a feedback controlled dosing system, as known in the art.
  • the methods of the invention are performed for the detection of metabolite, hormone or other biomarker indicative of organ function, health, or disease status, for example, glucose, creatinine, Cortisol, or A and B-type natriuretic peptides.
  • the recognition element polypeptide selectively binds a biomarker of a condition, a biological process, or disease state and the methods of the invention are performed for the assessment of such condition, biological process, or disease state.
  • the recognition element polypeptide comprises a receptor protein and the methods of the invention are performed for the detection of an activator, modulator, or repressor species.
  • the recognition element polypeptide comprises an epitope to which antibodies, activated T-cells, or other immune system elements may bind and the methods of the invention are performed for the detection of such immune system elements.
  • Such sensing elements may be used, for example, to detect and quantify antibody titers against a selected target.
  • the recognition element polypeptide selectively binds a pathogenic cell or a factor secreted by a pathogenic cell and the methods of the invention are performed for the diagnosis of infection or disease.
  • the methods of the invention are performed for the detection of a pollutant or contaminant and the sample comprises an environmental sample or a food sample.
  • Electrochemical impedance spectroscopy employs an electrode-bound receptor such that the steric or electrostatic "bulk” of the target molecule impedes the ability of an added redox moiety (e.g., ferrpcyanide) to approach and generate a current.
  • EIS suffers, however, from overwhelming background effects when challenged with realistically complex samples due its extreme sensitivity to non-specific adsorption.
  • an electrochemical sensing approach has been pursued, utilizing a double-stranded nucleic acid "scaffold" modified on one end to present both a protein-recognizing polypeptide or small molecule and a redox reporter and covalently attached to gold electrode via a flexible linker via the other.
  • the previous approach is extended by utilizing sensors that, rather than using a double-stranded DNA scaffold and a relatively low molecular weight recognition element (e.g., a polypeptide), instead employ full-length proteins as both the recognition element (receptor) and the scaffold, expanding the range of analytes that the approach can be used to detect.
  • a relatively low molecular weight recognition element e.g., a polypeptide
  • CheY a response regulator protein from the E. coli chemotaxis signal transduction system was selected.
  • CheY into a single-step electrochemical sensor a family of CheY variants was generated containing a carboxy-terminal hexa-His-tag with each exposing a single cysteine side chain for conjugation to a maleimide-functionalized methylene blue.
  • the sensor architecture responds quantitatively when challenged with the appropriate target molecule.
  • the redox reporter In the absence of either of CheY's binding partners the redox reporter is relatively free to collide with the electrode surface, producing a large faradaic current at the redox potential expected for methylene blue when the system is interrogated using cyclic voltammetry. This peak is reduced in the presence of the protein's binding partners.
  • the current falls 22% upon the addition 100 ⁇ of the ligand CheA-P2, a 74- residue protein that is part of the bacterial chemotaxis system, with a time constant of 2.8 ⁇ 0.7 min "1 .
  • the observed signal change increases monotonically with increasing ligand concentration until it approaches saturation at a change of 24%.
  • the resultant binding curve is well fitted with a Langmuir isotherm, producing a dissociation constant of 14 ⁇ 4 ⁇ , which is within error of the value previously reported for this interaction for the proteins when free in solution.
  • the sensor also performs well when challenged in clinically realistic samples. For example, the sensor's gain is only slightly reduced, to 17% (reporter at position 97), when it is instead challenged in 20% blood serum.
  • the signal gain (the relative signal change seen upon the addition of saturating target) seen for this sensing architecture depends on both the attachment- position of the methylene blue and the structure of the target. To illustrate the former, seven sensors were fabricated differing only in the residue on which the methylene blue was attached using the variants M17C, E37C, T71 C, A80C, G89C, K91 C, K97C and E117. The signal gain observed for these when CheA-P2 was employed as the target ranged from 4% to 30%, with the later value larger than the signal change typically seen in fluorescence polarization assays.
  • the largest gain is seen when the redox-reporter is placed closest to the CheA-binding site (position 97), demonstrating that signal change arises due to steric blocking of the redox reporter by the target protein. Consistent with this, the gain of the sensor is abated when the reporter is positioned farther from the binding site (e.g., at E37C or A80C).
  • the sensor's ability to detect a second naturally occurring binding CheY binding partner was also tested, the 16-residue peptide FliM 16 , and behavior similar to that for the detection of CheA-P2 was observed. Specifically, a sensor modified with methylene blue near the binding site (at residue 91 ) exhibits the greatest gain (14%). The signal gain observed upon FliM 16 binding, however, is generally less than that observed upon CheA-P2 binding. This demonstrates the consequence of the larger bulk of CheA-P2, which hinders the approach of the reporter to the electrode.
  • This new sensor architecture readily detects changes in binding affinity associated with the phosphorylation of CheY. Phosphorylation induces allosteric communication between the phosphorylation site (D57) and the target-binding site, which in turn facilitates CheY's dissociation from CheA while strengthening its interactions with the flagellar motor switch protein FliM. Consistent with this, significant decreases and increases in affinity for CheA-P2 and FliM 16 , respectively, were observed upon phosphorylation of the surface-bound CheY. Specifically, the phosphorylation of surface-bound CheY enhances its affinity for FliM 16 by a factor of ca.
  • HBsAg hepatitis B surface antigen
  • HBsAb anti-hepatitis antibodies
  • This new protein-based, electrochemical sensing platform provides an alternative means to fluorescence polarization for probing protein-macromolecular interactions and thus provides the art with a promising tool for point-of-care clinical applications. Fluorescence polarization assays have seen widespread adoption at the point of care for the measurement of markers of heart attacks and drug overdose despite producing signal changes of only 10-20%.
  • the sensors of the invention produce similar signal gains and are similarly label-free, providing a system that is faster, simpler, and, less costly than ELISAs and western blots.
  • the transition from optical to electrochemical read-outs offers potential advantages, including its relatively inexpensive supporting electronics, its ability to perform well in relatively high concentrations of blood serum, and the ready multiplexing of electrochemical approaches.
  • the receptor proteins used were obtained as follows. The gene encoding wild-type E. coli Che Y (residues 1 -129) was cloned into pET28a at the Ncol and Xhol sites in frame with the carboxy-terminal hexahistidine tag. Single-cysteine variants (M17C, E37C, T71 C, A80C, G89C, K91 C, K97C and E1 17) were generated with a site-directed mutagenesis kit and transformed into E. coli BL21 (DE3). The mutagenesis results were checked via sequencing.
  • IPTG isopropyl ⁇ -D-l -thiogalactopyranoside
  • the culture was harvested after 3 hr, resuspended in 50 mM sodium phosphate, 10 mM imidazole, 300 mM sodium chloride, pH 8.0 buffer and lysed by French pressure cell press. Cell debris was removed by centrifugation 30,000 g for 30 min before application of the supernatant to an immobilized metal ion (nickel) affinity chromatography column.
  • the column was washed with 50 mM sodium phosphate, 300 mM sodium chloride and 35 mM imidazole, pH 8. His-tagged CheY was eluted with 150 mM imidazole in the same buffer. The collected fractions were dialyzed into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.9 and concentrated to ⁇ 1 mM (CheY D57A variants were concentrated to ⁇ 0.5 mM). Purity of all variants was >95% as determined by SDS-PAGE electrophoresis. His-tagged emerald green fluorescent protein (GFP) was expressed from pRSET-EmGFP expression plasmid in E.
  • GFP His-tagged emerald green fluorescent protein
  • the target proteins and peptides were obtained as follows.
  • the CheA P2 domain (residues 156-229) was expressed from the pTM22 plasmid in E coli strain K38 and purified by ion-exchange chromatography and size-exclusion fast performance liquid chromatography. Purity was >95% as determined by SDS-PAGE electrophoresis.
  • the maleimide-functionalized methylene blue used as the redox reporter and was synthesized starting with 5 mg monocarboxy-methylene blue NHS ester dissolved in 150 ⁇ _ DMSO. 15 mg N-(2-aminoethyl) maleimide trifluoroacetate salt and 10 ⁇ _ triethylamine were added followed by 12 hr stirring at room temperature. 50 ⁇ _ 0.5 M sodium bicarbonate solution was added to this crude reaction mixture and incubated for 2 hr. The final mixture was dried under reduced pressure and purified using silica thin layer chromatography (CHCI 3 : methanol 10:1 ).
  • the relevant receptor protein was modified with a methylene-blue redox reporter at either cysteine or lysine.
  • cysteine either single-cysteine variants of CheY or the wild-type cysteines of GFP and HBsAg were used. These were first reduced by treatment with 5 mM dithiothreitol for 2 hr prior to removal using a spin column) immediately followed by the addition of maleimide-modified methlyene blue (as 1 -2% v/v solution in DMSO) at a 10:1 reagent- to-protein molar ratio.
  • the CheY D57A variants were labeled with MB2 maleimide- modified methylene blue.
  • reaction mix was incubated at room temperature for 12 hr, and a spin column was then used to remove unreacted methylene blue and exchange the sample into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.9.
  • a spin column was then used to remove unreacted methylene blue and exchange the sample into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.9.
  • the receptor protein on the epsilon amine group of random lysines was modified.
  • Wild-type protein was mixed with methlyene blue NHS-ester (as 1 -2% v/v solution in DMSO) at a 2:1 reagent-to-protein molar ratio in 0.5 M sodium bicarbonate solution (pH 8.5) for 4 hr in dark at room temperature, and then a spin column was used to remove unattached methylene blue and exchange the sample into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.9.
  • methlyene blue NHS-ester as 1 -2% v/v solution in DMSO
  • a spin column was used to remove unattached methylene blue and exchange the sample into 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.9.
  • Sensors were prepared by analogy to previously described E-DNA sensors.
  • gold disk electrodes 2.0 mm diameter, were cleaned both mechanically (by successively polishing with 1 ⁇ diamond and 0.05 ⁇ aluminum oxide slurries) and electrochemically (through successive scans in 0.5 M sulfuric acid and 0.1 M sulfuric acid 0.01 M KCI). Proteins were grafted onto these electrodes using Cu-NTA/His-tag complexation as follows.
  • NTA-thiol ( ⁇ -[ ⁇ , ⁇ - bis(carboxymethyl)-lysine]-12-mercaptodocanamide) was dissolved in 3 mM 6- mercapto-1 -hexanol methanol solution at 10 ⁇ (NTA-thiol/6-mercapto-1 -hexanol ratio is 1 :300). Freshly cleaned electrodes were incubated in this solution for 1 hr at room temperature, rinsed with methanol, and then incubated in 10 mM 6-mercapto-1 -hexanol in methanol overnight (-16 hr) at 4°C to form a continuous, mixed, self-assembled monolayer on the gold electrode surface.
  • the NTA-modified electrodes were then rinsed with methanol and incubated with 100 ⁇ copper sulfate in deionized water for 20 min.
  • the electrodes were then incubated with the methylene blue (MB) modified protein for 30 min at room temperature, using 2 to 10 ⁇ CheY (based on concentration determined by ultraviolet-visible spectroscopy immediately prior to modification with methylene blue) and GFP or 0.2 mg/ml HBsAg.
  • the electrodes were then washed with 150 mM imidazole in 50 mM sodium phosphate, 500 mM sodium chloride, pH 7.0.
  • the CheY coated electrode was rinsed with 50 mM sodium phosphate, 100 mM sodium chloride buffer, and incubated in the buffer for 30 min immediately prior to use.
  • the GFP and HBsAg coated electrodes were washed with 2% bovine serum albumin (BSA) and 0.05% Tween 20.
  • BSA bovine serum albumin
  • CheY phosphorylation was carried out using 10 mM acetyl phosphate in 50 mM sodium phosphate, 5 mM MgCI 2 , 100 mM NaCI. The freshly prepared CheY electrodes were incubated in 10 mM acetyl phosphate in buffer for 30 min before test. CheY was phosphorylated by acetyl phosphate on the surface immediately prior to measuring binding.

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Abstract

L'invention concerne de nouveaux capteurs électrochimiques basés sur des polypeptides modifiés par un rapporteur redox. Les capteurs détectent les courants faradiques entre les polypeptides modifiés par un rapporteur redox et un substrat d'électrode. Lors de la liaison de l'espèce cible, l'interférence stérique modifie le signal en fonction de la concentration. La conception des capteurs décrits dans la présente invention permet l'utilisation de sondes à base de protéines pour détecter une large gamme de cibles chimiques et biologiques. Les capteurs peuvent être avantageusement déployés dans des échantillons complexes, tels que du sang total, et peuvent être déployés in vivo.
PCT/US2017/045810 2016-08-09 2017-08-07 Détection sans réactif à une seule étape par biocapteurs électrochimiques à base de protéines utilisant l'interférence stérique Ceased WO2018031497A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108535343A (zh) * 2018-04-01 2018-09-14 桂林理工大学 亚甲基蓝-金复合纳米微粒修饰电极的制备方法及其应用
CN108690870A (zh) * 2018-05-15 2018-10-23 北京工业大学 三联吡啶钌电化学发光淬灭生物传感器的制备方法
WO2020176792A1 (fr) * 2019-02-27 2020-09-03 California Institute Of Technology Approche de détection électrochimique pour la quantification de molécules dans des fluides corporels
CN113711040A (zh) * 2019-02-27 2021-11-26 加州理工学院 用于体液中分子量化的电化学传感方法
US11549934B2 (en) 2019-02-27 2023-01-10 California Institute Of Technology Electrochemical sensing approach for molecule quantification in body fluids
CN114222921A (zh) * 2019-06-12 2022-03-22 加利福尼亚大学董事会 E-dna型传感器中的调节电子转移动力学
JP2024501571A (ja) * 2021-01-14 2024-01-12 オプトレーン テクノロジーズ インコーポレイテッド スイッチングペプチド、それを含む免疫分析装置及びそれを用いた免疫分析方法
JP2024501570A (ja) * 2021-01-14 2024-01-12 オプトレーン テクノロジーズ インコーポレイテッド スイッチングペプチド及びそれを用いたマルチプレックス免疫分析方法
JP2024501572A (ja) * 2021-01-18 2024-01-12 オプトレーン テクノロジーズ インコーポレイテッド スイッチングペプチド及びそれを用いた免疫分析方法
WO2022154632A1 (fr) * 2021-01-18 2022-07-21 (주)옵토레인 Peptide de commutation et dosage immunologique l'utilisant
US11513097B1 (en) 2021-05-21 2022-11-29 PERSOWN, Inc. Methods of obtaining and using electrochemical diagnostic results
US11525799B1 (en) 2021-05-21 2022-12-13 PERSOWN, Inc. Electrochemical diagnostic system
EP4392183A4 (fr) * 2021-08-23 2025-05-21 Universal Biosensors PTY Limited Détecteurs électrochimiques
CN116297756A (zh) * 2022-12-23 2023-06-23 江苏大学 一种检测黑色素瘤标志物s100b蛋白的电化学传感器及其制备方法

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