Electrode for electrochemical measurement and measurement method

Abstract

To provide an electrode for electrochemical measurement capable of detecting a target substance without bonding an index substance, and a measuring method.SOLUTION: A measurement method includes a binding step of bringing a specimen solution 8 containing a target substance 7 into contact with an electrode 2 in which a naturally denatured protein 22 having a naturally denatured region that is folded by specifically binding to the target substance 7 is immobilized as an identifier on a surface of a conductive layer 21 to specifically bind the target substance 7 to the naturally denatured protein 22, and a measurement step of bringing the electrode 2 brought into contact with the specimen solution 8 in the binding step 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 a surface exposure area of the conductive layer 21 caused by folding of the naturally denatured protein 22 by bonding with the target substance 7.SELECTED DRAWING: Figure 3

Description

[Technical Field]

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

[0002] Biosensors using electrodes with molecular recognition elements such as antibodies, enzymes, or microorganisms immobilized on the surface of the electrode 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 in which a capture electrode, to which an identifier for capturing abnormal prions (target substance) is immobilized, is immersed in a specimen solution to be tested, an indicator attachment step in which the capture electrode is immersed in an indicator solution containing an indicator reagent that adsorbs to abnormal prions and is electrochemically detectable, and a measurement step in which the capture electrode is immersed in an electrolyte solution and the electrochemical response is measured.

[0003] However, conventional biosensors have the problem that they require a process of binding an electrochemically detectable indicator substance to a target substance or an identifier, which makes pre-processing complicated. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-98342 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made to improve this current situation, and aims to provide an electrode for electrochemical measurement and a measurement method that can detect 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 measurements, 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.

[0007] Another aspect of the present invention is a measurement method including: a binding step of contacting a sample solution containing a target substance with an electrode having an intrinsically unfolded region that specifically binds to and folds a target substance, the sample solution having the target substance, immobilizing the intrinsically unfolded protein as an identifier on the surface of a conductive layer, thereby specifically binding the target substance to the identifier; and a measurement step of contacting the electrode that has been contacted with the sample solution in the binding step with an electrolyte solution containing a redox substance, thereby performing electrochemical measurement; wherein an increase in the exposed surface area of ​​the conductive layer, which is caused by the identifier folding upon binding with the target substance, is detected or quantified, thereby measuring 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, for example, the intrinsically disordered protein is human Praja1, and the target substance is α-synuclein or tau protein. As used herein, "intrinsically unfolded proteins" include proteins in which the entire molecule or a certain region of the molecule does not adopt a specific three-dimensional structure but adopts an irregular three-dimensional structure. In addition, "intrinsically unfolded proteins" include intrinsically unfolded proteins having an artificially designed amino acid sequence. [Effects 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 explanation of the drawings]

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

[0011] Embodiments of the electrode and 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 for containing an electrolyte solution 6. The measurement device 5 is a potentiostat.

[0012] In the electrochemical measurement system 1, the working electrode 2, counter electrode 3, and reference electrode 4 are immersed in an electrolyte solution 6 containing a redox species. The potential of the working electrode 2 is swept using the reference electrode 4 as a reference, and the value of the current obtained when the redox species in the electrolyte solution 6 are 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 unfolded 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 substrate 23. In this embodiment, the conductive layer 21 is made of gold (Au), the intrinsically unfolded protein 22 is human Praja1 (E3 ubiquitin ligase Praja1), and the substrate 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 with carbon, or elemental carbon, etc. Furthermore, the conductive layer 21 may be a single-layer film, or a multilayer film in which multiple films are stacked.

[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 immobilized as an identifier on 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 proteins associated with neurodegenerative diseases, and examples of target substances include α-synuclein, as well as tau protein (Tau), ataxin-3 (ATXN-3), TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), and superoxide dismutase (SOD1).

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

[0018] Next, a method for measuring a target substance 7 in a sample solution 8 using the electrochemical measurement system 1 will be described. First, as shown in Fig. 2, the sample solution 8 containing the target substance 7 is brought into contact with a working electrode 2 on which an intrinsically unfolded protein 22, which has 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 then brought into contact with an electrolyte solution 6 containing a redox substance, and electrochemical measurement is performed (measurement step). For example, the redox substance in the electrolyte solution 6 is ferricyanide ions (oxidant Ox) and ferrocyanide ions (reduced 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 the 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 sample solution 8 can be measured by electrochemically detecting or quantifying the increase in the exposed surface area of ​​the conductive layer 21 that occurs when the intrinsically unfolded protein 22 binds to the target substance 7 and folds. This allows the target substance 7 to be measured electrochemically 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 in which the intrinsically denatured protein 22 is immobilized on the conductive layer 21 will be described.

[0023] <Example> The working electrode 2 was fabricated by vacuum deposition of gold on a silicon substrate (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 disordered protein 22) on the conductive layer 21. Non-adsorbed components were removed by rinsing with deionized water and dried with nitrogen. The purified human Praja1 protein was purified according to the procedure described in 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). Human Praja1 protein immobilized on the conductive layer 21 may also be purified using other procedures.

[0024] Next, sample solution 8 containing α-synuclein (target substance 7) in Tris-HCl (pH 8.0) 150 mM NaCl solution was added dropwise 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.

[0025] Using this substrate as the working electrode 2, an electrochemical measurement system 1 was used to investigate changes in the surface of the conductive layer 21 (gold layer) of the working electrode 2 due to the interaction between human Praja1 and α-synuclein by cyclic voltammetry. The measurement device 5 (potentiostat) was an Interface 1010T (Gamy Instruments, USA). The counter electrode 3 was a platinum wire, and the reference electrode 4 was a silver-silver chloride electrode (3M NaCl). A conventional three-electrode electrochemical cell was used. The electrolyte solution 6 was a 5 mM potassium ferricyanide (K3 [Fe(CN)6] solution, with a supporting electrolyte of 100 mM KCl. Cyclic voltammetry was performed at a scan rate of 20 mV / s over a 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 α-synuclein), 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 higher oxidation and reduction peak currents. This indicates that the presence of α-synuclein (target substance 7) in sample solution 8 causes many human Praja 1 (intrinsically disordered protein 22) molecules on conductive layer 21 to fold, increasing the exposed surface area of ​​conductive layer 21, 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). Furthermore, the electrochemical measurement system 1 may be a two-electrode system that does not include 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 used as an identifier immobilized on the conductive layer 21 of the working electrode 2 is not limited to Praja1, but may be any intrinsically unfolded protein having an intrinsically unfolded region that specifically binds to and folds a target substance. Note that the target substance here includes molecules and ions other than proteins.

[0031] For example, combinations of a naturally denatured protein as an identifier to be fixed on the conductive layer 21 and a target substance that specifically binds to the naturally denatured protein include Ptc2 (one of the protein phosphatase 2C group proteins) and carbon dioxide, granulin and zinc ions, prothymosin α (ProTα) and zinc ions, a surface membrane specific protein of magnetic particles (Mms6, magnetosome membrane specific protein) 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 disordered protein, Ox oxidized form (redox substance), Red reduced form (redox substance)

Claims

1. An electrode used in electrochemical measurements, an intrinsically disordered protein having an intrinsically disordered region that specifically binds to a target substance and folds is immobilized on the surface of the conductive layer as an identifier; Electrodes for electrochemical measurements.

2. a binding step of contacting a sample solution containing a target substance with an electrode having an intrinsically disordered protein, which has an intrinsically disordered region that specifically binds to and folds a target substance, as an identifier, and causing the target substance to specifically bind 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 electrochemical measurement, the presence or concentration of the target substance in the sample solution is measured by detecting or quantifying an increase in the exposed surface area of ​​the conductive layer caused by the identifier folding upon binding with the target substance; Measurement method.

3. the intrinsically disordered protein is human Praja1; The target substance is α-synuclein or tau protein. The electrode according to claim 1 or the measurement method according to claim 2.

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