WO2015028811A2 - Procédés de détection de protéines - Google Patents

Procédés de détection de protéines Download PDF

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Publication number
WO2015028811A2
WO2015028811A2 PCT/GB2014/052620 GB2014052620W WO2015028811A2 WO 2015028811 A2 WO2015028811 A2 WO 2015028811A2 GB 2014052620 W GB2014052620 W GB 2014052620W WO 2015028811 A2 WO2015028811 A2 WO 2015028811A2
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Prior art keywords
protein
reagent
sample
reaction
immobilised
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WO2015028811A3 (fr
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Jonathan Cooper
Julien Reboud
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University of Glasgow
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University of Glasgow
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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
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/726—Devices
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/725—Haemoglobin using peroxidative activity

Definitions

  • Figure 3 shows typical responses of an electrochemical immunoassay of the invention at different concentrations of Hb (equivalent to a range between 0 and 8.0 mg/mL Hb), measured in vitro using model analytical solutions.
  • the protein may comprise a polypeptide, and the protein is capable of catalysing the reaction of the first reagent and the second reagent.
  • references to a protein may also include references to the glycosylated versions of that protein.
  • the methods described herein may be used to directly detect such proteins if the glycosylation does not interfere with the ability of the protein to catalyse the reaction of the reagents.
  • the haemoglobin is a glycosylated haemoglobin.
  • haemoglobin may be taken as a reference to a single haemoglobin type as described above, or a plurality of haemoglobin types. Binding moieties are available that are capable of selectively binding one haemoglobin type over other of haemoglobin types.
  • Such detection and analysis is based on the catalytic activity of the protein label. It follows that the protein, whilst it is associated with the analyte, retains its ability to catalyse the reaction of the reagents. The analyte itself does not catalyse the reaction of the reagents.
  • the protein may itself be associated with an analyte of interest as noted above. This association does not result in the loss of the catalytic activity of the protein for the reaction of the reagents.
  • the increase in the reaction rate of the reagents is an indicator for the presence of the protein, bound to the binding moiety.
  • the present case uses the intrinsic activity of the immobilised protein to catalyse the reaction of the reagents.
  • the sandwich assays rely on the activity of the additional binding moiety to provide a reporter activity for the immobilised protein.
  • the protein is not associated with an additional binding moiety.
  • the catalytic improvement in the reaction rate of the reagents is associated with the protein itself, and not any binding moiety, label or analyte that is connected to it.
  • the methods for the detection of the protein involve the step of measuring the reaction catalysed by the protein.
  • the method of the invention may include the step of determining the contribution of the protein to the change in the reaction rate.
  • the protein is capable of acting as a catalyst for the reaction of the first reagents and the second reagent.
  • the binding moiety, the surface and so on have a negligible catalytic activity.
  • the present invention uses the catalytic activity of the protein as an identifiable characteristic to allow the presence of that protein to be detected and quantified.
  • the method of the invention may be used to detect the presence or absence of a protein in a sample.
  • the protein is the analyte of interest.
  • the protein is associated with the analyte of interest, and therefore acts as a label.
  • the sample may be a biological sample.
  • the sample may be obtained from a subject such as a mammal, including a human, rodent, canine, feline, bovine, avian or equine subject, such as described further below.
  • the sample may be a faecal sample, a saliva sample, a hair sample, a blood sample, a urine sample, or a skin sample, amongst others.
  • the sample is a faecal sample.
  • the sample may be a liquid sample or a solid sample, or it may be a sample having solids and liquids, for example a fluid, such as a liquid, having solids suspended within it.
  • a faecal sample is an example of a sample having solid and liquid parts.
  • the method is for detecting and optionally quantifying the amount of a biological molecule in a sample.
  • That biological molecule may be a polypeptide (protein), polynucleotide, or polysaccharide.
  • the biological molecule is a protein, such as a metalloprotein.
  • the method may use the inherent catalytic ability of that protein as the means for detecting and quantifying the protein.
  • the biological molecule is labelled with a protein that is capable of catalysing the reaction the first and second reagents.
  • the present invention is particularly suitable for analysing stool samples, and may be used to detect blood within that sample, for example by detecting the presence of haemoglobin.
  • the sample may be obtained from a subject who is known or is suspected to have a clinically relevant disease or injury.
  • the subject is one suspected or known to have a disease associated with intestinal bleeding.
  • the subject is a human having Crohn's disease, ulcerative colitis, ulcers and/or cancer, such as bowel cancer.
  • a stool sample may be tested.
  • the presence of blood, as indicated by the presence of haemoglobin in the sample may be an indication of intestinal bleeding.
  • the stool sample is taken from the inner portion of the stool, and is not limited to samples taken from the outer surface of the stool.
  • the presence of blood on the surface of the stool sample is not necessarily indicative of a disease as described above.
  • the presence of blood within a core of the stool has a greater association with the diseases described above.
  • the subject is one suspected or known to have a disease associated with a blood disorder, for example diabetes or haemophilia.
  • the sample such as a faecal (stool) sample
  • the sample is processed in order to ensure that material within the core and the surface of the sample is accessible.
  • SAW surface acoustic wave actuation
  • the sample may be diluted for analysis, for example with water. The purpose of the dilution step is to reduce the concentration of the protein in the sample thereby to minimise non-specific binding of the protein to the surface.
  • the concentration of the protein, such as haemoglobin, in the sample is at most 6 mg/mL.
  • concentrations above this are associated with there is a saturation in the detectable signal, which may be associated with transport of the catalysis reaction product, which is likely to be diffusions-limited at higher concentrations. Additionally or alternatively, the saturation may be linked to the depletion of a reagent, such as the second reagent.
  • the subject from which a biological sample is taken may be an animal, a mammal, a placental mammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orang-utan, gibbon), or a
  • the subject may be in any of its forms of development, for example, a foetus.
  • the subject is a human.
  • the protein is specifically attached to a surface, i.e. the surface has a high specificity for the particular protein for detection.
  • the surface is provided with a binding moiety that is suitable for binding the protein with high specificity.
  • the binding moiety is immobilised to the surface. The binding of the protein to the binding moiety therefore also immobilises the protein to the surface.
  • the specificity can be achieved by an antibody immobilised on the surface, the antibody having specificity for the analyte as described above.
  • the binding moiety is attached directly to the surface.
  • the binding moiety is attached to the surface via a linker. This may be appropriate where the binding moiety is not suitably functionalised to attach to the surface, for example where the surface is an electrically conductive surface, such as a metal surface.
  • a thiol-containing linker may provide a connection between the binding moiety and the gold surface.
  • the modification or patterning of surfaces in this way for connection to binding moieties is well known in the art. Described herein, by way of example, is a gold surface having a monolayer of mercaptoundecanoic acid linkers (see also Bioanalytical Applications of Self Assembled Monolayers, J.M. Cooper and B. Leidberg. BioAnalytical Techniques, 1999 Ed. A. E.G. Cass, Oxford University Press).
  • the blocking agent comprises milk protein and/or BSA, which are standard blocking agents known in the art.
  • the blocking agents where they include proteins, are not specifically bound to the surface. Moreover, these proteins do not substantially catalyse the reaction of a reagent, such as a first reagent with a second reagent.
  • the analyte can also be specifically attached to the surface via a microbead which has a specific binding moiety, for example an anti-analyte antibody, immobilised on its surface.
  • the microbeads can then be dispersed in the sample enabling a high volume coverage thus increasing capture efficiency.
  • Such dispersion can be achieved by using microfluidic mixers, magnetic handling of magnetic beads or using surface acoustic waves.
  • the beads may then be gathered next to the electrochemical sensor. This can be achieved in the same way as for dispersion, i.e. using hydrodynamics (for example, weir type structures or valves), magnetic fields or acoustics (for example, particle concentration in rotating flow).
  • hydrodynamics for example, weir type structures or valves
  • magnetic fields for example, particle concentration in rotating flow.
  • acoustics for example, particle concentration in rotating flow
  • Bead-like structures which comprise rough surfaces or porous structures (for example, zeolites) can be used as these advantageously increase the available surface for specific binding moieties.
  • mesoporous spheres in an electrochemical Hb-containing sensor.
  • Such spheres may be adapted to include a binding moiety specific for a protein such as Hb.
  • the specific binding moiety may also be incorporated into a porous structure such as a membrane or matrix, which may be part of an electrode itself or the structure may be located at or in close proximity to an electrode.
  • a binding moiety may be provided on a membrane, such as a cellulose membrane, for use in the immobilisation of a protein. That membrane, once suitably exposed to protein, may be placed on or close to an electrode surface.
  • the porous nature of the structure permits a reaction product, such as the product of the reaction between the first reagent and the second reagent catalysed by the immobilised protein, to pass through the structure and to become exposed to the electrode surface.
  • the method of the present invention makes use of a binding moiety that is immobilised on a surface.
  • the surface may be an electrically conductive surface. That binding moiety is specific for a protein, and may be referred to as a specific binding moiety.
  • the binding moiety is therefore capable of holding the protein close to the surface.
  • the binding moiety immobilises the protein by non-covalent interactions.
  • the binding moiety is bound to a surface, either covalently or non-covalently, preferably covalently.
  • the binding moiety may be connected to the surface using techniques described in the art. In one embodiment the binding moiety is covalently bound to the surface.
  • the binding moiety may be directly bonded to the surface or the binding moiety may be indirectly connected to the surface via a linker.
  • the linker is not a catalyst for the reaction of the first reagent and the second reagent. In one embodiment the linker is not or does not contain a polypeptide.
  • the analyte is specifically attached to a surface by a binding moiety, the binding moiety being specific for the analyte.
  • the binding moiety may be an antibody specific for the protein.
  • the binding moiety may be an anti-Hb antibody, such as an anti-human Hb.
  • the binding moiety is an antibody for any one of the proteins mentioned in the Protein and Analyte section above.
  • the binding moiety may itself be a protein having specificity for the protein.
  • the binding moiety may be haptoglobin, a strong
  • Hb-binding protein The use of haptoglobin to bind Hb proteins is described, for example, by Stollner er a/.
  • the binding moiety is a polypeptide, such as an antibody or a protein
  • the binding moiety may be connected to the surface via free amino, thiol hydroxy or carboxy groups present within the binding moiety, such as on the surface of the binding moiety.
  • the binding moiety has a specificity, or K D , for the protein of at least 10 15 M, at least 10 "12 M, at least 10 "10 M, at least 10 "9 or at least 10 "6 M.
  • the binding moiety preferentially binds to protein in the mixture, and has essentially no affinity for any other components in the mixture.
  • the specificity or KD for each of the other components is at most 10 -5 M, at most 10 3 M, or at most 10 _1 M.
  • the binding moiety is an antibody
  • the preferred binding affinity for the protein is simply the inverse of the K D values given above.
  • Antibodies for binding metalloproteins are well known in the art.
  • antibodies for haemoglobin are readily available from commercial sources, such as Santa Cruz
  • the electrochemical sensor is suitable for use in the electrochemical detection of the reaction catalysed by the protein, such as the reaction of the first reagent with the second reagent.
  • the electrochemical sensor includes a working electrode.
  • the working electrode is not particularly limited and may be or include Pd, Au, Ag, Pt, Fe and mixtures thereof.
  • the working electrode may be or comprise steel. Other materials that are suitable for use are carbon or graphite, and include these materials as matrix forms.
  • the electrode is of a material suitable for modification.
  • Au and Ag electrodes are suitable for modification.
  • Pd and Pt electrodes are less suitable, and electrodes having these metals may be used together with another surface for immobilising the binding moiety.
  • a surface such as cellulose, may be used to immobilise the binding moiety, and the surface may be placed in close proximity to, including on, the electrode.
  • the electrode may be in the form of a plate, disc, mesh or wire, amongst others.
  • Exemplified in the present case is an Au electrode.
  • the electrode is provided with a self-assembled monolayer of thiol-containing linker for connection of the electrode to the binding moiety.
  • the working electrode is stable. It does not degrade substantially over time or degrade substantially on prolonged exposure to the sample or the test solution.
  • the working electrode may be stored for at least 14 days, at least 28 days, or at least 6 months without significant loss of electrode activity.
  • the electrode is stored for this time in a dry state to minimise degradation. It is preferred that the working electrode, and optionally the other components of the sensor, is stored in an atmosphere of an inert gas, such as nitrogen or argon.
  • the activity of the working electrode may be gauged by electrochemical analysis of a standard sample solution comprising a protein.
  • a loss of electrode activity corresponds to a fall in the average recorded current recorded at the working electrode in comparison to a recorded current at a control working electrode.
  • the fall in average recorded current is about 50 % or less, about 30 % or less, about 10 % or less, or about 5 % or less.
  • the environment against which the stability of the working electrode is tested may include a biological sample as described herein.
  • the working electrode may also be tested against a sample approximating the conditions to which the working electrode is intended to be exposed.
  • the sample may be a simulated intestinal fluid, for example, or a simulated stool sample.
  • the working electrode is part of an electrochemical circuit which includes a counter electrode.
  • the counter electrode is connectable to a power source.
  • the counter electrode is in electrical communication with the working electrode.
  • Preferred electrode materials include steel and platinum. Steel is the most preferred electrode material for use in disposable and one shot sensors and apparatus owing to its relatively low cost.
  • a reference electrode may be included in the electrochemical sensor of the invention.
  • the reference electrode may be a standard silver / silver chloride (Ag/AgCI) electrode.
  • the reference electrode may be a pseudo reference electrode, which is operable as a reference electrode in the presence of a suitable buffer comprising appropriate ions.
  • the pseudo reference electrode may be a silver-based electrode that is obtained, or is obtainable from, a silver electrode that is treated with about 1 % aqueous FeC solution.
  • the first reagent is reactable with the second reagent in the presence of the analyte, such as a protein.
  • the second agent is hydrogen peroxide, or a precursor thereof.
  • the first reagent is preferably a compound that reacts with hydrogen peroxide in the presence of the protein.
  • the first reagent is, or comprises, a compound selected from
  • the first reagent is, or comprises, tetramethylbenzidine.
  • the first reagent is, or comprises, 3, 3', 5, 5' tetramethylbenzidine.
  • the first reagent is, or comprises, tetramethylbenzidine.
  • This reagent is particularly suitable for use in the invention as it is not believed to be harmful to the user.
  • TMB is also used within other assay systems, and will therefore be familiar to those of skill in the art. For example, TMB is described by Liem ei al. ⁇ Analytical
  • TMB is also a common reagent for use in HRP-based assays, such as described by Josephy et al. ⁇ J. Biol. Chem., 1982, 257, 3669-3675).
  • the methods of the invention measure the oxidation product of TMB. In one embodiment, the one electron oxidation product of TMB is measured.
  • the second reagent is an oxidising agent or a precursor thereof. It is reactable with the first reagent in the presence of the protein.
  • the second reagent is or comprises hydrogen peroxide or a precursor thereof.
  • the hydrogen peroxide is reactable with the first reagent in the presence of the protein.
  • the second reagent is hydrogen peroxide.
  • the second reagent is, or comprises, a compound selected from urea peroxide, a perborate compound and a periodate compound. In one embodiment, the second reagent is, or comprises, urea peroxide. In another embodiment, the second reagent is, or comprises, a perborate compound, preferably sodium perborate. Test Solution
  • the method of the present invention includes the step of permitting the immobilised protein to catalyse a reaction of a reagent, such as a first reagent with a second reagent.
  • a reagent such as a first reagent with a second reagent.
  • the reagents are then made available to the immobilised protein.
  • the reagents may be provided in a test solution.
  • the test solution is also an electrolyte suitable for electrochemistry.
  • the reagent is present in the test solution at a concentration of about 2 mM.
  • the test solution contains a buffer to substantially maintain the pH of the test solution during the analysis steps.
  • the test solution is acidic. In one embodiment, the pH of the test solution is at most 5 or at most 6. In one embodiment, the pH of the test solution is at least 2, is at least 3, or is at least 4. In one embodiment the pH of the test solution is in a range selected from the upper and lower pH values mentioned.
  • the test solution may include other components, such as one or more salts.
  • the salt is present in the test solution at a concentration of at least 1 mM, at least 5 mM, at least 10 mM or at least 50 mM. In one embodiment, the salt is present in the test solution at a concentration of at most 0.2 M, at most 0.5 M or at most 1 M. In one embodiment the concentration is in a range selected from the upper and lower values mentioned. Optionally other component may be included in the test solution, for example to improve electrochemical analysis.
  • the method of the invention is performed within a microfluidic device.
  • a microfluidic device Such devices carry out laboratory assays in a miniaturised format.
  • the microfluidic device may be generally small enough to fit on a laboratory bench, and in exemplary embodiments is small enough to be carried by a user.
  • the device may be a "lab-on-a-chip" device.
  • a microfluidic device can comprise a support in which one or more channels are formed to provide a channel network capable of directing flow, and optionally controlling flow, of liquid through part, or all, of the network.
  • the channel network will have multiple channel portions.
  • the microfluidic device is configured to interact with a meter in order to provide the results of the method.
  • channels and channel portions are generally enclosed spaces defined by surrounding walls.
  • the channel can have any cross-sectional shape (e.g.
  • Channels can be in fluid communication with the atmosphere external to the microfluidic device by means of apertures (e.g., inlets, outlets or vents) formed in the channel network.
  • Channels or channel portions can be open to the atmosphere for part or all of their length, e.g. by not having an enclosing lid.
  • Channels or channel portions can comprise a capillary, i.e. a channel of small internal diameter capable of holding or transporting liquid by capillary action, wherein capillary action is (at least in part) the effect of surface tension that draws a liquid into or along the channel.
  • the present inventions provides a method of detecting an immobilised protein by using the intrinsic activity of that protein to catalyse the reaction of the first reagent with a second reagent.
  • the method includes the step of immobilising the protein.
  • the method comprises the steps of:
  • reaction of the first reagent with the second reagent (iii) detecting the reaction of the first reagent with the second reagent.
  • the reaction of the first reagent and the second reagent may be detected electrochemically.
  • the method includes the step of immobilising the protein.
  • the method comprises the steps of:
  • the method of the invention comprises the preliminary step of treating the sample with surface acoustic waves, such as described herein. After a protein is immobilised to a surface, the surface may be washed thereby to remove components of the sample that are not immobilised.
  • electrochemical methods are used to detect the presence of an immobilised protein.
  • the sample is exposed to an immobilised binding moiety which has specificity for the protein.
  • the binding moiety is permitted to immobilise the protein.
  • the protein-depleted sample is subsequently separated from the immobilised protein, for example by washing.
  • the binding moiety is attached to a surface, as described herein.
  • the surface may be a surface of a working electrode.
  • the methods of the invention includes the step of providing the surface, after immobilisation of the protein, at or in close proximity to a working electrode.
  • the immobilised protein is then permitted to catalyse the reaction of a reagent, such as the reaction of a first regent with a second reagent.
  • the reaction provides a detectable signal at the working electrode.
  • the working electrode is held at a bias as described herein.
  • the change in current within the electrochemical cell is monitored over time, and is attributable to the electrochemical reaction of a reaction product or a reagent in the catalysis reaction.
  • the change in current over time may be directly linked to the concentration of the reaction product or reagent, which may then be directly linked to the concentration of protein in the original sample.
  • the immobilised protein is haemoglobin
  • the immobilised haemoglobin catalyses the reaction of TMB with hydrogen peroxide.
  • the oxidised TMB product is reduced at the working electrode to give rise to a detectable signal.
  • a sample such as a biological sample and particularly a blood-containing sample, may be process with SAW methods prior to the exposure of that sample to the binding moiety.
  • SAW Surface acoustic wave actuation
  • a SAW system may be used to lyse blood cells without the need for chemical reagents.
  • SAWs Surface acoustic waves
  • Rayleigh waves Rayleigh waves
  • SAWs can be used to rapidly homogenise a sample, for example, a stool sample, to increase the confidence in the results of the diagnostic test, and can be readily integrated with other microfluidic functions.
  • a stool sample (1-10 mg) is deposited on the surface of piezoelectric wafer, onto which an IDT has been patterned to drive a SAW at a specific frequency (>1 MHz).
  • a drop (20 ⁇ _) of buffer (MOPS or HEPES for example) is then added to the stool sample. This can be driven by the SAW.
  • the buffer may include a lysis agent or solubilising agent (such as saponin for example), but using SAW may allow the use of such agents to be avoided.
  • the sample can be homogenised in situ, i.e. when the sample is already in contact with the specific binding moiety.
  • the working electrode can then detect the Hb as it is released from the sample.
  • a sample Prior to the processing of the stool, a sample may be taken from the core of the stool, and a sample may also be taken from an outer portion. Testing of both samples can show what proportion of blood in the total sample is derived from non-serious bleeding (blood from the outer part of the stool, which may be associated with e.g. haemorrhoids) and serious bleeding (blood from the core, which may be associated with e.g. bowel cancer).
  • a sample volume of 20 ⁇ _ is sufficient for the methods of the present invention.
  • an immobilised protein for the detection of an analyte that is a binding partner for that protein.
  • the protein is capable of catalysing the reaction of a reagent, such as a first and second reagent, to give rise to a detectable signal.
  • a binding partner is present, the protein binds to the binding partner, thereby rendering the protein incapable of catalysing the reaction of the reagent.
  • a change in the reaction rate of the reagents may be associated with the presence of the binding partner, and the amount of change may be correlated to the quantity of binding partner that is present.
  • the inherent catalytic activity of the protein may be used as an indicator for the presence of components that bind to the protein.
  • the protein may be immobilised, for example it may be specifically immobilised by a binding moiety, to a surface.
  • the present invention provides a method for detecting an analyte in a sample, which analyte is a binding partner for a protein, the method comprising the steps of:
  • the analyte may be or comprise a polypeptide for binding to the immobilised protein.
  • the analyte may itself be or comprise a protein.
  • the analyte is a receptor for the protein.
  • the receptor may be located on a cell surface.
  • the cell may be part of a pathogen, including a microorganism, such as a bacterium such as S. aureus.
  • haemoglobin proteins such as haemoglobin, for example as a source of iron or its ions (such as Fe 2+ or Fe 3+ ), or amino acids.
  • the present methods may be used to detect those pathogens that target proteins such as haemoglobin.
  • IsdB hemoglobin receptor from S. aureus that binds Hb via the N-terminal NEAT domain (lsdB-N1).
  • IsdB is a cell wall-anchored protein, and therefore an immobilised protein that is capable of binding to this receptor may act as a reporter for the presence of S. aureus.
  • the analyte comprises a NEAT (near iron transporter) domain.
  • the analyte is or comprises an antibody ("antibody analyte").
  • the antibody may be specific for the protein.
  • the antibody analyte is a bispecific antibody.
  • the antibody analyte has specificity for the immobilised protein and specificity for another component, such as another protein, which may be a receptor, such as a receptor on a cell surface.
  • the another protein may be present on a pathogen, such as a virus.
  • the bispecific antibody analyte may be associated with that other component prior to its exposure to the immobilised protein. In this way, the bispecific antibody may be used to allow the indirect detection of the other component.
  • the method of the invention may comprise the step of exposing the analyte, which analyte has specificity for the protein and another component, to the other component thereby to bind the analyte to the other component, and subsequently exposing the immobilised protein to the analyte, thereby to bind the analyte to the immobilised protein.
  • the methods of the invention may be used to detect components to which the protein cannot bind, or binds to with low affinity.
  • the analyte is bispecific for the protein and the other component. The analyte therefore acts as a bridge to allow the detection of the component using the methods of the invention.
  • the bispecific analyte may be a bispecific antibody. Examples of bispecific antibodies are described by May et al. (Biochemical Pharmacology 2012, 84, 1105- 112).
  • Hb is used as the analyte of interest.
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • HRP/H2O2 The mechanism of TMB catalysed oxidation by HRP/H2O2 is well established and generates a blue-coloured intermediate complex (following a one electron oxidation) and subsequently, when fully oxidised, a yellow di-imine product (as a two electron oxidation).
  • the catalysed oxidation of TMB by Hb occurs in a similar manner, and, in fact, TMB has previously been used as a cytochemical indicator for the optical detection of Hb in cells. In this study, TMB was the mediator of choice due to its low reactivity with H2O2.
  • the immunoassay involved the immobilisation of anti-human Hb (Abeam, UK) on the surface of an Au-disc electrode using a standard immobilisation based upon N-hydroxysuccinimide activation of a self-assembled monolayer of 11-mercaptoundecanoic acid (leading to reaction with free amine groups on the surface of the antibody).
  • the immunosensor electrode was then immersed in a solution of Hb.
  • the bound-Hb antibody construct was then immersed in a solution containing TMB (2 mM), H 2 0 2 (2 mM) and KCI (0.1 M) in phosphate-citrate buffer (pH 4.5). Current time transients were measured for various Hb concentrations between 0.0 and 8.0 mg/mL (w/v), covering a clinically significant range.
  • the immunoassay may also be performed using sheep anti-human Hb antibody, such as the affinity purified (A80-135A) product available from Bethyl Laboratories Inc.
  • the immunoassay accommodates the clinically relevant levels of Hb in fluids from the lower Gl tract (ca. > 0.5 mg/mL). Importantly, the results also show that Hb retains its intrinsic peroxidase activity once bound by anti-Hb on the electrode surface.
  • the assay not only provides a better biosensing route for determining Hb in turbid or opaque samples, when compared with its optical counter-part, but as a label-free assay it requires fewer liquid handling steps (making it a promising candidate for successful miniaturisation onto a microfluidic platform).

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Abstract

Cette invention concerne un procédé pour la détection d'une protéine, telle qu'Hb, dans un échantillon d'essai, le procédé comprenant les étapes consistant : (i) à exposer l'échantillon à une surface portant un fragment de liaison et à laisser le fragment de liaison immobiliser spécifiquement la protéine dans l'échantillon : (ii) à laisser la protéine immobilisée catalyser une réaction d'un premier réactif avec un second réactif; et (iii) à détecter, par exemple par un moyen électrochimique, la réaction du premier réactif avec le second réactif. Le fragment de liaison peut être fourni sur la surface d'une électrode.
PCT/GB2014/052620 2013-08-30 2014-08-29 Procédés de détection de protéines Ceased WO2015028811A2 (fr)

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WO2018177961A1 (fr) * 2017-03-31 2018-10-04 Syddansk Universitet Purification d'échantillons de sang avec hémolyse
CN110244050A (zh) * 2019-06-11 2019-09-17 中央民族大学 一种细胞裂解原位光学传感检测芯片及其制备和使用方法

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US5506114A (en) * 1992-02-07 1996-04-09 Osborn Laboratories Methods and kits for detecting the presence or concentration of biological analytes
GB0820817D0 (en) * 2008-11-13 2008-12-24 Wireless Biodevices Ltd Electrode, electrochemical sensor and apparatus, and methods for operating the same
GB0914762D0 (en) * 2009-08-24 2009-09-30 Univ Glasgow Fluidics apparatus and fluidics substrate

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018177961A1 (fr) * 2017-03-31 2018-10-04 Syddansk Universitet Purification d'échantillons de sang avec hémolyse
CN110244050A (zh) * 2019-06-11 2019-09-17 中央民族大学 一种细胞裂解原位光学传感检测芯片及其制备和使用方法

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