Solid nanopore-based protein detection system and application thereof
Through the solid-state nanopore binding diabodyne sandwich method, the problem of insufficient detection sensitivity and specificity of retinol-binding protein 4 was solved, and high sensitivity, rapid and trace detection effects were achieved, which were suitable for clinical samples and cell secretion analysis.
Patent Information
- Application Number
- CN202411289653.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art has insufficient sensitivity and specificity when detecting retinol-binding protein 4 (RBP4), especially in samples with lower concentrations, which are difficult to achieve efficient detection.
Using a protein detection system based on solid-state nanopores, combined with a dual-antibody sandwich method, the retinol-binding protein 4 monoclonal antibody and capture antibody were used to voltage-driven through nanopores on the silicon nitride chip (pore size is 8-30nm). High sensitivity detection is achieved using transmembrane osmotic pressure gradient.
Single-molecular-level detection of retinol-binding protein 4 is achieved, with significantly improved sensitivity, shortened detection time and low sample volume requirements, which is suitable for the analysis of clinical samples and trace cell secretions.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protein detection, and in particular to a protein detection system based on solid-state nanopores and applications thereof. Background Art
[0002] A nanopore is a nanoscale channel with a diameter of 1-100nm. It can be used as a highly sensitive biosensor to detect and analyze various biological molecules such as DNA, RNA and protein. When the molecule to be tested passes through the nanopore, it causes a change in the ionic current in the channel. By analyzing the current signal, the qualitative and quantitative analysis of the molecule can be achieved. Nanopores can be divided into two categories: solid-state nanopores and biological nanopores. Solid-state nanopores are made of inorganic materials (such as silicon nitride, silicon dioxide and graphene), and biological nanopores are composed of membrane proteins (such as α-hemolysin and Mycobacterium smegmatis membrane protein A MspA).
[0003] Solid-state nanopores have unique advantages in protein molecule detection. Solid-state nanopores are made of inorganic materials and have excellent mechanical strength, thermal stability and chemical stability. In contrast, biological nanopores are relatively unstable due to their composition of lipid bilayers and transmembrane proteins, and are easily affected by factors such as temperature and pH. The high stability of solid-state nanopores is conducive to the reliable detection of proteins. In addition, the size and shape of solid-state nanopores can be precisely controlled through microfabrication technology and can be customized according to the size of the protein structure, thereby optimizing the detection performance. The size and properties of biological nanopores are limited by natural transmembrane proteins and are less customizable.
[0004] Nanopore technology has been widely studied and applied in the field of protein detection. Nanopores can be used to detect proteins at the single molecule level, providing a powerful tool for proteomics research and disease marker screening. Combining nanopore technology with specific recognition molecules such as aptamers and antibodies can achieve high-sensitivity detection of low-abundance protein markers in clinical samples, demonstrating its potential for application in early clinical diagnosis.
[0005] In addition to nanopore technology, ELISA, colloidal gold and chemiluminescence are also commonly used technologies in the field of protein detection. ELISA has high sensitivity and specificity, but the sample volume is large and the detection time is long; the colloidal gold method is easy to operate and the results are intuitive, suitable for rapid on-site detection, but the sensitivity and specificity are low; the chemiluminescence method has high sensitivity, a wide dynamic range, and can achieve quantitative analysis, but it requires special instruments and is relatively expensive.
[0006] The double antibody sandwich method is a commonly used immunological detection technology. Through the combination of capture antibody and detection antibody with target antigen, a sandwich complex of "antibody-antigen-antibody" is formed to achieve high sensitivity and high specificity detection of antigen. The sandwich structure has strong specificity. The two antibodies bind to different epitopes of the antigen respectively, which greatly improves the specificity of the detection and reduces the interference of non-specific binding. It has high sensitivity. The sandwich structure sandwiches the target antigen between the two antibodies to form a stable complex, which effectively increases the detection signal and improves the detection sensitivity. Since the volume of the sandwich structure is significantly larger than that of a single antigen or antibody molecule, it helps to amplify the signal and further improve the detection sensitivity.
[0007] Retinol binding protein 4 (RBP4) belongs to the lipocalin family and is a carrier protein for serum vitamin A. Studies have shown that RBP4 may be involved in the pathogenesis of type 2 diabetes, and is closely related to the occurrence and development of many liver diseases. RBP4 is also closely related to sarcopenia. However, the concentration of RBP4 in body fluids is low, and the specificity and sensitivity of current methods for detecting RBP4 still need to be improved. Summary of the invention
[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a highly sensitive protein detection system based on solid-state nanopores and its application.
[0009] To achieve the above object, the technical solution adopted by the present invention is:
[0010] In a first aspect, the present invention provides a protein detection system based on solid-state nanopores, wherein the protein detection system comprises a detection device and a detection reagent;
[0011] The detection device comprises an electrode cavity, electrodes and a data acquisition system;
[0012] The electrode cavity comprises a cis cavity and a trans cavity, a silicon nitride chip is arranged between the cis cavity and the trans cavity, and the cis cavity and the trans cavity are connected through nanopores on the silicon nitride chip;
[0013] Electrodes are provided in the cis cavity and the trans cavity and are connected to a data acquisition system;
[0014] The detection reagent contains a double antibody sandwich paired retinol binding protein 4 monoclonal antibody, which is obtained by immunizing mice with retinol binding protein 4 as an immunogen.
[0015] The retinol binding protein 4 monoclonal antibody of the present invention includes a retinol binding protein 4 detection antibody and a retinol binding protein 4 capture antibody, which forms a double antibody sandwich with the retinol binding protein 4 to be detected, thereby improving the detection sensitivity, being able to detect samples with low retinol binding protein 4 content, and realizing real-time detection.
[0016] Furthermore, the pore diameter of the nanopore is 8-30 nm.
[0017] Furthermore, the protein detection system also contains a buffer solution, and the buffer solution contains KCl.
[0018] Furthermore, the concentration of KCl is 1-3M.
[0019] Furthermore, the cis cavity contains a cis cavity buffer solution, the trans cavity contains a trans cavity buffer solution, and the concentration of KCl in the trans cavity buffer solution is lower than the concentration of KCl in the cis cavity buffer solution.
[0020] In a specific embodiment of the present invention, the concentration of KCl in the cis-cavity buffer solution is 3M, and the concentration of KCl in the trans-cavity buffer solution is 1M.
[0021] Furthermore, the cis-cavity buffer solution and the trans-cavity buffer solution also contain Tris and EDTA.
[0022] Furthermore, the concentration of Tris in the cis-cavity buffer solution is 15-45 mM, preferably 30 mM. The concentration of Tris in the trans-cavity buffer solution is 5-15 mM, preferably 10 mM.
[0023] Furthermore, the concentration of EDTA in the cis-cavity buffer solution is 1.5-4.5 mM, preferably 3 mM. The concentration of EDTA in the trans-cavity buffer solution is 0.5-1.5 mM, preferably 1 mM.
[0024] Furthermore, the pH value of the buffer solution is 7-9, preferably 8.
[0025] Furthermore, the electrode is an Ag / AgCl electrode.
[0026] In a second aspect, the present invention provides the use of the protein detection system in the preparation of a product for detecting retinol binding protein 4.
[0027] The method of using the system of the present invention is as follows:
[0028] S1: adding 50 μL of cis-cavity buffer solution to the cis-cavity, adding 50 μL of trans-cavity buffer solution to the trans-cavity, applying a voltage of 100 to 200 mV, and detecting and recording blank signals through a data acquisition system;
[0029] S2: Add the test sample, RBP4 protein capture antibody and RBP4 protein detection antibody into the cis cavity as the test liquid; apply a voltage of 100 to 200 mV to drive the test liquid in the trans cavity through the nanopore, and detect and record the signal generated when the test liquid passes through the nanopore through the data acquisition system.
[0030] In a specific embodiment of the present invention, the applied voltage is 200 mV. The generated signal is stable and pore blocking is not likely to occur.
[0031] Compared with the prior art, the present invention has the following beneficial effects:
[0032] The present invention combines solid-state nanopore technology with the protein double antibody sandwich method. First, it can achieve single-molecule-level detection sensitivity, which is much higher than ELISA and other methods. Secondly, the solid-state nanopore can monitor the process of retinol binding protein 4 passing through the nanopore in real time, without the need for complex signal amplification and reading steps, greatly shortening the detection time. Finally, the solid-state nanopore has very low requirements for sample volume, usually only a few microliters of protein sample are needed to complete the detection, which is particularly important for the analysis of trace samples such as clinical samples and cell secretions. The use of solid-state nanopores can achieve single-molecule level detection of protein molecules, providing a new technical means for studying protein structure, function and interaction.
[0033] In summary, the present invention provides a new system and method with high sensitivity, rapidity and trace amount for the analysis of retinol binding protein 4. Compared with the traditional method, the system and method of the present invention have the advantages of single molecule sensitivity, real-time detection and low sample amount requirement. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 Schematic diagram of the RBP4 dual antibody sandwich detection method using silicon nitride nanopores.
[0035] Figure 2 Schematic diagram of the detection device of the protein detection system based on solid-state nanopores.
[0036] Figure captions: 1-cis cavity; 2-trans cavity; 3-silicon nitride chip; 4-nanopore; 5-Ag / AgCl electrode; 6-data acquisition system.
[0037] Figure 3 The influence of the ion concentration difference between the cis-cavity and trans-cavity buffer on the detection effect, where A is the isotropic ion concentration system and B is the asymmetric ion concentration system.
[0038] Figure 4 The influence of different applied voltages on the detection effect.
[0039] Figure 5 The effects of RBP4 protein capture antibody and monoclonal antibody (RBP4 protein sample to be tested + RBP4 protein capture antibody) on the detection effect. A is the time domain diagram of RBP4 protein capture antibody; B is the time domain diagram of monoclonal antibody; C is the peak characteristic scatter diagram of RBP4 protein capture antibody; D is the peak characteristic scatter diagram of monoclonal antibody.
[0040] Figure 6 Schematic diagram of the RBP4 protein double antibody sandwich structure permeation signal.
[0041] Figure 7 This is a time domain diagram when using double antibody sandwich to detect different concentrations of RBP4 protein.
[0042] Figure 8 It is a sensitivity statistics chart.
[0043] Fig. 9 Specific experimental results. DETAILED DESCRIPTION
[0044] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.
[0045] Example 1 A protein detection system and detection method based on solid-state nanopores
[0046] like Figure 1 Shown is the schematic diagram of the RBP4 double antibody sandwich detection method using silicon nitride nanopores.
[0047] 1. Protein detection system based on solid-state nanopores
[0048] The protein detection system based on solid-state nanopore of the present invention comprises a detection device and a detection reagent;
[0049] like Figure 2 As shown, the detection device includes an electrode cavity, electrodes and a data acquisition system;
[0050] The electrode cavity includes a cis cavity 1 and a trans cavity 2, a silicon nitride chip 3 is arranged between the cis cavity 1 and the trans cavity 2, and the silicon nitride chip 3 is connected through a nanopore 4 on the silicon nitride chip 3. The diameter of the nanopore 4 is 8-30nm, so that the sample to be tested and the antibody can enter the trans cavity 2 from the cis cavity 1 through the nanopore 4;
[0051] Ag / AgCl electrodes 5 were respectively connected to the cis cavity 1 and the trans cavity 2, and connected to an external power supply to form a closed loop, thereby constructing a detection loop of a data acquisition system 6. The data acquisition system 6 was a patch clamp amplifier (Axopatch 200B, Molecular Devices, California, US), which collected data at a sampling frequency of 500 kHz and performed low-pass filtering at 10 kHz.
[0052] The detection reagents include a cis-cavity buffer solution, a trans-cavity buffer solution, a retinol binding protein 4 (RBP4 protein) capture antibody and an RBP4 protein detection antibody.
[0053] The components of the cis-cavity buffer solution are 3M KCl, 30mM Tris and 3mM EDTA, the solvent is water, and the pH is 8; the components of the trans-cavity buffer solution are 1M KCl, 10mM Tris and 1mM EDTA, the solvent is water, and the pH is 8.
[0054] 2. Detection method
[0055] (1) Clean the cis cavity and trans cavity of the solid-state nanopore detection system with pure water, add 50 μL of cis cavity buffer solution to the cis cavity to rinse the cis cavity; add 50 μL of trans cavity buffer solution to the trans cavity to rinse the trans cavity. Then add 50 μL of cis cavity buffer solution to the cis cavity and 50 μL of trans cavity buffer solution to the trans cavity, adjust the power supply to apply 200 mV voltage, and measure the blank signal through the data acquisition system.
[0056] In the protein detection system of the solid-state nanopore of the present invention, the ion concentration difference between the cis-cavity buffer solution in the cis-cavity and the trans-cavity buffer solution in the trans-cavity generates a transmembrane osmotic pressure gradient. The transmembrane osmotic pressure gradient helps to overcome the steric hindrance of the antigen-antibody complex through the narrow nanopore channel, promotes its efficient and rapid passage through the nanopore, thereby achieving sensitive and accurate single-molecule level detection.
[0057] (2) Incubate the RBP4 protein capture antibody and the RBP4 protein detection antibody in a centrifuge tube at 37°C for 30 min, and dilute the test sample, the RBP4 protein capture antibody and the RBP4 protein detection antibody with the cis-chamber buffer solution to obtain the diluted test sample, the RBP4 protein capture antibody (5.5 nM) and the RBP4 protein detection antibody (5.5 nM).
[0058] The diluted test sample, RBP4 protein capture antibody (5.5 nM) and RBP4 protein detection antibody (5.5 nM) were added to the trans cavity, and the power supply was adjusted to apply a voltage of 200 mV to drive the test sample and antibody through the nanopore. Data was collected through the data acquisition system, and the blocking pulse signal generated when the test sample and antibody passed through the nanopore was recorded using the pCLAMP software.
[0059] Example 2 Preparation of RBP4 protein capture antibody and RBP4 protein detection antibody
[0060] The mRNA9 fragment of RBP4 protein (NCBI accession number NM_001323518.2) was cloned into the pCOLDⅡ vector to construct the pCOLDⅡ-RBP4 plasmid. The plasmid was transformed into Escherichia coli BL21 (DE3), and the recombinant RBP4 protein was induced to express with 1.0mM IPTG (isopropyl-β-D-thiogalactoside) and 2.0mg / mL L-arabinose. The recombinant protein was purified with Ni-NTA agarose. The molecular weight of RBP4 protein was about 24.5kD, and the purity of the purified RBP4 protein was above 90%. The purified recombinant RBP4 protein was mixed with an equal volume of Freund's complete or incomplete adjuvant and fully emulsified. The above emulsified mixture was injected subcutaneously and intradermally into 3 healthy Balb / c mice. Immunization was performed 3 times every 2 weeks, and 50μg RBP4 protein was injected subcutaneously and intradermally into each mouse for each immunization. The mouse serum titer was determined by indirect ELISA (enzyme-linked immunosorbent assay), and the mouse with the highest titer was selected for perforation immunization. Five days later, the immunized mouse with the highest anti-RBP4 protein antibody titer in the serum was sacrificed. Mouse spleen cells were immediately collected and fused with mouse osteoma cells SP2 / 0 using 50% (v / v) polyethylene glycol in a 50 ml centrifuge tube. The cells were distributed in a 96-well microplate (10 cells per plate). 7Before the fusion of spleen cells, HAT medium (selective medium containing hypoxanthine, aminopterin and thymidine) was added for hybridoma screening. 10 days after cell fusion, the supernatant of 96-well plates was collected and the antibody secretion was detected by ELISA. The positive well hybridoma was cloned by limiting dilution method. After 3 rounds of subcloning, the hybridoma cell line with the highest absorbance at 450nm was screened to obtain anti-RBP4 monoclonal antibody. The monoclonal antibody was purified and enriched with protein G resin, and verified by SDS-PAGE (polyacrylamide gel electrophoresis) staining, and finally 6 purified anti-RBP4 monoclonal antibodies (5-A2, 5-C4, 5-E2, 5-E4, 8-B1 and 8-B5) were determined. The 6 purified anti-RBP4 monoclonal antibodies were paired according to the double antibody sandwich principle. The results showed that the OD value of the 5-A2 and 8-B5 antibody pairs was the highest, and the best antibody pair was 5-A2 and 8-B5. 5-A2 antibody was used as RBP4 protein detection antibody, denoted as Ab1; 8-B5 antibody was used as RBP4 protein capture antibody, named Ab2 (Guo W, Yu Z, Li T, Lu L, Lin H, Liao Y, Zheng Y, Liu Y, Glaskovich MA, Barysavets DS, Chen J, Zan J, Lu J. Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of retinol-binding protein 4 in urine. Microchim Acta. 2024; 191(311): 1-13. https: / / doi.org / 10.1007 / s00604-024-06381-6.).
[0061] Example 3 Effect of different detection parameters on detection effect
[0062] 1. Experimental Methods
[0063] 1. Effect of the ion concentration difference between the cis-cavity buffer solution and the trans-cavity buffer solution on the detection effect The pore size of the silicon nitride nanopore is set to 10 nm, and two ion concentration differences are set:
[0064] (1) Plasma concentration system: The components of the buffer solution of the cis cavity and the trans cavity are: 1M KCl, 10mM Tris and 1mM EDTA, the solvent is water, pH = 8;
[0065] (2) Asymmetric ion concentration system: The components of the buffer solution of the cis cavity are: 3M KCl, 30mM Tris and 3mM EDTA, the solvent is water, and the pH is 8; the components of the buffer solution of the trans cavity are: 1M KCl, 10mM Tris and 1mM EDTA, the solvent is water, and the pH is 8.
[0066] Other conditions were the same as those in Example 1, and the RBP4 protein capture antibody (5.5 nM) was detected.
[0067] 2. Effect of different applied voltages on detection results
[0068] The applied voltages were set to 100, 200, 300, 400 and 500 mV, respectively. The cis-cavity and trans-cavity buffer solutions were set to the asymmetric ion concentration system in this embodiment. Other conditions were the same as in Example 1, and the monoclonal antibody was detected.
[0069] 3. Use RBP4 protein capture antibody and monoclonal antibody (RBP4 protein sample to be tested + RBP4 protein capture antibody) for detection respectively
[0070] The pore size of the silicon nitride nanopore is set to 10 nm, the cis-cavity and trans-cavity buffer solutions are set to the asymmetric ion concentration system in this embodiment, the applied voltage is set to 200 mV, and the other conditions are the same as in Example 1, and the RBP4 protein capture antibody and monoclonal antibody (RBP4 protein sample to be tested + RBP4 protein capture antibody) are detected. The concentrations of the RBP4 protein capture antibody and the RBP4 protein detection antibody are both 5.5 nM, and the antibodies are diluted with the cis-cavity buffer solution to reach the corresponding concentration.
[0071] 4. Detection using double antibody sandwich (RBP4 protein + RBP4 protein capture antibody + RBP4 protein detection antibody)
[0072] Since the signal differentiation between the RBP4 protein capture antibody and the monoclonal antibody (RBP4 protein sample to be tested + RBP4 protein capture antibody) is not obvious, it is necessary to bind another antibody to the different binding site of the antigen to form an antibody-antigen-antibody RBP4 double antibody sandwich structure to further amplify the volume of the analyte.
[0073] The pore size of the silicon nitride nanopore was set to 28 nm, the cis-cavity and trans-cavity buffer solutions were set to the asymmetric ion concentration system of this embodiment, the applied voltage was set to 200 mV, and the following were detected: (1) blank solution; (2) RBP4 protein capture antibody; (3) RBP4 protein detection antibody + RBP4 protein capture antibody; (4) monoclonal antibody (RBP4 protein + RBP4 protein capture antibody); (5) double antibody sandwich (RBP4 protein + RBP4 protein capture antibody + RBP4 protein detection antibody)
[0074] The concentrations of RBP4 protein capture antibody and RBP4 protein detection antibody were both 5.5 nM.
[0075] 2. Experimental Results
[0076] 1. Asymmetric ion concentration system ( Figure 3 B) signal peak ratio plasma concentration system ( Figure 3 A) significantly increased and the number increased, indicating that the osmotic pressure caused by the ion concentration difference drove the protein through the nanopore.
[0077] 2. The voltage is 100 and 200mV, the signal generated is stable, and it is not easy to block the hole. When the voltage exceeds 300mV, the protein will pass through the hole faster and it is easy to block the hole. Therefore, 200mV is selected as the voltage applied to the hole, where the signal is more obvious and it is not easy to block the hole. Figure 4 ).
[0078] 3. RBP4 protein capture antibody ( Figure 5 A and Figure 5 C) and monoclonal antibody (RBP4 protein + RBP4 protein capture antibody) ( Figure 5 B and Figure 5 D) signal differentiation is not obvious.
[0079] 4. If Figure 6 As shown, when detecting the double antibody sandwich (RBP4 protein + RBP4 protein capture antibody + RBP4 protein detection antibody), there is an obvious detection signal compared to detecting other antigens and antibodies.
[0080] Example 4 Sensitivity Evaluation
[0081] 1. Experimental Methods
[0082] 1. Sensitivity: The concentrations of RBP4 protein capture antibody and RBP4 protein detection antibody were set to 5.5 nM, and other conditions were the same as in Example 1. RBP4 protein solutions of different concentrations (0.055 pM, 0.275 pM, 0.55 pM, 2.75 pM and 5.5 pM) were tested to evaluate the sensitivity of the method. The number of occurrences of the signal was counted by the Transalyzer toolbox in MATLAB, and the curve was fitted using Origin software.
[0083] 2. Specificity: The concentrations of RBP4 protein capture antibody and RBP4 protein detection antibody were set to 5.5 nM, and other conditions were the same as in Example 1. Different antigens were detected to evaluate the specificity of the method.
[0084] The antigens are RBP4, SAA1 (serum amyloid A1), NGAL (neutrophil gelatinase-associated lipocalin) and S100β (central nervous system-specific protein), all with a concentration of 4.1 pM, and are diluted and prepared with cis-cavity buffer solution.
[0085] When counting the number of signals in the Transalyzer toolbox in MATLAB, the baseline is set to 55 nA, the minimum duration of the peak is set to 0.01 ms, the maximum duration is set to 200 ms, and the current detection threshold is set to 2.413 nA.
[0086] 2. Experimental Results
[0087] 1. If Figure 7 and Figure 8 As shown, the detection limit of the method in Example 1 is 55 fM.
[0088] 2. If Fig. 9 As shown, the method of Example 1 has good specificity, and the detection results of SAA1, NGAL and S100β have no significant difference with those of the blank group, which effectively avoids the influence of other interfering proteins on the detection results.
[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.
Claims
1. A protein detection system based on solid-state nanopores, characterized in that: The protein detection system comprises a detection device and a detection reagent; The detection device comprises an electrode cavity, electrodes and a data acquisition system; The electrode cavity comprises a cis cavity and a trans cavity, a silicon nitride chip is arranged between the cis cavity and the trans cavity, and the cis cavity and the trans cavity are connected through nanopores on the silicon nitride chip; Electrodes are provided in the cis cavity and the trans cavity and are connected to a data acquisition system; The detection reagent contains a double-antibody sandwich paired retinol binding protein 4 monoclonal antibody, which is obtained by immunizing mice with retinol binding protein 4 as an immunogen.
2. The protein detection system according to claim 1, characterized in that The pore diameter of the nanopore is 8-30 nm.
3. The protein detection system according to claim 1, characterized in that: The system also contains a buffer solution, wherein the buffer solution contains KCl.
4. The protein detection system according to claim 3, characterized in that: The concentration of KCl is 1-3M.
5. The protein detection system according to claim 4, characterized in that: The cis cavity contains a cis cavity buffer solution, and the trans cavity contains a trans cavity buffer solution. The concentration of KCl in the trans cavity buffer solution is lower than the concentration of KCl in the cis cavity buffer solution.
6. The protein detection system according to claim 5, characterized in that: The concentration of KCl in the cis-cavity buffer solution is 3M, and the concentration of KCl in the trans-cavity buffer solution is 1M.
7. The protein detection system according to claim 3, characterized in that: The cis-cavity buffer solution and the trans-cavity buffer solution also contain Tris and EDTA.
8. The protein detection system according to claim 1, characterized in that: The electrode is an Ag / AgCl electrode.
9. Use of the protein detection system according to any one of claims 1 to 8 in the preparation of a product for detecting retinol binding protein 4.