CZ2017483A3 - Method for determining the presence of an analyte in a liquid sample, an analyte detection electrode in a liquid sample, a method of its preparation, a kit for determining the presence of an analyte and its use - Google Patents

Abstract

The present invention relates to a method for determining the presence of an analyte in a liquid sample comprising the steps of: (i) incubating the working electrode surface containing the immobilized particles for affinity interaction with the analyte, and binding the sample to the immobilized if the analyte is to be assayed particles on the electrode surface to form an immobilized analyte-immobilized particle complex on the electrode surface; (ii) contacting the working electrode with the immobilized analyte-immobilized particle complex with a solution of a redox substance having an opposite charge than the analyte to be determined; (iii) measuring the electrochemical parameter to determine the impedance at the interface of the working electrode surface and the redox solution relative to the supplemental electrode present in the liquid sample; (iv) an impedance change is evaluated from the measured value of the electrochemical parameter, the presence of the analyte in the sample results in a decrease in the impedance value at the electrode surface and redox solution. The presence of an analyte in a liquid sample is also determined qualitatively and / or semi-quantitatively and / or quantitatively from the impedance change. The present invention further relates to an electrode for detecting an analyte in a liquid sample, a method for its preparation, a kit for determining the presence of an analyte in a liquid sample, and their use.

Description

Technical field

The present invention relates to a method for determining the presence of an analyte in a liquid sample, a kit for determining the presence of an analyte, and their use, in particular for determining bacterial antigens or corresponding antibodies in the body fluids of test subjects suspected of disease. in blood serum.

BACKGROUND OF THE INVENTION

Most current commercial immunosensors are based on lateral flow immunoassays (LFIA) with visual or optical detection. Electrochemical detection methods for immunosensors are not widespread.

Previously, affinity electrochemical immunosensors were based on the labeling of one of the interacting partners of the antigen-antibody pair. Enzymes or other redox substances such as ferrocene could be used as labels (Okochi, Ohta, Tanaka, Matsunaga, Biotechnol Bioeng 90 (2005) 14). Today's approaches most often use the monitoring of the change of sensor electrochemical impedance in dependence on the affinity interaction of the recognitive component with the analyte.

Affinity interactions monitored by electrochemical impedance exploit complex formation between different molecules. These may be, for example, interactions between aptamers and adenosine (Zayats, Huang, Gill, Ma, Willner, J Am Chem Soc 128 (2006) 13666) or antibodies and antigens (Susmel, Guilbault, O'Sullivan, Bosens Bioelectron 18 (2003) 881). Susmel et al. measured the decrease in the diffusion coefficient of the redox probe when bacteria (food pathogens) were captured on the electrode surface.

There is a mass increase on the surface of biosensors based on immunoaffinity interactions, which is why the most common variant of detection is the measurement of impedance increase (measure of the resistance of charged substances through this layer of biomolecules). Impedance measurement was also used to detect Salmonella sp. (Kim, Moon, Hahm, Morgan, Bhunia, Om, Food Science Biotechnol 18 (2009) 89) or Escherichia coli (Yang, Li, Erf, Anal Chem 76 (2004) 1107, Maalouf, FournierWerth, Coste, Chebib, Sailkali, Vittori, Errachid, Cloarec, Claude Martelet, Jaffrezic-Renault, Anal Chem 79 (2007) 4879). The Escherichia coli assay was based on precipitation of a non-conductive layer, the amount of precipitate shielding the electroactive surface being proportional to the amount of bound bacteria (Ruan, Zang, Li, Anal Chem 74 (2002) 4814). The signal - impedance increase - was thus amplified.

Nanomaterials for affinity diagnostics are widespread (Wang, Analyst 130 (2005) 421), but are mainly used as signal amplification labels (Wan, Su, Zhu, Liu, Fan, Biosens Bioelectron 47 (2013) 1; Mwilu, Aluoch, Miller, Wong, Sadik, Fatah, Arciles, Anal Chem 81 (2009) 7561).

The principle of prior art impedance immunoreaction assays is to block or "isolate" the electrode surface during a detection reaction between a specifically modified electrode and an analyte. The passage of the redox tag through the modification layer on the surface of the detection electrode is reduced by the generated affinity pair (eg, antibody-antigen, receptor-ligand or other combination of complementary biomolecules), thereby imparting so-called diffusion limitations on faradic current generated by the redox tag electrode the redox tag penetration through the protein / modification layer on the electrode is reduced). In some cases, this principle is strengthened by various means. They may be membranes, e.g.

- 1 CZ 2017 - 483 A3 aluminum matrices (Koh, Agarwal, Cheow, Toh, Electrochim Acta 53 (2007) 803), where the detection principle used a pore block whose walls were modified by one of the immunoreaction partners.

For electrochemical measurements were used lithographically prepared electrodes with subsequent galvanization primarily for the common method of measurement in the setting: Au working, respectively. auxiliary electrode and Ag reference electrode (La Belle, Shah, Reed, Nandakumar, Alford, Wilson, Nickerson, Josh, Electroanalysis 21 (2009) 2267; La Belle, Gerlach, Svarovsky, Josh, Anal. Chem. 79 (2007) 6959; Bhavsar, Fairchild, Alonas, Bishop, La Belle, Sweeney, Alford, Joshi, Biosensor, Bioelectron, 25 (2009) 506). The electrochemical measurement was then carried out in the usual way by setting the potential on the working electrode against that of the reference electrode. The most commonly used electrochemical measurement methods are impedance and voltammetric measurements.

Lyme disease is caused by infection with Borrelia sp. In Europe, B. afzelii and B. garinii are the predominant infection, and in B. America, B. burgdorferi. This bacterial infection is currently most commonly diagnosed by ELISA, where the time for diagnosis is several hours. More accurate methods are Western blot or PCR, where the minimum time for diagnosis is a 14-hour analysis. However, sampling, transport, sample processing, communication and interpretation of results are not taken into account in this period. As the determination is performed only in specialized laboratories, the time of one analysis is at least 3 days. Despite the existing diagnostic system for detecting infection by Borelia sp. there is no possibility of rapid and cheap determination at home or by a general practitioner who would not need to analyze the sample in a specially equipped laboratory.

The lengthy, complicated and costly nature of an analyte assay, in particular anti-Lyme borreliosis antibodies, of the prior art is solved by the present invention, which allows pre-screening in a physician or at home (suspected of infection, eg in the initial immune response of an organism after sucking an infected tick). or insect bites) that is still missing from the market.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for determining the presence of an analyte in a liquid sample, comprising the following steps:

(i) determining the value of the electrochemical parameter of a working electrode surface comprising immobilized particles for affinity interaction with an analyte immersed in a solution of a redox having an opposite charge to the analyte being determined, wherein the solvent of the redox is preferably water, an aqueous buffer solution , preferably at a pH of 4 to 9, or saline, and wherein preferably the concentration of the redox in the solution is in the range of 1 µΜ to 1 M, preferably from 1 mM to 20 mM; optionally, the surface of the working electrode is washed, preferably with distilled water or saline, and subsequently freed of excess liquid;

(ii) the working electrode surface containing the immobilized particles for affinity interaction with the analyte is incubated with the liquid sample, and if the sample contains the analyte to be determined, the analyte binds to the immobilized particles, preferably antigen or antibody, on the electrode surface to form an affinity an analyte-immobilized particle complex on the electrode surface; the incubation time is preferably in the range of 5 to 120 minutes at a temperature of 10 to 40 ° C and a pH of 4 to 9, optionally the surface of the working electrode with the immobilized affinity complex is washed, preferably with distilled water or saline, removes excess liquid;

(iii) a working electrode with the immobilized analyte-immobilized particle affinity complex is

A3 - 483 A3 is contacted with an equally concentrated solution of the same redox as in step (i) having the opposite charge to the analyte being determined, wherein the solvent of the redox is preferably water, an aqueous buffer solution, preferably pH 4- 9, or saline, wherein the concentration of the redox in the solution is in the range of 1 μΜ to 1 M, preferably 1 mM to 20 mM;

(iv) determining a value of an electrochemical parameter selected from direct current, direct voltage, resistance, alternating current, alternating voltage, impedance, at the interface of the working electrode surface and the redox substance solution relative to the supplemental electrode present in the liquid sample; an opposite charge to the analyte being determined, preferably the measured electrochemical parameter is an electric current or voltage from which the system impedance is subsequently determined;

determining from the value of the determined electrochemical parameter in step iv) the change in the electrochemical parameter relative to the electrochemical parameter of the redox solution determined in step i), wherein the presence of the analyte in the sample leads to a change in the electrochemical parameter at the interface of the electrode surface and the redox solution; if the electrochemical parameter is a resistance or impedance or voltage, it will drop, and if the electrochemical parameter is current, it will increase. From the change in the electrochemical parameter, preferably impedance, the presence of the analyte in the liquid sample is qualitatively and / or semi-quantitatively and / or quantitatively determined.

In this way, the analyte can be determined over a range of concentrations in the liquid sample from ng / ml to mg / ml.

Potentiometry, voltammetry, amperometry or biamperometry, preferably biamperometry, and their respective electrode arrangement can be used to determine the electrochemical parameter. A person skilled in the art would be able to determine the appropriate electrode arrangement, types and number of electrodes without resorting to the invention.

Preferably, the liquid sample is dropped directly onto the working electrode, in the case of biamperometry to the electrode system, and the measurement takes place in the dropped sample. The same sample is also dropped onto the complementary / reference electrode to conduct the electrodes electrically connected to the sample being measured. This reduces the instrumental complexity of this method of measurement as well as the measurement error that could arise, for example, when transferring a sample to a measuring vessel, electrode rinsing, etc. A liquid sample is, for example, a blood serum or subject blood sample.

The optimal (most sensitive) detection technique is to determine the impedance change of the working electrode or, in the case of biamperometry of the electrode system, in a redox solution before and after incubation with a liquid sample. It is most advantageous to measure the impedance at lower frequency values (most often in the range up to 1000 Hz), where both the analyte's influence on the AC and DC components of the excitation signal, respectively, is applied. determined impedance. The measurement is done by inserting an alternating voltage between two electrodes in the solution and measuring the passed current - magnitude and its phase offset against the inserted voltage, from which the impedance value is then calculated. The output signal is measured. impedance, meaning the effect of the reaction of the immobilized particle with the analyte on the excitation signal. The alternating component of the excitation signal is affected by the bi-directional movement of ions towards and away from the electrode; the DC component is affected by the unidirectional ion flux to or from the electrode. Other non-impedance techniques, such as various voltammetric or amperometric methods for monitoring the patency of ions through the modification layer or voltage variations generated by the presence / absence of charged antibodies, may also be used as an electrochemical detection method.

Another method of measurement is also the method where the sample is deposited on an electrode system where an immobilized particle (complementary immunoreaction partner) and a solid redox substance are present. This electrode system (using biamperometry) or the working electrode (using the other aforementioned determination methods) is prepared by working the working electrode,

A3, optionally an electrode system comprising immobilized particles for affinity reaction with the analyte is contacted with a redox solution and an electrochemical parameter is determined (step i)). Subsequently, the working electrode / electrode system is allowed to air dry, thereby adsorbing the redox material to the surface of the working electrode / electrode system. Addition of the liquid sample to the analyte simultaneously interacts with the analyte with the modified surface (step ii)) and dissolves the redox substance (step iii)), in which solution the electrochemical quantity is measured. As the electrochemical parameter of the working electrode / electrode system surface is altered by the binding of the analyte, the redox signal is also altered, from which the presence and possibly also the concentration of the analyte in the sample can be determined in subsequent steps iv) and v). Thus, the entire assay procedure is performed without the need to rinse the electrode and the result is known in a minimum time after sampling. In order to maintain the condition of the same redox concentration in step (i) and step (iii), the volume of the sample that is dropped onto the electrode system in step (ii) must be the same as the volume of the redox solution in step (i) that was After measuring the electrochemical parameter, allow it to dry on the electrode system. For example, if in step (i) a 5 μΐ solution of a redox solution of known concentration was applied to an electrode / electrode system, the electrochemical was measured and the drop was subsequently dried (thereby adsorbing the redox substance to the electrode surface). electrode also 5 µl of liquid sample. This produces the same concentration of redox in the liquid sample potentially containing the analyte to be determined.

In a preferred embodiment, the immobilized particle for affinity interaction with the analyte on the working electrode surface is an antibody or antigen, preferably the immobilized particle is a whole cell antigen of a Borrelia sp.

In a preferred embodiment, the analyte is an antibody or an antigen, and when the immobilized particle is an antibody, the analyte is an antigen and more versa, preferably the analyte is an antibody against Borrelia sp.

In one embodiment, the redox is a negatively charged potassium hexacyanoferrate and potassium ferrocyanide. It is also possible to use other types of redoxactive substances preferably carrying a positive or negative charge. Positive charges include, for example, hexaaminruthenium (II / III) chloride, cobaltocene, ferrocene, or aminopherocene, and negatively charged substances such as Γ '/ Τ or ferrocarboxylic acid.

In one embodiment, measuring the electrochemical parameter to determine the impedance at the interface of the working electrode surface and the redox solution relative to the complementary electrode present in the liquid sample in step (iv) is measuring electrical current and / or electrical voltage by amperometry, biamperometry, potentiometry and / or voltammetry, the most preferred is the use of biamperometry.

Preferably, the measurement of electric current and / or electric voltage is performed at AC / voltage frequency values ranging from Hz to hundreds of kHz. It is advantageous to limit the measurement to one or two frequencies, which greatly simplifies, speeds up and cheaper the whole assay, which is particularly suitable for low-cost detection.

In a preferred embodiment, the electrochemical parameter is measured in step (iv) by a biamperometric method using two identical working electrodes with immobilized particles for affinity interaction with the analyte. In the case of biamperometry, the working electrodes are modified in the same way (the same surface of the same size is the same modified potential between these electrodes is therefore zero), thus having the same electrochemical properties and measurement can be performed at zero potential input, simplifying analysis. Another factor favoring the measurement arrangement used is that without the use of biamperometry, where impedance measurement (insertion of 5 to 10 mV AC excitation signal) is performed at zero offset of the DC voltage component, it would not be possible to measure small differences in charge distribution change at the interface electrode / solution. When measuring in a classical arrangement with working and

The reference electrode inserts either the potential suitable for measuring the redox mediator, thereby deflecting the system due to the potential difference of the system of the two electrodes embedded in the solution (electrode / solution-solution / electrode), or in the second case the system is not biased due to the potential difference of the electrode / solution-solution / electrode, the system is biased due to the redox potential of the redox mediator pair. These differences in applied offset potential do not occur when measuring biamparometry.

The working electrode for detecting the analyte in the liquid sample is preferably an electrode selected from the group consisting of a metal and carbon electrode, preferably an electrode selected from the group consisting of copper, silver, platinum, gold, stainless and composite carbon electrodes, and affinity interaction with the analyte, preferably the immobilized particles are an antigen or an antibody. Advantageously, the working electrode can be prepared by screen printing or photolithography according to generally known methods (patent 291411 and La Belle, Gerlach, Svárovský, Joshi, Analytical Chemistry 79 (2007) 6959). The immobilized particles for interaction with the analyte are bound, preferably covalently, to a polymer layer carrying activatable functional groups. This polymer layer is adhered to the surface of the working electrode. The polymer layer may be, for example, poly-L-lysine, poly-allylamine, polyethyleneimine, chitosan, polyacrylic acid and others.

In a preferred embodiment, the working electrode for detecting an analyte in a liquid sample is a metal or carbon electrode that contains adsorbed poly-L-lysine and / or polyallylamine and / or polyethyleneimine to which the particles are covalently bound for affinity interaction with the analyte , preferably an antigen or antibody. Most preferably, the working electrode is gold or platinum coated with a 20-50 nm layer of poly-L-lysine (Colville, Tompkins, Rutenberg, Jericho, Langmuir 2010 26 (4), 2639) with a covalently bound whole cell antigen of Borrelia sp.

The present invention further provides a method of preparing a working electrode for detecting an analyte in a liquid sample by the method of the present invention, comprising the steps of:

(i) the metal or carbon electrode is incubated with a positively charged polymeric carrier carrying free / conjugatable amino groups (i.e., the polymeric carrier is characterized by such amino group representation in its structure that it has a total charge positive at physiological pH, meaning that the pI value of such carrier it is above pH 7.4), in particular with poly-L-lysine and / or polyallylamine and / or polyethyleneimine, preferably having a molecular weight of from 30 to 70 kDa, for at least 30 minutes; then rinse with water or buffer, or excess excess rinse solution is blown off in a stream of compressed air.

(ii) the resulting electrode coated with a layer of positively charged polymeric carrier containing free amino groups, preferably coated with a layer of poly-L-lysine and / or poly-allylamine and / or polyethyleneimine, is incubated with a solution of low molecular weight molecular weight up to 1000 kDa two carboxyl groups, at least one of which is activated to conjugate to the amino groups of the polymeric carrier, preferably the low molecular weight substance is citric acid with EDC / NHS activated by at least one COOH group for at least 15 minutes to form the carboxyl modified surface as a mono layer; carboxyl groups for binding to poly-L-lysine and / or poly-allylamine and / or polyethyleneimine are activated with carbodiimide, preferably EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), to form a covalent bond between a carboxyl group of a low molecular weight substance carrying at least two carboxyl groups and an amino group of poly-L-lys and / or poly-allylamine and / or polyethyleneimine.

(iii) for binding to the particles for affinity interaction, the remaining free carboxyl groups on the electrode surface are activated with carbodiimide, preferably EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), and N-hydroxysuccinimide for at least 20 minutes in

A3 of formation of active esters on the surface of the polymer (preferably poly-L-lysine) layer;

(iv) the analyte affinity interaction particles react with N-hydroxysuccinimide activated carboxyl groups on the surface of the polymeric support during at least 30 minutes of incubation to form a covalent bond between the polymeric support coated with the electrode surface and the analyte affinity reaction particle.

In a most preferred embodiment of the method for preparing the working electrode, the positively charged polymeric carrier is poly-L-lysine, the low molecular weight carrying at least two carboxyl groups is citric acid, and the analyte affinity particle is a whole cell antigen of Borrelia sp.

The present invention also provides a kit for determining the presence of an analyte in a liquid sample according to the method of the present invention comprising at least one working electrode for detecting an analyte in a liquid sample selected from the group consisting of metal and carbon electrodes, preferably an electrode selected from copper. a silver, platinum, gold, stainless and paste carbon electrode comprising surface-immobilized particles for affinity interaction with an analyte, preferably the immobilized particles are antigen or antibody, for connection to a meter for quantifying an electrochemical parameter selected from impedance, current and / or voltage ; and at least one additional electrode for connection to a meter for quantifying an electrochemical parameter selected from impedance, current and / or voltage; wherein the kit further comprises a measuring device for quantifying an electrochemical parameter selected from impedance, current and / or voltage; a redox substance having an opposite charge in solution to the analyte to be determined, or a solution thereof in water, buffer or saline; optionally, the kit further comprising an analyzer for connection to the measuring instrument and / or a control unit (eg, computer or mobile phone) for connection to the measuring instrument or analyzer; and / or a display which may be an integral part of the measuring instrument or the analyzer or control unit; and / or a light emitting diode, which may be an integral part of the measuring instrument or analyzer or control unit. The measuring instrument is an instrument capable of measuring and quantifying the electrochemical parameter, preferably the measuring instrument is a potentiostat, an amperometer, a voltmeter. The light emitting diode may be set to illuminate at a predetermined specific threshold value of the electrochemical parameter, preferably impedance.

The electrode of the present invention can be manufactured at low cost by using the screen printing method (manufacturing screen printing electrodes) or the photolithographic methods (printed circuit board forming method). The lithographically generated shapes (most often copper) can then be modified, for example by electroplating, where noble metals such as gold, platinum, silver, palladium or combinations thereof (a mixture or different coverage of different electrodes) are used as materials deposited on lithographically generated copper structures. ). Electrodes made by other means (eg, paste carbon) may also be used. Due to the low cost, the entire electrode can be used for only one measurement, eliminating the problem of sample infectivity.

The output signal can be displayed on a connected computer screen, an integrated display, or a light emitting diode signal at a certain signal threshold. When setting the signal limit between the positive and negative values of the analyte in the sample, the signaling can be simplified to a simple LED - for example, the negative signal - green, the positive signal - red.

The control unit can be, for example, a mobile phone, which serves as a power supply and / or as a measuring device for measuring and displaying the impedance signal, or, by means of a built-in camera module, quantitatively read the light intensity of the indicator light.

The present invention further provides the use of a kit for determining the presence of an analyte in a liquid sample and / or a method for determining the presence of an analyte in a liquid sample according to the invention.

The present invention for the qualitative and / or quantitative and / or semi-quantitative detection of the presence of antibody and / or antigen and / or bacteria in a liquid sample.

In a preferred embodiment, the use is for detecting the presence of antibodies against Borrelia sp. in a liquid sample.

The present invention allows rapid determination of an analyte in a liquid sample through an affinity interaction between a specifically modified electrode surface and a detected analyte substance. The principle is the detection of an immunoaffinity interaction, where the binding of, for example, an antibody to an antigen-modified surface causes a decrease in impedance in the environment of a negatively charged redox, the rate of decrease in impedance corresponding to the amount of analyte bound (e.g. concentration of antibody in the sample. A condition for proper functioning is a combination of an oppositely charged redox tag and an analyte binding to the detection surface, e.g., a negatively charged redox tag and a positively charged analyte (protein) at a given pH (e.g., physiological pH 7.4 or lower pH).

This assay principle differs from the state of the art, where the detection of immunoaffinity interactions increases the impedance of protein binding to the surface - the protein layer reduces the permeability of the electrode modification layer to the permeating substances, resulting in increased mass transfer resistance and thus increased resistance / impedance .

The method for determining the presence of an analyte in a liquid sample according to the present invention is performed by a direct assay method (no competition approach is used, etc.) and the measurement is performed without the use of a label.

Brief description of the drawings

Giant. 1: Block diagram of an impedance analyzer consisting of a computer 1, a USB converter 2, a microcontroller 3, an amplifier 4, a sensor 5, an I / U converter 6 and a user interface 7. Reference numeral 8 denotes the impedance analyzer itself.

Giant. 2: Determination of an analyte, anti-HSA antibodies (HSA, human serum albumin), in an environment of 1 mg / ml ballast protein BSA (bovine serum albumin) by a validated method. Representation of real component impedance measured at 2 Hz, 10 mV amplitude in 5 mM ferri / ferrocyanide.

Giant. 3: Example of a change in the real component of electrochemical impedance for a negative and positive sample of the presence of anti-Borrelia antibodies in blood serum. The real component of impedance for the whole measured frequency spectrum of 6 identical lithographically prepared electrodes - electrodes with the same initial properties is shown. Three were incubated with the positive and three were incubated with the negative blood serum sample.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Construction of an electrode, connection of a measuring instrument and determination of the concentration of a model anti-HSA antibody analyte in a sample

The impedance measurement can be performed with any impedance measuring device. Preferably, a miniaturized impedance analyzer can be used (block diagram in Fig. 1).

Electrode modification was performed by combining poly-L-lysine with subsequent antigen binding

-7 CZ 2017 - 483 A3 (human serum albumin, HSA) via citrate activated EDC (carbodiimide). The modified surface was then incubated with an analyte (anti-HSA antibody) sample. Finally, the electrode was immersed in a solution of redox (ferri / ferrocyanide) and its electrochemical impedance was measured in this solution. The analyte concentration (anti-HSA) in the sample was determined from the impedance changes. As the impedance decreases, the antibody concentration in the sample increases (Fig. 2).

Detailed description of the methodology:

The electrodes were covered with a layer of poly-L-lysine after 30 min incubation in a solution of 50 µg / ml poly-Llysine, 50 mM PBS pH 7.4 (30-70 kDa).

Surface activation with 50 mM citric acid with 150 mM EDC / NHS in 500 mM MES buffer pH 5.6 for 30 min. Activated molecules with carboxyl groups allow subsequent binding of the protein molecules.

Immediately after the previous incubation step of 60 min with 1 mg / ml HSA / 100 mM MES pH 5.6, shaken 150 rpm. This will bind the protein to the electrode surface. 30-45 min incubation with anti-HSA antibody in 1 mg / ml BSA medium, 50 mM PBS pH 7.4, shaken 150 rpm, 4 µl

- Each step is followed by rinsing with distilled water and drying in a stream of compressed air.

- Electrode impedance measurement in 2.5 mM ferricyanide and 2.5 mM ferrocyanide in 50 mM PBS buffer, pH 7.4.

In FIG. 2 shows the determination of three concentrations of anti-HSA (0, 50, 200 pg / ml) in a 1 mg / ml solution of BSA (bovine serum albumin), which represents a large amount of ballast specific non-interacting proteins present in real samples; signal background pattern. Each concentration value was determined in four replicates - each value representing one separate electrode system modified and measured according to the methodology description.

Example 2: Determination of anti-Borrelia antibody concentration in a sample

In the same manner as in Example 1, the amount (presence) of anti-Borrelia antibodies in the blood serum sample was determined. In this case, as in Example 1, only the whole cell Borrelie antigen (0.1 mg / ml in 100 mM MES buffer pH 5.6) was bound instead of the HSA protein. After antigen modification, the electrode is incubated with a diluted serum sample that potentially contains anti-Borrelia antibodies. The presence of these antibodies in the serum can be determined from changes in impedance measured in ferri / ferrocyanide solution (Fig. 3). Two human serum samples (one positive, the other negative for the presence of anti-Borrelia antibodies) were measured with six different electrode systems prepared as described. The curves in FIG. 3 are records of impedance measurements of individual electrodes at different frequencies (range from 2 Hz to 100,000 Hz). The data is in the form of a Nyquist graph. The positive sample contains 3.04 IP antibodies, which is the amount of antibodies expressed in the ELIS A positivity index (positivity index is the ratio of the optical density of the sample to the optical density of the border control). Negative serum contains no anti-Borrelia antibodies.

Industrial applicability

The invention is particularly suitable for the rapid and inexpensive determination of bacterial antigens or corresponding antibodies in the body fluids of test subjects suspected of having a disease,

CZ 2017 - 483 A3 eg determination of antibodies against bacteria of Borelia sp. in blood serum. The invention can be used for any serological assay - antibodies raised against a pathogen, toxin or other foreign substance are determined.

PATENT CLAIMS

Claims (14)

A method for determining the presence of an analyte in a liquid sample, comprising the steps of: (i) determining the value of the electrochemical parameter of the redox solution having an opposite charge to the analyte to be determined; (ii) the working electrode surface containing the immobilized particles for affinity interaction with the analyte is incubated with the liquid sample, and if the sample contains the analyte to be determined, the analyte binds to the immobilized particles on the electrode surface to form an immobilized analyte-immobilized particle surface electrodes; (iii) contacting the working electrode with the immobilized analyte-immobilized particle complex with a solution of a redox substance having an opposite charge to the analyte being determined; (iv) determining the electrochemical parameter at the interface between the working electrode surface and the redox solution in relation to the supplemental electrode present in the liquid sample; (v) determining from the value of the determined electrochemical parameter the change in the electrochemical parameter relative to the electrochemical parameter of the redox solution determined in step i), wherein the presence of the analyte in the sample results in a change in the electrochemical parameter value if the electrochemical parameter is a resistance or impedance or voltage, it will drop, if the electrochemical parameter is current, it will increase; the presence of the analyte in the liquid sample is determined qualitatively and / or semi-quantitatively and / or quantitatively from the change in the electrochemical parameter. Method according to claim 1, characterized in that steps ii) and iii) are carried out simultaneously in a sample dropped onto a working electrode, which contains immobilized particles for interaction with the analyte and also adsorbed redox. Method according to claim 1 or 2, characterized in that the immobilized particle for affinity interaction with the analyte on the working electrode surface is an antibody or an antigen, preferably the immobilized particle is a whole cell antigen of a Borrelia sp. Method according to claim 1 or 2, characterized in that the analyte is an antibody or an antigen, and if the immobilized particle is an antibody, the analyte is an antigen or more versa, preferably the analyte is an antibody against Borrelia sp. A process according to any one of the preceding claims wherein the redox is selected from the group consisting of potassium hexacyanoferrate, potassium hexacyanoferrate, hexaaminruthenium (n / III) chloride, cobaltocene, ferrocene, aminoferocene, I / I3, ferrocarboxylic acid. Method according to any one of the preceding claims, characterized in that the measurement of the electrochemical parameter for determining the impedance at the interface of the working electrode surface and the redox substance solution relative to the reference electrode present in the liquid sample in step (iv) A3 is a measurement of electrical current and / or electrical voltage by amperometry, biamperometry, potentiometry, and / or voltammetry, most preferably by biamperometry using two identical immobilized particle working electrodes for affinity interaction with the analyte. Method according to any one of the preceding claims, characterized in that the working electrode for detecting the analyte in the liquid sample is selected from the group consisting of a metal and a carbon electrode, preferably the electrode is selected from the group comprising copper, silver, platinum, gold, stainless and composite carbon. an electrode comprising immobilized particles on the surface for affinity interaction with the analyte. An electrode for detecting an analyte in a liquid sample, characterized in that it is selected from the group consisting of a metal and carbon electrode, preferably an electrode selected from the group consisting of a copper, silver, platinum, gold, stainless and composite carbon electrode containing a polymer surface. a layer of a positively charged polymeric carrier containing free amino groups on which are immobilized particles for affinity interaction with the analyte via N-hydroxysuccinimide-activated carboxyl groups of low molecular weight substances up to 1000 kDa bound to the polymeric carrier. An electrode according to claim 8, characterized in that it consists of a metal or carbon electrode which contains on its surface adsorbed poly-L-lysine and / or poly-allylamine and / or polyethyleneimine to which it is covalently bonded via covalently bonded citrate molecules. whole cell antigen of Borrelia sp. Method for preparing a working electrode for detecting an analyte in a liquid sample by the method according to claims 1 to 7, characterized in that it comprises the following steps: (i) incubating the metal or carbon electrode with a positively charged polymeric carrier carrying free amino groups, preferably having a molecular weight of from 30 to 70 kDa, for at least 30 minutes; (ii) the resulting electrode coated with a layer of positively charged polymeric carrier containing free amino groups is incubated with a solution with a low molecular weight molecular weight solution up to 1000 kDa carrying at least two carboxyl groups, at least one of which is activated to conjugate with the amino groups of the polymer carrier. at least 15 minutes to form a carboxyl-modified electrode surface; (iii) the carboxyl groups on the electrode surface are activated with EDC / N-hydroxysuccinimide for at least 20 minutes to form active esters on the surface of the polymer layer; (iv) the analyte affinity interaction particles react with N-hydroxysuccinimide activated carboxylic acid groups on the polymer carrier surface at least 30 minutes of incubation to form a covalent bond between the polymeric carrier that is coated with the electrode surface and the affinity reaction particle with the analyte. The method of claim 10, wherein the positively charged polymeric poly-Llysine carrier, the low molecular weight substance carrying at least two carboxyl groups is citrate, and the analyte affinity reaction particle is a whole cell antigen of Borrelia sp. 12. A kit for determining the presence of an analyte in a liquid sample according to claim 1, characterized in that it comprises at least one working electrode for detecting the analyte in the liquid sample selected from the group consisting of a metal and a carbon electrode, preferably an electrode selected from the group comprising a copper, silver, platinum, gold, stainless and composite carbon electrode comprising surface-immobilized particles for affinity interaction with the analyte, for connection to a meter for quantifying an electrochemical parameter selected from impedance, current and / or voltage; and at least one additional electrode for the - 10GB 2017 - 483 A3 connection to a meter for quantifying an electrochemical parameter selected from impedance, current and / or voltage; wherein the kit further comprises a measuring instrument for quantifying an electrochemical parameter selected from impedance, current and / or voltage; a redox substance having an opposite charge in solution to the analyte to be determined, or a solution thereof; wherein optionally the kit further comprises an analyzer and / or a control unit and / or a display and / or a light emitting diode. Use of a kit for determining the presence of an analyte in a liquid sample according to claim 12 and / or a method for determining the presence of an analyte in a liquid sample according to any one of claims 1 to 7 for qualitative and / or quantitative and / or semi-quantitative detection of antibody and / or antigen. / or bacteria in a liquid sample. Use according to claim 13 for detecting the presence of antibodies of Borrelia sp. in a liquid sample.