A branched functional polypeptide, an optoelectrochemical biosensor for detecting cardiac troponin I and a preparation method and application thereof

Abstract

This invention discloses a branched functional polypeptide, a photoelectrochemical biosensor for detecting cardiac troponin I, its preparation method, and its applications, belonging to the field of biosensor technology. The branched functional polypeptide provided by this invention includes a main chain and branched chains. The main chain is a recognition polypeptide capable of recognizing the target analyte, and the branched chains are antifouling polypeptides capable of resisting biocontamination. The photoelectrochemical biosensor for detecting cardiac troponin I provided by this invention includes a ZIS / PDA photoelectrode. The branched functional polypeptide is directly anchored to the ZIS / PDA photoelectrode. The detection of cardiac troponin I is achieved by utilizing the significant steric hindrance effect after cardiac troponin I specifically binds to the main chain, which hinders charge transfer in the photoelectrochemical biosensor and causes a change in the photocurrent signal. This invention improves both the specificity and sensitivity of target analyte detection, as well as the accuracy of detection.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, and relates to a branched functional polypeptide, a photoelectrochemical biosensor for detecting cardiac troponin I, its preparation method and application. Background Technology

[0002] Biosensors are analytical and detection devices primarily constructed from signal conversion elements and molecular recognition elements, serving as a crucial method for the in vitro diagnosis of common and serious diseases. Photoelectrochemical biosensors are a new generation of sensing technology that organically combines photoelectrochemical technology with electrochemical analysis. Their detection principle relies on the specific recognition reaction between a biorecognition probe and the target analyte, which induces a change in photocurrent signal in the sensing system. Photoelectrochemical biosensors not only possess advantages such as simple device design, convenient operation, low sample preparation cost, and fast signal response, but also exhibit low background interference and high sensitivity due to the different energy forms of the excitation and detection signals. These advantages make photoelectrochemical biosensors more suitable for accurate, rapid, and high-throughput on-site detection.

[0003] Cardiac troponin I (cTnI) is a cardiac tissue-specific regulatory protein that inhibits the binding of myosin to actin, playing a crucial role in myocardial contraction. cTnI exhibits high myocardial specificity and is considered the "gold standard" for diagnosing myocardial injury. It is one of the most specific and sensitive biomarkers for the early diagnosis and prognosis of acute myocardial infarction. Therefore, rapid, sensitive, and accurate measurement of cTnI in human blood is of great significance for diagnosing acute myocardial infarction, risk stratification of acute coronary syndrome, monitoring myocardial injury caused by various factors, and assessing the prognosis of cardiac events. Currently, the most commonly used method for detecting cTnI is enzyme-linked immunosorbent assay (ELISA). This method is relatively simple to operate and uses inexpensive equipment, but it has drawbacks such as a relatively long measurement cycle, relatively low sensitivity, and a narrow detection range.

[0004] In light of this, various analytical methods for the quantitative detection of cTnI have emerged in recent years, such as radioimmunoassay, chemiluminescence immunoassay, fluorescence immunoassay, gold-labeled immunoassay, and electrochemical immunoassay. Although these cTnI detection methods have made significant progress, they still have some drawbacks, such as being time-consuming, expensive, requiring large sample volumes, having low detection accuracy, and limited sensitivity.

[0005] Biocontamination is a challenge for many biosensors in their practical application because complex samples such as serum and cell tissue fluid contain multiple components of biomolecules, including proteins, which can easily and non-specifically adsorb onto the sensor surface. To prevent false positive signals caused by the non-specific adsorption of biomolecules such as proteins onto the electrode surface, thereby reducing the specificity and sensitivity of the detection, the anticontamination properties of peptides have begun to attract attention.

[0006] Relevant data have shown that the polypeptide sequence CPPPPEKEKEKEK, which contains alternating negatively charged glutamic acid (E) and positively charged lysine (K), can effectively prevent non-specific biomolecules from adsorbing onto the sensor surface through hydrophobic and electrostatic interactions due to its strong hydrophilicity and electroneutrality, thus demonstrating excellent anti-biofouling ability. Summary of the Invention

[0007] To address the problems existing in the prior art, one of the objectives of this invention is to provide a branched functional polypeptide that specifically recognizes anti-biocontamination peptides or proteins. This polypeptide can not only improve the specificity of target analyte detection but also serve as an anti-biocontamination element, effectively preventing the non-specific adsorption of biomolecules such as proteins on the electrode surface and thus preventing false positive signals, thereby improving the accuracy of detection.

[0008] A second objective of this invention is to provide a photoelectrochemical biosensor for detecting CTnI. The main chain portion of a branched functional polypeptide is used as a specific recognition probe to bind to CTnI, achieving specific detection of the target analyte CTnI and improving detection specificity. A ZIS / PDA photoelectrode is used as a signal conversion element; this ZIS / PDA photoelectrode has the characteristic of generating a high photocurrent signal effect, which can further improve the detection sensitivity of the photoelectrochemical biosensor for detecting CTnI. The branched chain portion of the branched functional polypeptide is used as an anti-biocontamination element, which can effectively prevent the non-specific adsorption of biomolecules such as proteins on the electrode surface, thus preventing false positive signals and improving detection accuracy.

[0009] The third objective of this invention is to provide a method for preparing a photoelectrochemical biosensor for detecting CTnI, which has simple preparation steps, mild conditions, easy operation, and low cost.

[0010] The fourth objective of this invention is to provide an application of a photoelectrochemical biosensor for detecting CTnI in in vitro diagnostic devices, which consumes a small amount of biological samples, requires no complex separation, and avoids the problem of non-specific adsorption of biological macromolecules such as proteins.

[0011] To achieve one of the above objectives, the technical solution of the present invention is as follows:

[0012] A branched functional polypeptide of a biofouling-specific recognition polypeptide or protein, the branched functional polypeptide comprising a main chain portion and a branched chain portion, wherein the main chain portion is a recognition polypeptide capable of recognizing a target analyte, and the branched chain portion is an antifouling polypeptide capable of resisting biofouling.

[0013] Furthermore, the main chain portion is a recognition polypeptide capable of recognizing CTnI.

[0014] Further, the amino acid sequence of the branched functional polypeptide is (CKEKEKEKE)2KEPPPPFYSHSFHENWPSK, wherein the amino acid sequence of the main chain portion is KEPPPPFYSHSFHENWPSK, and the amino acid sequence of the branched chain portion is (CKEKEKEKE)2.

[0015] To achieve the second objective mentioned above, the present invention adopts the following technical solution:

[0016] A photoelectrochemical biosensor for detecting CTnI includes a ZIS / PDA photoelectrode. The ZIS / PDA photoelectrode comprises ZnIn2S4 nanosheets grown in situ on fluorine-doped SnO2 conductive glass via a hydrothermal method and polydopamine modified on the ZnIn2S4 nanosheets. A branched functional polypeptide is directly anchored to the ZIS / PDA photoelectrode. The branched functional polypeptide includes a main chain portion and a branched chain portion. The main chain portion is a probe that specifically recognizes CTnI, and the branched chain portion is an antifouling polypeptide capable of resisting biofouling. The detection of CTnI is achieved by utilizing the significant steric hindrance effect of CTnI specifically binding to the main chain portion to hinder charge transfer in the photoelectrochemical biosensor, thereby causing a change in the photocurrent signal.

[0017] Furthermore, the concentration of the branched functional polypeptide is 0.2–0.5 mg / mL.

[0018] Further, the amino acid sequence of the branched functional polypeptide is (CKEKEKEKE)2KEPPPPFYSHSFHENWPSK, wherein the amino acid sequence of the main chain portion is KEPPPPFYSHSFHENWPSK, and the amino acid sequence of the branched chain portion is (CKEKEKEKE)2.

[0019] To achieve the third objective mentioned above, the present invention adopts the following technical solution:

[0020] A method for preparing a photoelectrochemical biosensor for detecting CTnI includes the following steps:

[0021] S1. ZnIn2S4 nanosheets are grown in situ on fluorine-doped SnO2 conductive glass by hydrothermal method. After modifying the ZnIn2S4 nanosheets with polydopamine by self-polymerization reaction, the ZIS / PDA photoelectrode is prepared.

[0022] S2. Anchor the branched functional polypeptide to the ZIS / PDA photoelectrode to obtain the photoelectrochemical biosensor for detecting CTnI.

[0023] Further, in S1: ZnCl2, InCl3, and C2H5NS are used as Zn source, In source, and S source, respectively. The concentration of ZnCl2 is 10-30 mM. According to the concentration of ZnCl2, a mixed solution is prepared according to the molar ratio of Zn source, In source, and S source of 1:2:4. After reacting for 40-80 min, a precursor solution is obtained. The precursor solution is then transferred to a high-pressure reactor, and the fluorine-doped SnO2 conductive glass is vertically placed into the high-pressure reactor. Under sealed conditions, the reaction is carried out at 150-180℃ for 5-10 h to obtain the ZnIn2S4 nanosheets. 20 μL of a 5-10 mM dopamine solution is dropped onto the ZnIn2S4 nanosheets and incubated at 4℃ in the dark for 30-60 min to allow the dopamine to self-polymerize into polydopamine, thus obtaining the ZIS / PDA photoelectrode.

[0024] Further, 15–25 μL of the branched functional polypeptide was dropped onto the ZIS / PDA photoelectrode for anchoring, and incubated at 4°C for 8–12 h.

[0025] To achieve the fourth objective mentioned above, the present invention adopts the following technical solution:

[0026] This invention provides the application of the aforementioned photoelectrochemical biosensor for detecting CTnI in the preparation of in vitro diagnostic devices.

[0027] As can be seen from the above technical solution, compared with the prior art, the present invention has the following superior effects:

[0028] 1) The high-performance photoelectrochemical biosensor for detecting CTnI provided by this invention has a surface modified with a novel branched functional polypeptide that integrates anti-biocontamination and specific recognition functions. This polypeptide includes a main chain and a branched chain. The branched chain is an antifouling polypeptide capable of resisting biocontamination, exhibiting better antifouling performance compared to linear polypeptides. This is because the unique spatial structure of the branched chain polypeptide results in larger intramolecular and intermolecular cavities, promoting water transport and enhancing the hydration layer. The main chain is a recognition polypeptide capable of recognizing the target analyte, serving as a biological probe for specifically recognizing CTnI. Compared to the traditional use of antibodies as biological probes, using a polypeptide sequence as a biological probe enables high-performance detection of CTnI by the photoelectrochemical biosensor. Furthermore, the biological probe for specifically recognizing CTnI is anchored to a ZIS / PDA photoelectrode, which has the characteristic of generating a high photocurrent signal effect, thus minimizing photocurrent signal response loss and further improving the detection sensitivity of the target analyte. However, no reports have yet been made of this high-performance photoelectrochemical biosensor for detecting CTnI based on a novel branched functional peptide that integrates both anti-biocontamination and specific recognition functions, which has the potential for practical application in the detection of CTnI in blood.

[0029] 2) The photoelectrochemical biosensor for detecting CTnI provided by this invention can detect the target CTnI without purification, which is convenient and fast. It not only provides an efficient sensing mode for photoelectrochemical biosensors for detecting CTnI, but also effectively improves the accuracy of detecting disease biomarkers in actual biological samples. It has profound significance for in vitro diagnosis of diseases.

[0030] 3) The method for preparing the photoelectrochemical biosensor for detecting CTnI provided by the present invention has simple and efficient preparation steps, mild conditions, easy operation and low cost. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 The current signal response diagram of the ZIS / PDA photoelectrode corresponding to different concentrations of Zn source precursor in Example 1 is shown.

[0033] Figure 2 The current signal response diagrams are for sensing electrodes anchored with different concentrations of branched functional peptides in Example 1.

[0034] Figure 3 This is a scanning electron microscope image of the ZIS / PDA photoelectrode fabrication process in Example 2.

[0035] Figure 4 The images show the X-ray diffraction pattern and infrared spectrum of the ZIS / PDA photoelectrode fabrication process in Example 2.

[0036] Figure 5 This is a current signal response diagram of the sensor fabrication process anchored by the branched functional peptide in Example 3.

[0037] Figure 6 This is a standard curve of the sensor anchored to the branched functional peptide in Example 4 for the detection of the target CTnI.

[0038] Figure 7 This is a fluorescence imaging image of the sensor anchored by the branched functional peptide in Example 5, demonstrating its resistance to biofouling.

[0039] Figure 8 This is a graph showing experimental data on sensor-specific recognition of branched functional peptides anchored in Example 6.

[0040] Figure 9 This is a photocurrent signal diagram of the branched functional peptide-anchored sensor in Example 7 detecting the target CTnI in serum.

[0041] Figure 10 This is a comparison chart showing the biofouling resistance of sensors anchored with branched functional peptides and straight-chain functional peptides in Example 8. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] First, it should be noted that the current signal response in the following examples was tested on a photoelectrochemical system, as detailed below:

[0044] Using an emission wavelength of 430nm and a light intensity of 350W / m 2 An LED light was used as the excitation source, switching on and off every 10 seconds. Photocurrent was recorded using an electrochemical workstation. A three-electrode system was used to test the current signal response: a photoelectrode with a modified area of ​​0.7 mm × 0.7 mm as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode; the system was self-powered, requiring no external voltage.

[0045] Example 1

[0046] Optimization of detection conditions

[0047] 1. ZnCl2 solution concentration

[0048] The magnitude of the current signal response of the ZIS / PDA photoelectrode has a significant impact on the detection sensitivity of the final fabricated photoelectrochemical biosensor for detecting CTnI. Therefore, the fabrication process parameters of the ZIS / PDA photoelectrode were optimized as follows:

[0049] Since the deposition amount of ZIS on the electrode can be adjusted by the concentration of the precursor solution, the concentration of the Zn source in the precursor solution was optimized under the condition that the molar ratio of Zn source, In source, and S source in the precursor solution was fixed at 1:2:4. Hydrothermal deposition was performed using ZnCl2, InCl3, and C2H5NS as the Zn source, In source, and S source, respectively. The concentrations of the ZnCl2 solution were 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, and 35 mM. Mixed solutions with different Zn source concentrations were prepared under the condition that the molar ratio of Zn source, In source, and S source was 1:2:4. The mixed solutions were then reacted under stirring for 60 min to obtain the precursor solution, which was then transferred to a high-pressure reactor. Fluorine-doped SnO2 (FTO) conductive glass was vertically placed into a high-pressure reactor and kept at 150–180 °C for 8 hours under sealed conditions to obtain ZnIn2S4 nanosheets (ZIS nanosheets). A dopamine solution was prepared using 10 mM tris-HCl buffer solution with pH = 8.5, and 20 μL of the 8 mM dopamine solution was dropped onto the ZnIn2S4 nanosheets. The solution was incubated at 4 °C in the dark for 30 minutes to allow dopamine to self-polymerize into polydopamine (PDA), thus obtaining the ZIS / PDA photoelectrode.

[0050] The results can be obtained by performing photocurrent characterization tests, as shown in the attached figure. Figure 1 As shown, the current signal response of the ZIS / PDA photoelectrode reaches its optimal level when the ZnCl2 concentration is 25 mM. This is because an appropriate thickness of ZIS can effectively improve the photoelectric conversion efficiency of the photoelectrode, while excessive ZIS thickness will significantly increase the probability of charge recombination, leading to electron annihilation. Therefore, a ZnCl2 solution concentration of 25 mM is selected as the optimal preparation process parameter.

[0051] 2. Incubation concentration of branched functional peptides

[0052] The amount of branched functional peptides modified in the sensing electrode has a significant impact on the quantitative detection range of the photoelectrochemical biosensor for detecting the target analyte CTnI. Therefore, the preparation process parameters for modifying the sensing electrode with branched functional peptides were optimized.

[0053] Since the amount of modification on the sensing electrode can be reflected by its incubation concentration on the electrode, the incubation concentration of the branched functional peptides was optimized. 20 μL of branched functional peptides at concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, and 0.5 mg / mL were added to the ZIS / PDA photoelectrode for anchoring. The electrodes were incubated at 4 °C for 10 h. After washing the electrodes with phosphate buffer (10 mM, pH 7.4), a photoelectrochemical biosensor for detecting CTnI was obtained.

[0054] The results can be obtained by performing photocurrent characterization tests, as shown in the attached figure. Figure 2 As shown, the incubation concentration of the branched functional peptide needs to be greater than or equal to 0.25 mg / mL to ensure that the branched functional peptide is fully anchored on the sensing electrode and to obtain the optimal quantitative detection range. Therefore, a branched functional peptide concentration of greater than or equal to 0.25 mg / mL is selected as the optimal incubation concentration.

[0055] Example 2

[0056] Fabrication of ZIS / PDA photoelectrode

[0057] 25 mM ZnCl2, 50 mM InCl3, and 100 mM CH3CSNH2 were used as Zn, In, and S sources, respectively, dissolved in deionized water, and reacted under stirring for 60 min to prepare a precursor solution. The precursor solution was then transferred to a 25 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor. FTO conductive glass was vertically placed inside the high-pressure reactor, and the reactor was sealed and maintained at 160 °C for 8 h to obtain the ZnIn2S4 nanosheets. A dopamine solution was prepared using 10 mM tris-HCl buffer solution (pH 8.5), and 20 μL of an 8 mM dopamine solution was added dropwise to the ZnIn2S4 nanosheets. The solution was incubated at 4 °C for 30 min in the dark to allow dopamine to self-polymerize into polydopamine, resulting in the ZIS / PDA photoelectrode.

[0058] Scanning electron microscope images are attached. Figure 3 As shown in Figure A, the ZnIn2S4 nanosheets are interconnected to form a network film. The nanosheets grow almost perpendicular to the substrate, and the nanosheet layer thickness is approximately 45 nm. The elemental mapping analysis in the inset also confirms the successful preparation of the ZnIn2S4 nanosheets. Scanning electron microscope images are attached. Figure 3 As shown in Figure B, the ZnIn2S4 nanosheets are covered with a tight polydopamine film, and the nanosheet layer is thicker. The elemental mapping analysis in the inset also shows that the polydopamine was successfully modified, proving that the ZIS / PDA photoelectrode was successfully prepared.

[0059] Furthermore, the X-ray diffraction of the ZnIn2S4 nanosheet-modified electrode is shown in the attached figure. Figure 4 As shown in Figure A, the diffraction peaks at 27.6°, 30.5°, and 47.3° correspond to the (102), (104), and (112) crystal planes of the hexagonal ZIS (PDF No. 72-0773), respectively. Apart from the characteristic diffraction peaks of the FTO conductive glass substrate, no diffraction peaks of other impurity phases were found, thus proving the successful fabrication of ZnIn2S4 nanosheets. The infrared spectrum of the ZIS / PDA photoelectrode is attached. Figure 4 As shown in Figure B, compared with simple ZnIn2S4 nanosheets, the modification with polydopamine resulted in a higher nanosheet size at 3498 cm⁻¹. -1 Stretching vibration peaks of OH and NH appear at 1611 cm⁻¹. -1 and 1556cm -1 A stretching vibration peak of the aromatic ring appears at 1262 cm⁻¹. -1 The presence of CN stretching vibration peaks, which are characteristic peaks of polydopamine, further confirms the successful fabrication of the ZIS / PDA photoelectrode.

[0060] Example 3

[0061] Fabrication of branched functional peptide-anchored ZIS / PDA photoelectrodes

[0062] 20 μL of a branched functional peptide at a concentration of 0.25 mg / mL was added to the ZIS / PDA photoelectrode prepared in Example 2 for anchoring. The electrode was incubated at 4 °C for 10 h. After washing the electrode with phosphate buffer (10 mM, pH 7.4), the branched functional peptide-anchored ZIS / PDA photoelectrode was obtained.

[0063] The successful fabrication of the ZIS / PDA photoelectrode anchored to branched functional peptides was verified using current signal response testing, as shown in the attached figure. Figure 5 As shown, the ZnIn2S4 nanosheet-modified electrode exhibits a strong current signal response (curve a). This is because ZIS has a narrow bandgap and good photostability, enabling efficient absorption and utilization of visible light. After dopamine self-polymerization to modify polydopamine, the current signal response decreases appropriately (curve b). This is because the relatively weak charge transfer rate of polydopamine hinders interfacial electron transfer. The purpose of polydopamine modification is mainly to form a uniform film on the electrode surface, providing a large number of binding sites for subsequent branched functional peptide anchoring. After the branched functional peptide is anchored to the photoelectrode, the photocurrent signal response further decreases (curve c), which is due to the weak charge conduction ability of the branched functional peptide. Thus, the current signal response test proves the successful preparation of the branched functional peptide-anchored ZIS / PDA photoelectrode.

[0064] Example 4

[0065] The prepared photoelectrochemical biosensor for detecting CTnI effectively detects the target CTnI.

[0066] The branched functional peptide-anchored sensing electrode prepared in Example 3 was incubated with 20 μL of target compound CTnI at different concentrations for 1.5 h at room temperature, allowing the target compound CTnI to undergo a specific recognition reaction with the main chain of the branched functional peptide. After washing with phosphate buffer (10 mM, pH = 7.4), the photoelectrochemical biosensor for detecting CTnI was finally incubated. The photocurrent signal was measured in phosphate buffer (pH 7.4, 0.1 M) containing 0.1 M ascorbic acid as an electron donor. The photocurrent signal was detected by utilizing the significant steric hindrance effect of the target compound CTnI on the charge transfer of the sensor.

[0067] The detection results show that as the concentration of the target analyte CTnI increases, the corresponding photocurrent detection signal gradually weakens. Furthermore, within the CTnI concentration range of 0.5 pg / mL to 50 ng / mL, the photocurrent detection signal exhibits a good linear relationship with the logarithm of the CTnI concentration, as shown in the attached figure. Figure 6 As shown, the linear fitting equation is I = 39.95 - 6.64 logC (ng / mL), the linear correlation coefficient is 0.9978, and the experimental limit of detection is 0.5 pg / mL, which indicates that the photoelectrochemical biosensor for detecting CTnI based on branched functional peptides prepared by the present invention has high sensitivity to the target substance.

[0068] Example 5

[0069] Anti-biocontamination performance test of the prepared photoelectrochemical biosensor for detecting CTnI

[0070] The ZIS / PDA photoelectrode anchored with the branched functional peptide prepared in Example 3 was incubated with 2.0 mg / mL fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) for 2 h at room temperature. After washing the electrode with phosphate buffer (pH 7.4, 10 mM), it was ready for fluorescence imaging characterization. For comparison, the ZIS / PDA photoelectrode without branched functional peptide anchorage was also incubated with FITC-BSA under the same conditions and then washed.

[0071] Fluorescence microscope images are attached. Figure 7As shown in Figure A, the ZIS / PDA photoelectrode exhibited obvious green fluorescence after incubation with FITC-BSA, indicating significant non-specific protein adsorption. However, the ZIS / PDA photoelectrode anchored with branched functional peptides did not show obvious green fluorescence under a fluorescence microscope after incubation in FITC-BSA for 2 hours (Figure B). This demonstrates that the sensing electrode anchored with branched functional peptides can effectively resist the adsorption of non-specific proteins and has excellent anti-fouling effect.

[0072] Example 6

[0073] Specific recognition performance test of the prepared photoelectrochemical biosensor for detecting CTnI

[0074] To demonstrate the excellent specificity of the photoelectrochemical biosensor for detecting CTnI, common blood protein macromolecules—human serum albumin (HSA), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), alpha-fetoprotein (AFP), and human immunoglobulin (HIgG)—were selected as typical interfering agents for specificity verification experiments. The specific procedures are as follows:

[0075] Take 10 ng / mL of CTnI, HSA, CEA, CA125, AFP, and HIgG, either individually or in combination, and add them to serum diluted 10 times. Use the photoelectrochemical biosensor for detecting CTnI prepared in this invention to detect them according to the above method. The changes in photocurrent signals are shown in the attached figure. Figure 8 As shown in the figure. The results indicate that, relative to the detection of the target CTnI, the changes in photocurrent generated by interfering proteins HSA, CEA, CA125, AFP, HIgG, and their mixtures are negligible. This demonstrates that the photoelectrochemical biosensor for detecting CTnI prepared in this invention has excellent specific recognition ability for the target CTnI and is unaffected by interfering proteins.

[0076] Example 7

[0077] Application of the prepared photoelectrochemical biosensor for detecting CTnI in serum

[0078] The practical application capability of the high-performance photoelectrochemical biosensor for detecting CTnI prepared in this invention was evaluated through a spiked recovery experiment in serum samples, the specific operation of which is as follows:

[0079] The serum was diluted 10-fold and divided into three groups. CTnI standard samples with concentrations of 0.1 ng / mL, 1 ng / mL, and 10 ng / mL were added to each group, respectively. 20 μL of serum spiked samples of different concentrations were incubated for 1.5 h at room temperature using the ZIS / PDA photoelectrode anchored to the branched functional peptide prepared in Example 3. After cleaning the electrode, photocurrent signals were measured in phosphate buffer (pH 7.4, 0.1 M) containing 0.1 M ascorbic acid as an electron donor, and the photoelectrochemical detection performance was compared with that of Example 4.

[0080] The results of the spiked recovery experiments in serum samples are shown in the attached figure. Figure 9 As shown in the figure, Serum and PBS represent serum and buffer solution, respectively. The recovery rate of spiked samples ranged from 96.8% to 104.1%, and the relative standard deviation of the test results was within 5%. This demonstrates that the prepared photoelectrochemical biosensor for detecting CTnI has excellent application potential for accurate detection of CTnI in actual complex biological samples, and can achieve rapid, sensitive, accurate and efficient detection of the target analyte.

[0081] Example 8

[0082] Comparison of the biofouling resistance of sensors anchored with branched functional peptides and straight-chain functional peptides

[0083] The linear functional polypeptide sequence is CPPPPKEKEKEKEFYSHSFHENWPSK, which, compared to the branched functional polypeptide, has the same specific recognition sequence and a similar anticontamination sequence. To verify that the branched functional polypeptide used in this invention has superior anticontamination ability compared to the linear functional polypeptide, a photoelectrochemical biosensor for detecting CTnI, anchored by both the branched and linear functional polypeptides, was prepared under identical experimental conditions and tested in actual serum samples at different dilution concentrations. The photocurrent signal change rate is shown in the attached figure. Figure 10 As shown in the figure. The results indicate that, in serum samples of different dilution concentrations, the photoelectrochemical biosensor for detecting CTnI anchored by branched functional peptides used in this invention exhibits a lower rate of change in current signal compared to the sensor anchored by linear functional peptides. This suggests that the photoelectrochemical biosensor for detecting CTnI anchored by branched functional peptides has a weaker non-specific adsorption capacity for biomolecules in actual serum samples, even exhibiting a lower rate of change in current signal in serum diluted to 25%. This directly proves that the branched functional peptides used in this invention have superior resistance to biomolecule contamination.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A photoelectrochemical biosensor for detecting cardiac troponin I, comprising a ZIS / PDA photoelectrode, said ZIS / PDA photoelectrode comprising ZnIn2S4 nanosheets grown in situ on fluorine-doped SnO2 conductive glass via a hydrothermal method and polydopamine modified on said ZnIn2S4 nanosheets, characterized in that, A branched functional polypeptide is directly anchored to the ZIS / PDA photoelectrode. The branched functional polypeptide consists of a main chain and branched chains. The main chain is a probe that specifically recognizes cardiac troponin I, and the branched chains are antifouling polypeptides resistant to biofouling. The detection of cardiac troponin I is achieved by utilizing the significant steric hindrance effect of cardiac troponin I specifically binding to the main chain, which hinders charge transfer in the photoelectrochemical biosensor. The main chain is a recognition polypeptide capable of recognizing the target analyte, and the branched chains are antifouling polypeptides resistant to biofouling. The amino acid sequence of the branched functional polypeptide is (CKEKEKEKE)2KEPPPPFYSHSFHENWPSK, wherein the amino acid sequence of the main chain is KEPPPPFYSHSFHENWPSK, and the amino acid sequence of the branched chain is (CKEKEKEKE)2.

2. The photoelectrochemical biosensor for detecting cardiac troponin I according to claim 1, characterized in that, The concentration of the branched functional peptide is 0.2–0.5 mg / mL.

3. A method for preparing a photoelectrochemical biosensor for detecting cardiac troponin I according to any one of claims 1 to 2, characterized in that, Includes the following steps: S1. The ZnIn2S4 nanosheets are grown in situ on the fluorine-doped SnO2 conductive glass by hydrothermal method. After modifying the ZnIn2S4 nanosheets with polydopamine by self-polymerization reaction, the ZIS / PDA photoelectrode is prepared. S2. Anchor the branched functional polypeptide to the ZIS / PDA photoelectrode to obtain the photoelectrochemical biosensor for detecting cardiac troponin I.

4. The method for preparing a photoelectrochemical biosensor for detecting cardiac troponin I according to claim 3, characterized in that, In S1: ZnCl2, InCl3, and C2H5NS are used as Zn source, In source, and S source, respectively. The concentration of ZnCl2 is 10-30 mM. According to the concentration of ZnCl2, a mixed solution is prepared with a molar ratio of Zn source, In source, and S source of 1:2:

4. After reacting for 40-80 min, a precursor solution is obtained. The precursor solution is then transferred to a high-pressure reactor, and the fluorine-doped SnO2 conductive glass is vertically placed into the high-pressure reactor. Under sealed conditions, the reaction is carried out at 150-180 °C for 5-10 h to obtain the ZnIn2S4 nanosheets. 20 μL of a 5-10 mM dopamine solution is dropped onto the ZnIn2S4 nanosheets and incubated at 4 °C in the dark for 30-60 min to allow the dopamine to self-polymerize into polydopamine, thus obtaining the ZIS / PDA photoelectrode.

5. The method for preparing a photoelectrochemical biosensor for detecting cardiac troponin I according to claim 4, characterized in that, The specific steps of S2 are as follows: 15-25 μL of the branched functional polypeptide is added to the ZIS / PDA photoelectrode for anchoring, and incubated at 4 ℃ for 8-12 h.

6. The application of the photoelectrochemical biosensor for detecting cardiac troponin I as described in any one of claims 1 to 2 in the preparation of in vitro diagnostic devices.

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