Electrode for electrochemical measurement and measurement method

Abstract

The present invention provides an electrode for electrochemical measurement and a measurement method capable of detecting a target substance without binding an indicator substance. [Solution] The measurement method includes a binding step in which a specimen solution 8 containing the target substance 7 is brought into contact with an electrode 2 having an intrinsically unfolded region that specifically binds to and folds a target substance 7 and is fixed as an identifier on the surface of a conductive layer 21, thereby specifically binding the target substance 7 to the intrinsically unfolded protein 22, and a measurement step in which the electrode 2 that has been brought into contact with the specimen solution 8 in the binding step is brought into contact with an electrolyte solution 6 containing redox substances Red and Ox to perform electrochemical measurement. The presence or concentration of the target substance 7 in the specimen solution 8 is measured by detecting or quantifying an increase in the exposed surface area of ​​the conductive layer 21 caused by the intrinsically unfolded protein 22 folding upon binding with the target substance 7.

Description

[Technical field]

[0001] The present invention relates to an electrode for electrochemical measurement and a measurement method. [Background technology]

[0002] Conventionally, biosensors using electrodes with molecular recognition elements such as antibodies, enzymes, and microorganisms immobilized on the surface of the electrodes have been proposed (see, for example, Patent Document 1). Patent Document 1 discloses a method for electrochemically detecting abnormal prions, including a specimen attachment step of immersing a capture electrode, to which an identifier for capturing abnormal prions (target substance) is fixed, in a specimen solution to be inspected, an indicator attachment step of immersing the capture electrode in an indicator solution containing an indicator reagent that adsorbs to abnormal prions and is electrochemically detectable, and a measurement step of immersing the capture electrode in an electrolyte solution to measure the electrochemical response.

[0003] However, conventional biosensors require a process for binding an electrochemically detectable indicator substance to a target substance or an identifier, and this process requires complicated pre-processing. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2006-98342 A Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made to improve this current situation, and has an object to provide an electrode for electrochemical measurement and a measurement method capable of detecting a target substance without binding an indicator substance. [Means for solving the problem]

[0006] One aspect of the present invention is an electrode for use in electrochemical measurement, in which an intrinsically disordered protein having an intrinsically disordered region that specifically binds to a target substance and folds is immobilized as an identifier on the surface of a conductive layer, the identifier covers the surface of the conductive layer, and the identifier is target substance When the identifier is folded by binding with the conductive layer, the area of ​​the identifier covering the surface of the conductive layer is reduced and the exposed surface area of ​​the conductive layer is increased.

[0007] In another aspect of the present invention, an intrinsically disordered protein having an intrinsically disordered region that specifically binds to a target substance and folds is immobilized as an identifier on the surface of a conductive layer. the identifier covers a surface of the conductive layer. The method includes a binding step of contacting an electrode with a specimen solution containing the target substance to specifically bind the target substance to the identifier, and a measurement step of contacting the electrode that has been contacted with the specimen solution in the binding step with an electrolyte solution containing a redox substance to perform electrochemical measurement, wherein the identifier is folded by binding with the target substance. The area of ​​the surface of the conductive layer covered by the identifier is reduced. The increase in the exposed surface area of ​​the conductive layer caused by the increase in the surface area of ​​the conductive layer is detected or quantified to measure the presence or concentration of the target substance in the sample solution.

[0008] In the above-mentioned one aspect and another aspect of the present invention, examples include the intrinsically disordered protein being human Praja1 and the target substance being α-synuclein or tau protein. As used herein, an intrinsically unfolded protein includes a protein whose entire molecule or a certain region of the molecule does not adopt a specific three-dimensional structure but adopts an irregular three-dimensional structure, and also includes an intrinsically unfolded protein having an artificially designed amino acid sequence. Effect of the Invention

[0009] The present invention can provide an electrode and a measurement method that enable electrochemical measurement without performing a process of binding an electrochemically detectable indicator substance to a target substance or an identifier. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of an electrochemical measurement apparatus. [Diagram 2] FIG. 2 is a diagram illustrating a working electrode according to the embodiment. [Diagram 3] FIG. 2 is a diagram illustrating a measurement principle of the embodiment. [Figure 4] 1 is a graph showing the relationship between the target substance concentration and the redox peak current. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An embodiment of the electrode and the measurement method of the present invention will be described with reference to the drawings. Fig. 1 is a schematic diagram showing an electrochemical measurement device. As shown in Fig. 1, the electrochemical measurement system 1 is, for example, a three-electrode system, and includes a working electrode 2, a counter electrode 3, and a reference electrode 4, a measurement device 5 to which these three electrodes are connected, and a container that contains an electrolyte solution 6. The measurement device 5 is a potentiostat.

[0012] In the electrochemical measurement system 1, the working electrode 2, the counter electrode 3, and the reference electrode 4 are immersed in an electrolyte solution 6 containing an oxidation-reduction substance (redox species). Then, the potential of the working electrode 2 is swept by a measuring device 5 with respect to the reference electrode 4, and the value of the current obtained when the oxidation-reduction substance in the electrolyte solution 6 is oxidized and reduced on the surface of the working electrode 2 is measured.

[0013] In this embodiment, the working electrode 2 constitutes an electrode for electrochemical measurement of the present invention. As shown in Fig. 2, the working electrode 2 includes a conductive layer 21 and an intrinsically denatured protein 22 immobilized as an identifier on the surface of the conductive layer 21. The conductive layer 21 is formed on, for example, an insulating base material 23. In this embodiment, the conductive layer 21 is formed of gold (Au), the intrinsically denatured protein 22 is human Praja1 (E3 ubiquitin ligase Praja1), and the base material 23 is a silicon substrate.

[0014] The material of the conductive layer 21 is not limited to gold, but may be, for example, silver, titanium, nickel, aluminum, ruthenium, tantalum, titanium, copper, platinum, niobium, zirconium, or an alloy of these elements, or an alloy of these elements and carbon, or elemental carbon, etc. Furthermore, the conductive layer 21 may be a single layer film, or may be a multilayer film in which multiple films are laminated.

[0015] Furthermore, the material of the base material 23 is not particularly limited, and examples thereof include polyimide (PI), polyethylene terephthalate (PET), single crystal silicon (silicon wafer), glass, methacrylic resin (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), polyoxymethylene (POM), ABS resin (ABS), ceramics, quartz, etc. Furthermore, the shape, thickness, and size of the base material 23 are not particularly limited.

[0016] In this embodiment, the intrinsically disordered protein 22 fixed as an identifier to the conductive layer 21 is human Praja1, and the target substance is, for example, α-synuclein (α-Syn). Human Praja1 is a protein that interacts with a protein related to a neurodegenerative disease, and examples of the target substance include tau protein (Tau), ataxin-3 (ATXN-3), TAR DNA binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), and superoxide dismutase (SOD1), in addition to α-synuclein.

[0017] A known electrode, for example a platinum electrode, can be used as the counter electrode 3. A known electrode, for example a silver-silver chloride electrode, can be used as the reference electrode 4.

[0018] Next, a method for measuring a target substance 7 in a specimen solution 8 using the electrochemical measurement system 1 will be described. First, as shown in Fig. 2, a specimen solution 8 containing the target substance 7 is brought into contact with a working electrode 2 on which an intrinsically unfolded protein 22 having an intrinsically unfolded region that specifically binds to and folds the target substance 7 is immobilized as an identifier on the surface of a conductive layer 21. This causes the target substance 7 to specifically bind to the intrinsically unfolded protein 22 (identifier) ​​(binding step).

[0019] The working electrode 2, which has been brought into contact with the specimen solution 8 in the binding step, is brought into contact with an electrolyte solution 6 containing a redox substance to perform electrochemical measurement (measurement step). For example, the redox substance in the electrolyte solution 6 is ferricyanide ions (oxidant Ox) and ferrocyanide ions (reducant Red).

[0020] 3, when the target substance 7 is not bound to the intrinsically unfolded protein 22 immobilized on the conductive layer 21, the presence of the intrinsically unfolded protein 22 covering the surface of the conductive layer 21 suppresses the oxidation of the reduced form Red (or the reduction of the oxidized form Ox) through the conductive layer 21. On the other hand, when the target substance 7 is bound to the intrinsically unfolded protein 22, the exposed surface area of ​​the conductive layer 21 increases due to folding of the intrinsically unfolded protein 22, allowing the oxidation of the reduced form Red (or the reduction of the oxidized form Ox) through the conductive layer 21.

[0021] That is, the presence or concentration of the target substance 7 in the specimen solution 8 can be measured by electrochemically detecting or quantifying an increase in the exposed surface area of ​​the conductive layer 21 caused by the intrinsically unfolded protein 22 binding to the target substance 7 and folding. This makes it possible to electrochemically measure the target substance 7 without performing a process of binding an electrochemically detectable indicator substance to the target substance 7 or the identifier (intrinsically unfolded protein 22).

[0022] Next, an example of a measurement result using the working electrode 2 having the intrinsically denatured protein 22 immobilized on the conductive layer 21 will be described.

[0023] <Example> As the working electrode 2, a gold film was formed by vacuum deposition on a silicon substrate as a base material 23 to form a conductive layer 21. A 2 mg / ml solution of purified human Praja1 protein was dropped onto the conductive layer 21 and incubated at room temperature for 1 hour to immobilize human Praja1 (intrinsically denatured protein 22) on the conductive layer 21. Non-adsorbed components were removed by rinsing with deionized water and dried with nitrogen blowing. The purified human Praja1 protein was purified according to the procedure of a previous study (E3 ligase Praja1 mediates ubiquitination and degradation of microtubule-associated protein Tau, Shiho Aoki et al., bioRxiv preprint doi: https: / / doi.org / 10.1101 / 2024.06.10.598176). The human Praja1 protein immobilized on the conductive layer 21 may be human Praja1 purified by other procedures.

[0024] Next, sample solution 8 containing α-synuclein (target substance 7) was dropped into Tris-HCl (pH 8.0) 150 mM NaCl solution and incubated at room temperature for 1 hour to react with human Praja 1. Unreacted α-synuclein components were removed by rinsing with deionized water and dried with nitrogen blow.

[0025] Using an electrochemical measurement system 1 with this substrate as the working electrode 2, changes in the conductive layer 21 (gold layer) surface of the working electrode 2 due to the interaction between human Praja1 and α-synuclein by cyclic voltammetry were examined. The measurement device 5 (potentiostat) used was an Interface 1010T (Gamry Instruments, USA), the counter electrode 3 was a platinum wire, the reference electrode 4 was a silver-silver chloride electrode (3M NaCl), and a conventional three-electrode electrochemical cell was used. In addition, the electrolyte solution 6 was a 5 mM potassium ferricyanide K3 [Fe(CN)6] solution (supporting electrolyte was KCl 100 mM). Cyclic voltammetry was measured at a scan rate of 20 mV / s in the scan range from 0.5 V to -0.5 V.

[0026] Working electrodes 2 were prepared for sample solutions 8 containing α-synuclein (target substance 7) at concentrations of 0 μg / ml (no concentration), 0.01 μg / ml, and 0.1 μg / ml, and the oxidation peak current and reduction peak current were measured by cyclic voltammetry.

[0027] The measurement results are shown in Figure 4. In Figure 4, the horizontal axis represents α-synuclein concentration, and the vertical axis represents current density. As shown in Figure 4, the presence of α-synuclein (target substance 7) resulted in high oxidation peak current and reduction peak current. This shows that the presence of α-synuclein (target substance 7) in sample solution 8 causes many human Praja1 (intrinsically denatured protein 22) on conductive layer 21 to fold, increasing the exposed surface area of ​​conductive layer 21, and enabling α-synuclein to be quantified.

[0028] The present invention is not limited to the above-described embodiment, and can be embodied in various forms. For example, the working electrode 2 may be formed on the same substrate together with the counter electrode 3 and the reference electrode 4 (electrode chip). The electrochemical measurement system 1 may be a two-electrode system not including the counter electrode 3.

[0029] Furthermore, the electrochemical measurement method is not limited to cyclic voltammetry (CV), but may be methods such as differential pulse voltammetry (DPV), chronoamperometry (CA), linear sweep voltammetry (LSV), and short waveform voltammetry (SWV).

[0030] Furthermore, the intrinsically unfolded protein as an identifier immobilized on the conductive layer 21 of the working electrode 2 is not limited to Praja1, and may be any intrinsically unfolded protein having an intrinsically unfolded region that specifically binds to a target substance and folds. The target substance here includes molecules and ions other than proteins.

[0031] For example, a natural Denaturation Proteins and their natural Denaturation Combinations of proteins and target substances that specifically bind to them include Ptc2 (a member of the protein phosphatase 2C family of proteins) and carbon dioxide, granulin and zinc ions, prothymosin α (ProTα) and zinc ions, magnetosome membrane specific protein (Mms6) and iron ions, silver-binding protein (SilE) and silver ions, and protein deacetylase sirtuin 1 (SIRT1) and insulin. [Explanation of symbols]

[0032] 2 working electrode (electrode), 6 electrolyte solution, 7 target substance, 8 sample solution, 21 conductive layer, 22 intrinsically denatured protein, Ox oxidized form (redox substance), Red reduced form (redox substance)

Claims

1. An electrode for use in electrochemical measurements, comprising: An intrinsically disordered protein having an intrinsically disordered region that specifically binds to a target substance and folds is immobilized as an identifier on the surface of a conductive layer; the identifier covers a surface of the conductive layer; When the identifier is folded by binding to the target substance, the area of ​​the identifier covering the surface of the conductive layer is reduced, and the exposed surface area of ​​the conductive layer is increased. Electrodes for electrochemical measurements.

2. a binding step in which an intrinsically unfolded protein having an intrinsically unfolded region that specifically binds to a target substance and folds is immobilized as an identifier on the surface of a conductive layer, and a sample solution containing the target substance is brought into contact with an electrode on which the identifier covers the surface of the conductive layer, thereby specifically binding the target substance to the identifier; a measuring step of contacting the electrode that has been brought into contact with the specimen solution in the binding step with an electrolyte solution containing a redox substance to perform an electrochemical measurement, the identifier is folded by binding with the target substance, and the area of ​​the identifier covering the surface of the conductive layer is reduced, resulting in an increase in the exposed surface area of ​​the conductive layer, and this increase is then detected or quantified to measure the presence or concentration of the target substance in the sample solution. Measurement method.

3. the intrinsically disordered protein is human Praja1, The target substance is α-synuclein or tau protein.

2. The electrode of claim 1.

4. The intrinsically disordered protein is human Praja1, The target substance is α-synuclein or tau protein. The measurement method according to claim 2.

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