A biosensor for Cyt c detection and its construction method and application

Abstract

The present invention discloses a biosensor for cytochrome c (Cyt c) detection, its construction method and application, which comprises dropping a solution comprising Cu3SnS4, chitosan and acetic acid onto an ITO surface, and then sequentially adding a coupling agent, anti-Cyt c, a blocker and Cyt c for incubation. The method is simple to operate, and the prepared photoelectrochemical immunosensor has the advantages of fast response, high sensitivity, good reproducibility, high specificity and good storage stability. When applied to the detection of Cyt c, it has a low detection limit and a wide linear range.

Description

Technical Field

[0001] The present invention relates to a biosensor for Cyt c detection, a construction method and application thereof, and in particular to a biosensor for Cyt c detection based on Cu3SnS4 nanomaterials, a construction method and application thereof, belonging to the technical field of novel functional materials and biosensor detection. Background Art

[0002] Human cytochrome c (Cyt c) is a water-soluble metalloprotein that plays a crucial role in electron transport in the mitochondrial respiratory chain and in apoptosis. Current research has established that Cyt c can serve as a biomarker for excessive or insufficient apoptosis in human cells, potentially aiding in the early prevention of cancer. Therefore, accurately measuring Cyt c levels is of great significance. Numerous methods have been developed for Cyt c detection, including enzyme-linked immunosorbent assay (ELISA), Western blot analysis, high-performance liquid chromatography (HPLC), spectrophotometry, and flow cytometry. While existing methods can address Cyt c content testing to some extent—for example, Zhang et al. developed a label-free nitrogen-doped carbon dot nanosensor for quantitatively measuring Cyt c release during apoptosis using fluorescence-activated cell imaging—Tan et al. created a molecularly imprinted polymer sensor by hybridizing an aptamer molecularly imprinted polymer with quantum dots. However, these methods suffer from cumbersome detection procedures and insufficient sensitivity.

[0003] As a simple, rapid, and sensitive new technology, photoelectrochemical immunosensors have attracted widespread attention and application in analytical detection technology. Photoelectrochemical (PEC) immunosensors effectively reduce background interference by separating the excitation signal (light) and the response signal (current), resulting in ultra-high sensitivity and excellent stability. The easily integrated conversion of photoelectric signals makes PEC immunosensors easy to operate and miniaturize, providing a foundation for intelligent, portable detection. Summary of the Invention

[0004] In view of the defects of the prior art, the first object of the present invention is to provide a biosensor for Cyt c detection, which has good reproducibility, high specificity and good storage stability.

[0005] A second object of the present invention is to provide a method for preparing a biosensor for Cyt c detection, which has simple operation and mild reaction conditions.

[0006] The third object of the present invention is to provide an application of a biosensor for Cyt c detection, which is applied to the detection of Cyt c and has a low detection limit and a wide linear range.

[0007] In order to achieve the above technical objectives, the present invention provides a method for constructing a biosensor for Cyt c detection, which comprises adding a solution containing Cu3SnS4, chitosan and acetic acid to an ITO surface, and then adding a coupling agent, anti-Cyt c and a blocking agent in sequence for incubation.

[0008] The present invention makes full use of Cu3SnS4 with a nanoflower structure as a bimetallic sulfide, which has the characteristics of excellent photostability, large specific surface area, narrow band gap (1.2-1.67eV) and high absorption coefficient, so that it has good visible light capture ability and can effectively improve the photoelectric conversion efficiency. Cu3SnS4 is used as a photoactive material in a photoelectrochemical immunosensor for detecting biological molecules, which has the advantages of fast response, high sensitivity and strong specificity. By modifying a mixture of chitosan (CS) and acetic acid, amino functional groups are formed on the electrode, and a coupling agent is added dropwise to provide aldehyde groups that are conducive to anchoring anti-Cyt c. Then, a blocker is added to block nonspecific active sites, thereby improving the detection accuracy and specificity of the sensor. After incubation, a photoelectrochemical sensor that can be used to detect Cyt c is obtained.

[0009] As a preferred solution, the coupling agent is glutaraldehyde. The coupling agent glutaraldehyde used in the present invention is connected to the amino groups on chitosan and anti-Cyt c through two aldehyde groups, thereby fixing the probe anti-Cyt c to the electrode.

[0010] As a preferred embodiment, the Cyt c and anti-Cyt c used in the present invention are prepared by the following method: an expression vector containing the Cyt c or anti-Cyt c gene is transformed into Escherichia coli BL21 (DE3) cells and selected with 100 g mL⁻¹ of ampicillin. The Cyt c and anti-Cyt c culture media are incubated at 37°C in a shaker at 2200 rpm for 24 hours. For anti-Cyt c, an appropriate amount of isopropyl-β-D-thiogalactopyranoside (IPTG) is added 12 hours after the incubation to promote protein folding. After 24 hours, the cells are collected by centrifugation and pulverized using an ultrasonic disruptor to obtain the target protein. The resulting target protein is further purified and separated using a molecular sieve gel column and an ion exchange column. Cyt c and anti-Cyt c samples meeting A₁₆ / A₂₀₀ values ​​of >4.0 and >3.0 are collected for future use.

[0011] As a preferred embodiment, the blocking agent is bovine serum albumin (BSA). The present invention uses BSA to block nonspecific active sites, ensuring the specificity of the sensor. The concentration of BSA is 1 wt%, and the amount added is 100-120% of the volume of the anti-Cyt c.

[0012] As a preferred solution, the volume ratio of the coupling agent, Cu3SnS4 and anti-Cyt c is 1:(5-6):1.

[0013] As a preferred solution, the concentration of the coupling agent is 0.5-1 wt%.

[0014] As a preferred embodiment, the Cu3SnS4 concentration is 1-5 mg / mL. The concentration of Cu3SnS4 added in the present invention has a direct impact on the performance of the sensor. Within the selected range, as the Cu3SnS4 concentration increases, the sensor photocurrent first increases and then decreases. A further preferred Cu3SnS4 concentration is 2-4 mg / mL.

[0015] As a preferred solution, the concentration of the anti-Cyt c is 80-100 nM.

[0016] The biosensor constructed by the present invention can be used to detect Cyt c at a concentration of 1 fM to 1000 nM. Within the preferred concentration range, the sensor has good performance.

[0017] As a preferred solution, the incubation time is 0.5 to 1 hour. Too low an incubation time can have a significant impact, resulting in incomplete material binding and reduced detection performance. Further increases in incubation time, on the one hand, do not further improve detection performance, and on the other hand, result in a waste of resources.

[0018] As a preferred solution, the Cu3SnS4 has a nanoflower structure.

[0019] As a preferred solution, the Cu3SnS4 is obtained by the following preparation method: tin salt, alkali and copper salt are mixed and reacted in an aqueous solution to obtain a precursor; the precursor is mixed with a sulfur source and a chelating agent and subjected to a hydrothermal reaction to obtain Cu3SnS4.

[0020] The present invention can prepare nano-flower-like Cu3SnS4 structure by controlling the ratio of precursor, sulfur source and chelating agent and adopting hydrothermal method. The flower-like Cu3SnS4 structure has a large surface area (23.49m 2 g -1 ) and has a narrow band gap (1.67 eV), which can provide more active sites when used in photoelectrochemical sensors.

[0021] As a preferred solution, the tin salt is SnCl4 and / or SnCl4 hydrate.

[0022] As a preferred solution, the copper salt is CuCl2.

[0023] As a preferred solution, the sulfur source is thioacetamide.

[0024] As a preferred solution, the chelating agent is ethylenediaminetetraacetic acid.

[0025] As a preferred solution, the base is sodium hydroxide.

[0026] As a preferred solution, the molar ratio of the tin salt, the alkali and the copper salt is (2-3):(4-6):1.

[0027] As a preferred solution, the molar ratio of the precursor, sulfur source and chelating agent is (0.5-1): (4-5): 4. When the molar ratio is too high or too low, by-products will be generated and nano-flower-shaped Cu3SnS4 material cannot be obtained.

[0028] As a preferred solution, the conditions of the hydrothermal reaction are: temperature of 180-200° C. and time of 2-5 h.

[0029] The present invention also provides a biosensor for Cyt c detection, which is obtained by the above-mentioned preparation method. The sensor is a photoelectric immunosensor prepared using Cu3SnS4 nanoflowers as the photoactive material. The logarithm of the Cyt c concentration and the photocurrent intensity show a good linear relationship in the range of 1 fM to 1000 nM. The linear regression equation is I (μA) = 14.148 ~ 0.86101 gC Cyt c (pM), correlation coefficient R2 = 0.9970. Under 3σ conditions, the detection limit is 0.35fM.

[0030] The present invention also provides an application of a biosensor for detecting Cyt c. Compared with other methods for detecting Cyt c, the photoelectrochemical immunosensor of the present invention has a lower detection limit and a wider linear range.

[0031] As a preferred solution, a phosphate buffer solution containing ascorbic acid is used as the electrolyte solution during the Cyt c detection.

[0032] As a preferred solution, the pH of the phosphate buffer solution is controlled to be between 6.5 and 8.0. The pH of the buffer solution is a key factor affecting the photocurrent; excessively high or low pH values ​​will reduce the photocurrent of the sensor. The phosphate buffer solution used in the present invention is a mixed solution of Na2HPO4·12H2O and KH2PO4.

[0033] As a preferred solution, the ascorbic acid concentration is controlled to be 0.05 to 0.30 mol / L. The concentration of ascorbic acid is also an important factor affecting the photocurrent. Within the preferred range of the present invention, as the concentration of ascorbic acid increases, the photocurrent first increases and then decreases. It is further preferred that the ascorbic acid concentration be controlled to be 0.05 to 0.15 mol / L.

[0034] Compared with the existing technology, the technical solution of the present invention brings the following beneficial technical effects:

[0035] 1) The present invention uses nanoflower-shaped Cu3SnS4 as a photoactive material to construct a photoelectrochemical immunosensor. The operation is simple, and the prepared immunosensor has good reproducibility, high specificity, and good storage stability. It also has good detection ability for Cyt c in clinical serum samples, with a low detection limit and a wide linear range.

[0036] 2) The present invention prepares Cu3SnS4 nanomaterials by hydrothermal method. The preparation method is simple and the prepared Cu3SnS4 nanoflowers have a large specific surface area (23.49m 2 g -1 ), narrow band gap (1.67 eV), and multiple active sites. It has the advantages of fast response, high sensitivity, and strong specificity when used in photoelectrochemical immunosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The SEM images of Cu3SnS4 nanomaterials prepared in Example 1 of the present invention at different magnifications are shown in FIG. Figure 1 (A) is the SEM image of Cu3SnS4 (500nm), Figure 1 (B) is a SEM image of Cu3SnS4 (100nm). It can be clearly seen from the figure that the Cu3SnS4 nanomaterial prepared by the present invention has a nanoflower-like structure, thus having a large specific surface area and a suitable band gap, and has good visible light absorption capacity, which is more conducive to photoelectric conversion.

[0038] Figure 2 This is an elemental mapping diagram of the Cu3SnS4 nanomaterial prepared in Example 1 of the present invention. As shown in the figure, the Cu3SnS4 nanoflowers are composed of evenly distributed Cu, Sn and S elements, indicating that the present invention successfully prepared the Cu3SnS4 nanomaterial.

[0039] Figure 3 The XRD spectra of Cu3SnS4 and precursor CuSn(OH)6 are respectively ( Figure 3 (A)), UV-visible-near infrared absorption spectrum ( Figure 3 (B)) and N2 adsorption-desorption isotherms ( Figure 3 (C)). Figure 3As shown in (A), the diffraction of Cu3SnS4 corresponding to Cu3SnS4 crystal (JCPDS No.33-0501) shows that the Cu3SnS4 material prepared by the present invention is relatively pure in crystal phase and has a high degree of fit with the standard spectrum. The UV-visible-near infrared absorption spectrum shows that the absorption intensity of Cu3SnS4 in the ultraviolet and visible light regions has increased. The specific surface area of ​​Cu3SnS4 prepared by the present invention is calculated to be 23.49m 2 g -1 .

[0040] Figure 4 The electrode photocurrent response diagrams of electrodes modified with different materials are respectively ( Figure 4 (A)) and electrochemical impedance spectroscopy ( Figure 4 (B) In the figure: (a) Cu3SnS4, (b) Cu3SnS4 / CS, (c) Cu3SnS4 / CS / anti-Cyt c, (d) Cu3SnS4 / CS / anti-Cyt c / BSA, (e) Cu3SnS4 / CS / anti-Cyt c / BSA / Cyt c (the inset is the electrochemical impedance spectroscopy equivalent circuit). Figure 4 (A) It can be seen that Cu3SnS4 (curve a) has a strong photocurrent conversion efficiency and good charge separation ability. Due to the low conductivity of CS and amino groups (GA), their cross-linking reaction on the electrode surface significantly reduces the photocurrent (curve b). After the aptamer is fixed, the photocurrent intensity is further reduced (curve c), which may be due to the inhibition of electron transfer by anti-Cyt c protein. The role of bovine serum albumin (curve d) is to prevent enzyme degradation and nonspecific adsorption. Finally, Cyt c is dropped onto the electrode, and the photocurrent is further reduced (curve f). Figure 4 In Figure B, the Rct value of the Cu3SnS4-modified electrode is low. However, this value increases with the condensation reaction of CS and GA (curve b). The Rct value further increases with the gradual modification of the electrode surface with anti-Cyt c (curve c), BSA (curve d), and Cyt c (curve e). The steric hindrance of these modified materials hinders the efficient electron transfer at the interface, resulting in a decrease in the photocurrent. The results of the electrode photocurrent response and electrochemical impedance spectroscopy demonstrate that the Cu3SnS4-based PEC immunosensor can be successfully used to detect Cyt c.

[0041] Figure 5 This is a performance diagram of the sensor based on Cu3SnS4 material prepared in Example 1 of the present invention. Figure 5(A) is the time-current curve of the ITO electrode modified with different concentrations of Cyt c. It can be seen that (the concentrations of Cyt c in a to j are: 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM, 1000nM) as the concentration of Cyt c gradually increases, the corresponding photocurrent decreases. Figure 5 (B) is the photocurrent-concentration standard curve. It can be seen that in the range of 1fM to 1000nM, a good linear relationship can be achieved between the logarithm of Cyt c concentration and the photocurrent intensity. In addition, the detection limit under 3σ conditions is 0.35fM. Figure 5 (C) is the photocurrent response diagram of 15 repeated on / off lighting cycles under the condition of 100 pM Cyt c. It can be seen from the figure that the photocurrent does not change significantly. The calculated relative standard deviation (RSD) is 1.32%, indicating that the Cu3SnS4-based PEC immunosensor has good stability. Figure 5 (D) is the selectivity performance diagram of the sensor based on Cu3SnS4 material. The figure shows that the interference of the selected substance on the photocurrent is negligible, indicating that the Cu3SnS4-based PEC immunosensor has good selectivity.

[0042] Figure 6 The repeatability of the Cu3SnS4 material sensor prepared in Example 1 of the present invention ( Figure 6 (A)) and storage stability diagram ( Figure 6 (B)), such as Figure 6 As shown in (A), under the same conditions, 6 PEC immunosensor samples containing 100 pM Cyt c were selected, and the RSD of the immunosensor was calculated to be 1.15%, indicating that the Cu3SnS4 material sensor prepared by the present invention has good repeatability. Figure 6 As shown in (B), the prepared immunosensor was stored at 4°C for 15 days, and the photocurrent remained at 91.06% of the initial value, indicating that the Cu3SnS4 material sensor prepared by the present invention has good storage stability.

[0043] Figure 7 This is a performance diagram of the real-time detection of Cyt c by the Cu3SnS4 material sensor constructed in Example 1 of the present invention. Figure 7 (A) Real-time photoelectric response diagram of Cyt c at different concentrations; Figure 7 (B) Real-time photoelectric response diagram of Cyt c at different concentrations; Figure 7 (C) is the calibration curve for real-time detection of Cyt c; Figure 7(D) is the stability of the electrode tested over a long period of time (Cyt c: 100 pM). As shown in the figure, the RSD of the Cu3SnS4 material sensor prepared by the present invention when testing the electrode is 1.03%, indicating that the Cu3SnS4 material sensor prepared by the present invention has high test accuracy.

[0044] Figure 8 This is a comparison diagram of the photocurrent of sensors with different concentrations of Cu3SnS4 in Example 2.

[0045] Figure 9 This is a comparison diagram of the photocurrent of the Cu3SnS4 sensor with different Cyt c incubation times in Example 3.

[0046] Figure 10 This is a comparison diagram of the photocurrent of the Cu3SnS4 sensor in Example 4 with Na2HPO4·12H2O and KH2PO4 buffer solutions at different pH values ​​in the electrolyte.

[0047] Figure 11 This is a comparison diagram of the photocurrent of the Cu3SnS4 sensor with different concentrations of ascorbic acid (AA) in the electrolyte in Example 5. DETAILED DESCRIPTION

[0048] In order to facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed manner below in conjunction with preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0049] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0050] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0051] Example 1

[0052] Preparation of Cu3SnS4 nanomaterials:

[0053] S1: Dissolve 1.58g of SnCl4·5H2O in 180ml of deionized water. Then, add 1.52g of NaOH while stirring.

[0054] S2: Add 100 ml of a 0.044 M CuCl₂ aqueous solution and stir for 15 minutes to form a blue precipitate. The mixture is then allowed to stand at room temperature for 6 hours. The resulting precipitate is separated, washed, and dried to yield CuSn(OH)₂ nanorods.

[0055] S3: Add 0.25 mmol CuSn(OH)6, 1.25 mmol thioacetamide and 1 mmol EDTA to 15 mL deionized water and ultrasonicate in a water bath for 15 min.

[0056] S4: The mixture was transferred to a 25 mL Teflon-lined reactor for hydrothermal reaction at 200°C for 3 h. Finally, the mixture was rinsed with deionized water and anhydrous ethanol several times, collected, and dried in a vacuum at 60°C to obtain Cu3SnS4 nanomaterials.

[0057] Preparation of sensor based on Cu3SnS4 material:

[0058] S1: ITO glass was pretreated with acetone, anhydrous ethanol, and ultrapure water for 10 min each by continuous ultrasonication, and then dried in an oven at 60°C.

[0059] S2: Drop 300 μL of 3 mg / mL Cu3SnS4 suspension on the ITO conductive surface and dry it in a vacuum oven at 60°C for 4 h.

[0060] S3: The electrode was modified with 50 μL of a 0.01% chitosan (CS)-0.1% acetic acid mixture to form amino groups. The electrode was then dried in a vacuum oven at 60°C for 1 hour. Then, 50 μL of 1 wt% glutaraldehyde was dripped onto the electrode surface and incubated at room temperature for 1 hour to obtain Cu3SnS4 / CS.

[0061] S4: Drop 50 μL of 100 nM anti-Cyt c solution on the electrode surface and incubate for 1 hour to obtain Cu3SnS4 / CS / anti-Cyt c. Then add 50 μL of 1% bovine serum albumin and incubate at room temperature for 1 hour to obtain Cu3SnS4 / CS / anti-Cyt c / BSA.

[0062] Sensor performance test:

[0063] 1) Configuration of series standard solutions:

[0064] Cyt c with known concentration was diluted with 0.01 M Na2HPO4·12H2O and KH2PO4 buffer solution at pH 7.4 to prepare a series of Cyt c standard solutions with the following concentrations: 1000 nM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM and 1 fM.

[0065] 2) Detection of Cyt c:

[0066] 50 μL of Cyt c solution diluted with 0.01 M Na2HPO4·12H2O and KH2PO4 buffer solution with concentrations of 1 fM, 1 pM, 100 pM, 10 nM and 1000 nM, respectively, was dropped onto the electrode surface and incubated at room temperature for 1 h to obtain Cu3SnS4 / CS / anti-Cyt c / BSA / Cyt c.

[0067] 3) Obtaining the photocurrent-concentration standard curve:

[0068] The time-current (it) curve was measured using Cu3SnS4 / CS / anti-Cyt c / BSA as the working electrode, platinum wire as the counter electrode, Ag / AgCl as the reference electrode, and a 0.1M, pH 7.4 Na2HPO4·12H2O and KH2PO4 buffer solution containing 0.1M ascorbic acid as the electrolyte. The analytical performance of the PEC immunosensor was verified by detecting the photocurrent signal of the ITO electrode modified with different concentrations of Cyt c. The results are shown in Figure 2. Figure 5 shown.

[0069] 4) Repeatability test of the prepared sensor based on Cu3SnS4 material was conducted. Under the same conditions, 6 PEC immunosensor samples containing 100 pM Cyt c were selected. Figure 6 As shown in (A), the RSD of the immunosensor was calculated to be 1.15%, indicating that the PEC immunosensor has excellent repeatability. Figure 6 As shown in (B), the prepared immunosensor was stored at 4°C for 15 days, and the photocurrent remained at 91.06% of the initial value, indicating that the PEC immunosensor has good storage stability. Figure 5 As shown in (C), under the condition of 100 pM Cyt c, the photocurrent response was repeated for 15 on / off lighting cycles, and no significant change was observed in the photocurrent. The calculated relative standard deviation (RSD) was 1.32%, indicating that the PEC immunosensor has good stability. Figure 5 (D) The selectivity of the immunosensor was investigated. Interfering substances, including l-valine, l-tyrosine, l-arginine, l-cysteine, l-alanine, l-lysine, glutamic acid, glutathione, and bovine serum albumin, were dripped onto the modified ITO electrode surface. Cyt c was detected at a concentration of 10 nM, which was 100 times higher than the concentration of 100 pM for Cyt c. Clearly, the interference of the selected substances on the photocurrent was negligible, demonstrating the excellent selectivity of the PEC immunosensor.

[0070] 5) The prepared Cu3SnS4, Cu3SnS4 / CS, Cu3SnS4 / CS / anti-Cyt c, Cu3SnS4 / CS / anti-Cyt c / BSA and Cu3SnS4 / CS / anti-Cyt c / BSA / Cyt c were subjected to electrode photocurrent response and electrochemical impedance spectroscopy. The results are as follows: Figure 4 shown.

[0071] Table 1 compares the performance of sensors made from different materials for detecting Cyt c. The comparison of methods in the table shows that the Cu3SnS4-based sensor employed in the present invention exhibits a wide linear range for detecting Cyt c and a low detection limit, making it suitable for clinical detection of low- to moderate-concentration Cyt c.

[0072] Table 1 Performance comparison of sensors prepared from different materials for detecting Cyt c

[0073]

[0074] Example 2

[0075] The only difference between this embodiment and embodiment 1 is that the concentration of Cu3SnS4 is replaced by 0.5 mg mL -1 , 1mgmL -1 , 2mg mL -1 , 4mg mL -1 and 5 mg mL -1 , the other conditions are the same, the photocurrent test results of the material are as follows Figure 8 As shown in the figure, it can be seen that in the range of 0.5 to 5 mg mL -1 Within the range, as the concentration of Cu3SnS4 increases, the photocurrent of the sensor gradually increases. When the concentration of Cu3SnS4 reaches 3 mg mL -1 When the photocurrent reaches its maximum value, the photocurrent decreases when the Cu3SnS4 concentration is further increased. This is mainly because when the Cu3SnS4 concentration is too high, it will hinder and limit the transmission efficiency of electrons on the sensor surface.

[0076] Example 3

[0077] The only difference between this embodiment and embodiment 1 is that the incubation time in step 5) is replaced with 10 min, 20 min, 30 min, 40 min, 50 min and 70 min respectively, and the other conditions are the same. The photocurrent test results of the material are as follows: Figure 9 As shown in the figure, it can be seen that with the increase of incubation time, the photocurrent gradually decreases and gradually decreases at 60 minutes and reaches a plateau at 60 minutes, indicating that anti-Cyt c has been fully combined with Cyt c.

[0078] Example 4

[0079] The only difference between this embodiment and embodiment 1 is that the pH values ​​of the Na2HPO4·12H2O and KH2PO4 buffer solutions in the electrolyte are replaced with 5.6, 6.2, 6.6 and 8.0 respectively, and the other conditions are the same. Figure 10 As shown in the figure, it can be seen that the pH value of the buffer solution is also an important factor affecting the photocurrent. As the pH value of the buffer solution increases, the photocurrent of the material first increases and then decreases. When the pH is 7.4, the photocurrent of the material is the highest.

[0080] Example 5

[0081] The only difference between this embodiment and embodiment 1 is that the ascorbic acid concentration in the electrolyte is replaced with 0, 0.05, 0.15, 0.2, 0.25 and 0.30 mol / L respectively, and the other conditions are the same. The photocurrent test results of the material are shown in Figure 2. Figure 11 As shown in the figure, it can be seen that when the ascorbic acid concentration is 0.1 mol / L, the photocurrent reaches a peak value, and the photocurrent decreases with further increase of the ascorbic acid concentration.

Claims

1. A method for constructing a biosensor for Cyt c detection, characterized in that: A solution containing Cu3SnS4, chitosan and acetic acid is dropped onto the ITO surface, and then a coupling agent, anti-Cyt c and a blocking agent are added in sequence for incubation to obtain the product.

2. The method for constructing a biosensor for Cyt c detection according to claim 1, wherein: The coupling agent is glutaraldehyde; The blocking agent is bovine serum albumin; The volume ratio of the coupling agent, Cu3SnS4 and anti-Cyt c is 1:(5-6):1; The concentration of the coupling agent is 0.5 to 1 wt%; The concentration of the Cu3SnS4 is 1 to 5 mg / mL; The concentration of the anti-Cyt c is 80-100 nM.

3. The method for constructing a biosensor for Cyt c detection according to claim 1, wherein: The incubation time is 0.5 to 1 hour.

4. The method for constructing a biosensor for Cyt c detection according to claim 1, wherein: The Cu3SnS4 has a nano-flower structure.

5. The method for constructing a biosensor for Cyt c detection according to any one of claims 1 to 4, characterized in that: The Cu3SnS4 is prepared by the following method: a tin salt, an alkali and a copper salt are mixed and reacted in an aqueous solution to obtain a precursor; and the precursor is mixed with a sulfur source and a chelating agent to carry out a hydrothermal reaction to obtain the Cu3SnS4.

6. The method for constructing a biosensor for Cyt c detection according to claim 5, characterized in that: The tin salt is SnCl4 and / or SnCl4 hydrate; The copper salt is CuCl2; The sulfur source is thioacetamide; The chelating agent is ethylenediaminetetraacetic acid; The molar ratio of the tin salt, the base and the copper salt is (2-3): (4-6): 1; The base is sodium hydroxide; The molar ratio of the precursor, the sulfur source and the chelating agent is (0.5-1): (4-5):

4.

7. The method for constructing a biosensor for Cyt c detection according to claim 5, wherein: The conditions of the hydrothermal reaction are: temperature of 180-200° C. and time of 2-5 hours.

8. A biosensor for Cyt c detection, characterized in that: Obtained by the construction method according to any one of claims 1 to 7.

9. The use of a biosensor for Cyt c detection according to claim 8, characterized in that: Applied to the detection of Cytc.

10. The use of a biosensor for Cyt c detection according to claim 9, characterized in that: During the Cyt c detection, a phosphate buffer solution containing ascorbic acid is used as the electrolyte solution; The pH of the phosphate buffer solution is controlled to be 6.5-8.0; The ascorbic acid concentration is controlled to be 0.05-0.30 mol / L; The concentration range of the Cyt c detection is 1 fM to 1000 nM.

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