An electrochemical sensor for detecting melanoma marker S100B protein and a preparation method thereof

Abstract

The application belongs to the technical field of electrochemical sensors, and discloses an electrochemical sensor for detecting melanoma marker S100B protein and a preparation method thereof. A capture probe is fixed to the surface of a gold electrode through gold-sulfur self-assembly, and after being blocked by MCH, a gold electrode modified with the capture probe is obtained. Then, the S100B protein is modified as a detection object through specific binding between the capture probe and the S100B protein. Finally, the signal amplification probe is specifically combined with the S100B protein through self-assembly in water due to hydrophilic and hydrophobic effects, so that the electrochemical biosensor for detecting the S100B protein is obtained. The application constructs an electrochemical biosensor for detecting the S100B protein based on the specific recognition between the sequence KRLRRSAHARKETEFLRLKRTRLGLE in the polypeptide and the S100B protein in a 2:1 mode. The performance and characteristics of the sensor are characterized by CV and SWV, and the change of the electrochemical signal is accurately captured. The sensor has the characteristics of high sensitivity, strong specificity, fast analysis speed and the like.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, specifically relating to an electrochemical sensor for detecting the melanoma marker S100B protein and its preparation method. Background Technology

[0002] S100B protein is a 21kDa dimer protein. Among melanoma biomarkers, lactate dehydrogenase is the most prominent biomarker used clinically, and S100B protein is even more effective than lactate dehydrogenase. Therefore, S100B protein, as a novel biomarker, plays a crucial role in the early detection and prevention of melanoma. Currently, the main methods for detecting S100B protein are enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry using antibodies. ELISA has poor specificity and sensitivity, is cumbersome, and has a long detection time; immunohistochemistry using antibodies has poor sensitivity, requires high-quality antigens, and makes it difficult to obtain various commercially available antigens and antibodies. Here, we suggest using peptides as a simplified equivalent of antibodies and measuring them electrochemically rather than spectroscopically. On the one hand, electrochemical techniques are known to be simple, rapid, and cost-effective. On the other hand, several advantages of peptides, such as synthetic accessibility and ease of modification, make them a compelling choice for targeting ligands in protein assays. Currently, there are no reports on methods combining peptides and electrochemistry. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide an electrochemical sensor for detecting the melanoma marker S100B protein and its preparation method. This sensor enables real-time detection of S100B protein, exhibiting high sensitivity, strong specificity, and is simple and inexpensive. To achieve the above objective, this invention employs the following technical means:

[0004] This invention provides a method for preparing an electrochemical biosensor for detecting the melanoma marker S100B protein, comprising the following steps:

[0005] (1) Preparation of S100B protein solution:

[0006] S100B protein lyophilized powder was dissolved in a Tris-HCl solution containing tris-(2-chloroethyl) phosphate (TCEP) to obtain an S100B protein solution.

[0007] (2) Preparation of the capture probe solution:

[0008] The lyophilized capture probe peptide A powder was dissolved in deionized water containing TCEP to obtain the capture probe solution.

[0009] (3) Preparation of signal amplification probe solution:

[0010] The signal amplification probe peptide B lyophilized powder was dissolved in deionized water and then self-assembled to obtain the signal amplification probe solution.

[0011] (4) Assembly of electrochemical biosensors:

[0012] Step 4.1: The pretreated gold electrode is placed in the capture probe solution obtained in step (2) for self-assembly modification, and then washed with ethanol and water.

[0013] Step 4.2: Place the modified and cleaned gold electrode from step 4.1 into a 6-mercapto-1-hexanol MCH solution to seal the blank sites on the electrode surface that are not occupied by the capture probe. After sealing, rinse the electrode surface with ethanol and deionized water.

[0014] Step 4.3: Place the gold electrode processed in step 4.2 into the S100B protein solution obtained in step (1). The gold electrode surface modified with S100B protein is obtained by the specific binding of the capture probe modified on the gold electrode surface with the S100B protein.

[0015] Step 4.4: Place the gold electrode modified with S100B protein in step 4.3 into the signal amplification probe solution obtained in step (3). Through the specific binding between S100B protein and signal amplification probe, a "sandwich" structure of polypeptide-protein-polypeptide is formed, which is the electrochemical biosensor for detecting S100B protein.

[0016] Furthermore,

[0017] In step (1), the concentration of TCEP in the Tris-HCl solution containing tris(2-chloroethyl) phosphate (TCEP) is 1 mM; the concentration of Tris-HCl is 20 mM; and the concentration of S100B protein solution is 0.2 nM-12.8 nM.

[0018] In step (2), the concentration of TCEP in the deionized water containing TCEP is 1 mM; the concentration of the capture probe solution is 30 μM; and the amino acid sequence of polypeptide A is: CP4-KRLRRSAHARKETEFLRLKRTRLGLE.

[0019] In step (3), the concentration of the signal amplification probe solution is 30 μM; the amino acid sequence of polypeptide B is: C 16 -GGG-KRLRRSAHARKETEFLRLKRTRLGLE-Fc.

[0020] In step 4.1, the conditions for self-assembly modification are: reaction in a dark environment at 4 degrees Celsius for 12-16 hours.

[0021] In step 4.2, the concentration of the MCH solution is 1 mM, and the blocking time is 0.5-1 h.

[0022] In step 4.3, the modification time of S100B protein is at least 2.5 hours.

[0023] In step 4.4, the modification time for the signal amplification probe is 2 hours.

[0024] In step 4.1, the pretreatment steps for the gold electrode include: immersing the gold electrode in a piranha solution to remove organic matter, rinsing it with deionized water, and polishing the electrode surface; placing the polished gold electrode in a sulfuric acid solution and scanning CV 20 times with a voltage ranging from -0.4mV to 1.6mV; then placing the gold electrode in a potassium ferricyanide / potassium ferrocyanide solution and scanning CV with a voltage ranging from -0.2V to 0.6V. The pretreated gold electrode in the potassium ferricyanide / potassium ferrocyanide solution has a ΔEP ≤ 90mV.

[0025] In addition, the present invention also provides a method for detecting the melanoma marker S100B protein, the steps of which are as follows: using an electrochemical biosensor for detecting S100B protein as the working electrode, using an Ag / AgCl reference electrode, and using a platinum wire as the counter electrode; placing the working electrode in an electrolytic cup containing PBS electrolyte, and performing electrochemical testing using square wave voltammetry, and characterizing the S100B protein concentration by the electrochemical signal state.

[0026] Furthermore, the parameters of the square wave voltammetry are as follows: scanning voltage from 0.2V to 0.8V, potential increment of 5mV, and amplitude of 25mV; NaClO4 is also added to the PBS electrolyte, and the concentration of NaClO4 is 0.1M.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] In this invention, the capture probe we designed is a polypeptide. The specific amino acid sequence of the polypeptide is CP4-KRLRRSAHARKETEFLRLKRTRLGLE. The polypeptide sequence we designed not only has the recognition sequence KRLRRSAHARKETEFLRLKRTRLGLE that specifically binds to S100B, but also the CP4 sequence that binds to the gold electrode. This is because the CP4 sequence on the capture probe has a thiol group, which can be used to fix the capture probe on the surface of the gold electrode through Au-S self-assembly.

[0029] In this invention, we propose a novel signal amplification method based on in-situ peptide self-assembly for the sensitive detection of S100B protein. Interestingly, the peptide plays multiple roles simultaneously in our biosensing platform. Specifically, the designed peptide possesses both a recognition region that binds to S100B and a self-assembly region, forming peptide nanospheres under mild conditions to increase the loading of the signal tag Fc. These two reactions occur concurrently, simplifying the operation and improving performance. The signal amplification probe C... 16 The specific binding ability of -GGG-KRLRRSAHARKETEFLRLKRTRLGLE-FC is not affected by self-assembly because C 16 The tail portion is packaged within a hydrophobic core, and the recognition fragment KRLRRSAHARKETEFLRLKRTRLGLE and the Fc tag are exposed on the self-assembled nanospheres. Since controllable peptide self-assembly can increase the loading of electroactive labels, this multifunctional peptide provides a general approach for preparing electrochemical biosensors with ideal sensitivity. The performance and characteristics of the sensor were characterized using CV and SWV, accurately capturing changes in electrochemical signals. It exhibits advantages of high specificity and high sensitivity. The electrochemical sensor provided by this invention has a certain promoting effect on the early detection of melanoma. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of an electrochemical sensor.

[0031] Figure 2 Cyclic voltammetry curves characterizing the electrochemical sensor construction process;

[0032] Figure 3 Square wave voltammetry plots for different concentrations of S100B protein;

[0033] Figure 4 The graph shows the linear relationship between SWV signal value and S100B protein concentration.

[0034] Figure 5 The graph shows the selectivity results of the electrochemical biosensor in detecting S100B protein. Detailed Implementation

[0035] This invention discloses an electrochemical sensor for detecting the melanoma marker S100B protein and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0036] Unless otherwise specified, the methods, equipment, and materials used in the following implementation examples are all conventional methods, equipment, and materials in the field and are all available on the market.

[0037] Example 1: Fabrication of an electrochemical sensor

[0038] (1) Pretreatment of gold electrodes:

[0039] The gold electrode was immersed in a piranha solution (a mixture of concentrated sulfuric acid and hydrogen peroxide in a 7:3 volume ratio) for 30 minutes. The electrode was then removed and rinsed thoroughly with deionized water to remove any remaining piranha solution. The electrode surface was polished to a mirror finish using white nylon and chamois polishing cloths with 0.3 μm and 0.05 μm alumina polishing powders, respectively. Subsequently, the electrode was immersed in a 0.5 M sulfuric acid solution and subjected to CV scanning at a voltage range of -0.4 to 1.6 mV for 20 cycles, ensuring the peak value remained below 89 mV. The electrode was then immersed in a 5 mM potassium ferricyanide / potassium ferrocyanide solution and subjected to CV scanning at a voltage range of -0.2 to 0.6 V, ensuring ΔEP ≤ 90 mV; this completed the pretreatment of the gold electrode.

[0040] (2) Modification of gold electrodes:

[0041] The pretreated gold electrode was placed in 300 μL of 1 mM tris(2-chloroethyl) phosphate (TCEP) and 30 μM capture probe in a deionized aqueous solution and reacted at 4 °C in the dark for at least 12 hours. The remaining solution was then washed away with ethanol and water.

[0042] The electrode was then immersed in an ethanol solution of 1 mM MCH to quench the remaining active sites on the gold electrode. The reaction was carried out in a fume hood at room temperature for 1 h.

[0043] After sealing, the gold electrode was inserted into 300 μL of S100B protein test solution with concentrations of 0.2 nM, 0.4 nM, 0.8 nM, 1.6 nM, 3.2 nM, 6.4 nM and 12.8 nM, and reacted at room temperature for at least 2.5 h.

[0044] Finally, the gold electrode modified with S100B protein was inserted into 300 μL of a 30 μM signal amplification probe in a deionized aqueous solution and modified for at least 2 hours, thus completing the entire modification process of the gold electrode in the electrochemical sensor.

[0045] Figure 1 This is a schematic diagram of an electrochemical sensor; such as... Figure 1 As can be seen, by sequentially modifying the pretreated gold electrode surface with a capture probe, MCH, S100B protein, and a signal amplification probe, a peptide-protein-peptide "sandwich" structure is formed, which is the electrochemical biosensor for detecting S100B protein. The signal amplification principle in this invention is achieved by the signal amplification probe in the final modification step self-assembling in water due to hydrophilic-hydrophobic interactions, thereby increasing the loading of signal molecules and achieving signal amplification, thus enabling ultrasensitive detection of S100B protein.

[0046] Figure 2 This is a cyclic voltammetry curve characterizing the electrochemical sensor construction process. The parameters for cyclic voltammetry are: scan voltage from -0.2V to 0.6V, scan rate 50mV; the cyclic voltammetry measurements are performed in a 5mM K3[Fe(CN)6] / K4[Fe(CN)6]SWV solution.

[0047] like Figure 2 As shown, the bare gold electrode exhibits the highest current, with a voltage difference ΔV between the redox peaks of approximately 90 mV. After modification with the trapping probe, the voltage difference significantly increases while the current decreases. This indicates that the trapping probe lacks conductivity, hindering electron transfer. Subsequent modification with MCH and S100B proteins also resulted in varying degrees of current reduction, demonstrating successful modification of the electrodes by MCH and S100B proteins. However, when the signal amplification probe specifically adsorbs onto the electrode, the current rebounds, possibly due to the electrostatic interaction between ferrocene molecules and potassium ferricyanide promoting electron transfer. Figure 2 This primarily demonstrates that the electrochemical sensor we constructed was modified layer by layer, and indirectly proves the formation of the "sandwich" structure of peptide-protein-peptide.

[0048] Example 2: Electrochemical sensor detection of S100B protein

[0049] In this embodiment, a three-electrode system was used to test the S100B protein:

[0050] The electrochemical sensor prepared in Example 1 was used as the working electrode, with an Ag / AgCl reference electrode and a platinum wire as the counter electrode. The working electrode was placed in a glass electrolytic beaker (CHI222) containing 0.1M NaClO4 in PBS electrolyte, and different concentrations of S100B protein were detected according to experimental requirements. The sensor's performance and characteristics were characterized using square wave voltammetry (SWV). The SWV parameters were: scan voltage from 0.2V to 0.8V, potential increment of 5mV, and amplitude of 25mV. All electrochemical tests were performed on a Chenhua electrochemical workstation (CHI660E). SWV was conducted in 0.1M NaClO4 in PBS (pH = 7.4).

[0051] According to the test results, the SWV signal value I p The S100B protein showed a linear relationship in the range of 0.2 nM to 12.8 nM. p = 1.172 + 1.042 log C(I) p Unit: μA C Unit: nM / LR 2 =0.995), the SWV signal values ​​corresponding to different concentrations of S100B protein are as follows: Figure 3 As shown in the figure, the linear relationship between SWV signal value and S100B protein concentration is as follows: Figure 4 As shown.

[0052] The construction process of the electrochemical sensor was monitored by cyclic voltammetry (CV), and the concentration of S100B protein was quantitatively detected by square wave voltammetry (SWV). As shown in Example 2, the SWV signal value and the S100B protein concentration have a linear relationship in the concentration range of 0.2 nM to 12.8 nM. Therefore, according to the detection limit calculation formula 3σ / S, the detection limit of the electrochemical biosensor for detecting S100B protein prepared in Example 2 can reach 0.027 nM, which shows that the electrochemical biosensor of the present invention has high sensitivity.

[0053] Example 3: Specificity Test

[0054] Using the electrochemical biosensor for detecting S100B protein prepared in Example 1 as the working electrode, the experimental conditions were the same as in Example 2 above. 0.2 nM S100B protein and 20 nM common interfering proteins: BSA, Thrombin, CEA, and lysozyme were detected. The results are as follows: Figure 5As shown in the figure. The results indicate that, except for the target protein S100B, the SWV signal values ​​of other interfering proteins showed very little change after interacting with the electrochemical biosensor. This demonstrates that 100-fold increases in common interfering proteins do not affect detection, and the biosensor of this invention exhibits good specificity. This is mainly because, after the biosensor interacts with the S100B protein, the S100B protein specifically recognizes the polypeptide sequence KRLRRSAHARKETEFLRLKRTRLGLE, causing the signal amplification probe containing the signal molecule Fc to adsorb onto the electrode surface, thereby increasing the SWV signal. Based on this, high specificity and high sensitivity detection of the S100B protein are achieved.

[0055] This invention expands the detection method for the melanoma biomarker S100B protein. Compared to ThT-based fluorescence analysis, this invention achieves simple, rapid, and low-cost monitoring of S100B protein through electrochemical technology, providing a reference for medical researchers to develop early detection methods for melanoma. This invention modifies a pretreated gold electrode with a capture probe, primarily utilizing the thiol group in the CP4 sequence of the capture probe, which allows for Au-S self-assembly to immobilize the capture probe on the gold electrode surface. Subsequently, 6-mercapto-1-hexanol (MCH) is used to block unoccupied sites on the electrode surface. Then, S100B protein and a signal amplification probe are modified separately. Based primarily on the 2:1 specific recognition between the peptide (KRLRRSAHARKETEFLRLKRTRLGLE) and S100B protein, an electrochemical sensor for detecting the melanoma biomarker S100B protein with a peptide-protein-peptide "sandwich" structure is constructed. The change in SWV signal value at the sensor interface after the interaction between the sensor and the S100B protein test solution is detected using Fc as a probe. The S100B protein concentration showed a good linear relationship with the SWV signal value in the range of 0.2 nM to 12.8 nM, with a detection limit of 0.027 nM.

[0056] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical sensor for detecting the melanoma marker S100B protein, characterized in that, Includes the following steps: (1) Preparation of S100B protein solution: S100B protein lyophilized powder was dissolved in a Tris-HCl solution containing tris-(2-chloroethyl) phosphate (TCEP) to obtain an S100B protein solution. (2) Preparation of the capture probe solution: The lyophilized capture probe peptide A powder was dissolved in deionized water containing TCEP to obtain the capture probe solution. In the deionized water containing TCEP, the concentration of TCEP was 1 mM; the concentration of the capture probe solution was 30 μM; and the amino acid sequence of polypeptide A was: CP4-KRLRRSAHARKETEFLRLKRTRLGLE. (3) Preparation of signal amplification probe solution: The signal amplification probe peptide B lyophilized powder was dissolved in deionized water and then self-assembled to obtain the signal amplification probe solution. The concentration of the signal amplification probe solution was 30 μM; the amino acid sequence of polypeptide B was: C 16 -GGG-KRLRRSAHARKETEFLRLKRTRLGLE-Fc; (4) Assembly of electrochemical biosensors: Step 4.1: The pretreated gold electrode is placed in the capture probe solution obtained in step (2) for self-assembly modification, and then washed with ethanol and water. Step 4.2: Place the modified and cleaned gold electrode from step 4.1 into a 6-mercapto-1-hexanol MCH solution to seal the blank sites on the electrode surface that are not occupied by the capture probe. After sealing, rinse the electrode surface with ethanol and deionized water. Step 4.3: Place the gold electrode processed in step 4.2 into the S100B protein solution obtained in step (1). The gold electrode surface modified with S100B protein is obtained by the specific binding of the capture probe modified on the gold electrode surface with the S100B protein. Step 4.4: Place the gold electrode modified with S100B protein in step 4.3 into the signal amplification probe solution obtained in step (3). Through the specific binding between S100B protein and signal amplification probe, a "sandwich" structure of polypeptide-protein-polypeptide is formed, which is the electrochemical biosensor for detecting S100B protein.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of TCEP in the Tris-HCl solution containing tris(2-chloroethyl) phosphate (TCEP) is 1 mM; the concentration of Tris-HCl is 20 mM; and the concentration of S100B protein solution is 0.2 nM-12.8 nM.

3. The preparation method according to claim 1, characterized in that: In step 4.1, the conditions for self-assembly modification are: reaction in a dark environment at 4 degrees Celsius for 12-16 hours.

4. The preparation method according to claim 1, characterized in that: In step 4.2, the concentration of the MCH solution is 1 mM, and the blocking time is 0.5-1 h.

5. The preparation method according to claim 1, characterized in that: In step 4.3, the modification time of S100B protein is at least 2.5 h; in step 4.4, the modification time of the signal amplification probe is 2 h.

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