Photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate and its preparation method and application

Abstract

The present invention discloses a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate and a preparation method and application thereof, and belongs to the field of biosensor technology. The present invention uses a polypeptide-nucleic acid conjugate for the first time to construct a photoelectrochemical aptamer sensor, including preparing a ZnO / HOF-101 / PDA photoelectrode, directly anchoring the polypeptide-nucleic acid conjugate to the photoelectrode, and utilizing the obvious steric hindrance effect after the target specifically binds to the nucleic acid aptamer to hinder the charge transfer of the aptamer sensor to cause a change in the photocurrent signal to achieve the detection of the target mucin 1. The sensor has an excellent ability to resist biological contamination in complex biological samples, improves the sensitivity, specificity and accuracy of target detection, and at the same time, the design of the polypeptide-nucleic acid conjugate solves the competitive problem of the modification process of a single polypeptide and a nucleic acid probe.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and relates to a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate, and a preparation method and application thereof. Background Art

[0002] Accurately and sensitively detecting disease-specific biomarkers in complex human physiological fluids is a huge frontier challenge for practical biosensors. This is due to the far-reaching significance of practical biosensors in promoting the early clinical diagnosis and timely treatment of major diseases, especially critical diseases and malignant tumors. In fact, in actual complex biological samples such as serum and cell tissue fluid, in addition to the target biomarkers to be detected, there are also multi-component proteins and other biological macromolecules. They can easily be non-specifically adsorbed on the sensor surface, resulting in biological contamination, destroying the sensor's ability to detect real signals, and ultimately leading to false positive or false negative test results. Therefore, there is an urgent need to explore advanced practical anti-contamination biosensors.

[0003] In the field of biosensors, photoelectrochemical sensors are a new generation of sensing technology that has been developed by organically combining photoelectrochemical technology with electrochemical analysis. The detection principle is achieved by causing the sensor system to produce a change in the photocurrent signal through a specific recognition reaction between the biorecognition probe and the target analyte. Photoelectrochemical sensors not only have the advantages of simple device, low equipment cost, convenient operation, fast signal response, easy miniaturization and integration, but also because the energy form of the excitation signal and the detection signal are completely different, the technology has the characteristics of low background interference and high sensitivity. These advantages make photoelectrochemical sensors easier to achieve fast, accurate, and high-throughput on-site detection.

[0004] Zwitterionic peptides are widely considered to be promising anti-biofouling materials, mainly based on their outstanding advantages such as excellent biocompatibility, simple synthesis, cost-effectiveness, stable structure and flexible design. Zwitterionic peptides have two key properties: hydrophilicity and electrical neutrality. These properties enable them to effectively construct a robust hydrophilic neutral biointerface, hindering the nonspecific adsorption of biofouling molecules to the electrode through hydrophobic interaction or electrostatic attraction. At present, the main method for preparing anti-fouling biosensors involves co-modifying zwitterionic peptides and capture probes on electrodes respectively. However, due to the competitive modification of zwitterionic peptides and capture probes on electrodes, if one is modified more, the other will inevitably be modified less. This method will inevitably impair the sensitivity or anti-biofouling ability of the biosensor. Therefore, the efficient preparation of zwitterionic peptide-capture probe conjugates and direct modification to the electrode can effectively solve this problem. In addition, compared with antibody or peptide recognition probes, nucleic acid aptamer probes are cheaper, which is conducive to saving sensor preparation costs; they have less steric hindrance, which is conducive to obtaining higher detection sensitivity of sensors; corresponding to a wider range of specific recognition targets, the sensors are more versatile. However, there are no reports on the application of photoelectrochemical sensors based on this type of zwitterionic peptide-nucleic acid aptamer probe conjugates (abbreviated as peptide-nucleic acid conjugates).

[0005] Therefore, the development of a photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugates can effectively solve the bottleneck problem of traditional anti-biological contamination sensors. It can not only resist the problem of nonspecific adsorption of biological macromolecules such as proteins in actual complex biological samples, but also give the sensor higher sensitivity and a simpler preparation process. It has potential practical application capabilities for the detection of targets in actual complex biological samples such as blood, and also has far-reaching significance for the precise diagnosis of diseases in vitro. Summary of the invention

[0006] In view of this, one of the purposes of the present invention is to provide a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate with high sensitivity, resistance to biological contamination and simple preparation, in order to solve the problems existing in the prior art.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The first technical purpose of the present invention is to provide a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate, wherein the biosensor is prepared by directly modifying the polypeptide-nucleic acid conjugate on a photoelectrode, and utilizing the obvious steric hindrance effect of the target biomarker to hinder the charge transfer of the sensor to realize photocurrent signal detection.

[0009] Furthermore, in the polypeptide-nucleic acid conjugate, the polypeptide is a branched zwitterionic polypeptide BZP, and the amino acid sequence is (CESKSESKS)2KSEPPPPEK-N3; the nucleic acid is a mucin 1 nucleic acid aptamer pDNA, and the base sequence is 5'-GCA GTTGAT CCT TTG GAT ACC CTG G-DBCO-3', and BZP and pDNA are formed by a fast and efficient click chemistry reaction.

[0010] More specifically, the sensor is prepared by directly modifying a conjugate formed by a branched zwitterionic polypeptide with anti-biological contamination and a nucleic acid aptamer that specifically recognizes mucin 1 on a photoelectrode, and utilizing the obvious steric hindrance effect of mucin 1 to hinder the charge transfer of the sensor to achieve photocurrent signal detection.

[0011] It should be noted that mucin 1 (MUC1) is a protein expressed in many tissues, and its detection is of great significance in medicine, especially in the diagnosis and treatment of cancer. MUC1 is overexpressed in many cancers, including breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, etc. Detecting the level of MUC1 can be used as a biomarker for cancer, which is helpful for early diagnosis and monitoring of the development of cancer. Therefore, the present invention uses MUC1 as a target detection object, which is representative.

[0012] In addition, relevant data have shown that branched zwitterionic peptides (BZP) have more stable interface fixation ability, increased molecular intracavity spatial repulsion and stronger water permeability than linear zwitterionic peptides (LZP), so BZP can more effectively prevent non-specific proteins and other biological macromolecules from being adsorbed on the electrode surface through hydrophobic interactions and electrostatic effects, showing excellent anti-biological contamination ability. However, there has been no report on the application of BZP-pDNA conjugates in the field of biosensors, and the detection of MUC1 in blood has more potential practical application capabilities.

[0013] The second technical purpose of the present invention is to provide a method for preparing the above-mentioned photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate, and the specific steps include:

[0014] (1) Preparation of PDA / HOF-101 / ZnO photoelectrode: Using a UV-absorbing ZnO nanoarray electrode as a substrate, two sensitizers, a visible-light-absorbing hydrogen-bonded organic framework material (HOF-101) and polydopamine (PDA), were modified on the surface of the substrate to prepare a PDA / HOF-101 / ZnO photoelectrode;

[0015] (2) Synthetic peptide-nucleic acid conjugates: The branched zwitterionic peptide (BZP) with anti-biological contamination and the nucleic acid aptamer (pDNA) that specifically recognizes the target MUC1 are coupled in one step through a fast and efficient click chemistry reaction to form a peptide-nucleic acid (BZP-pDNA) conjugate.

[0016] (3) The BZP-pDNA conjugate synthesized in step (2) is directly anchored to the PDA / HOF-101 / ZnO photoelectrode prepared in step (1), thereby obtaining the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

[0017] By adopting the above technical solution, the beneficial effects of the present invention are as follows:

[0018] The preparation method disclosed by the invention is simple, rapid, efficient, easy to operate, and suitable for promotion and application.

[0019] Preferably, in the step (1), a ZnO nanoarray is prepared by a wet chemical method, and a ZnO nanoarray electrode is obtained by treating cyclohexamethylenetetramine and a Zn(NO3)2 aqueous solution at 95°C; methanol is quickly added to an anhydrous DMF solution of 1,3,6,8-tetra[p-benzoyl]pyrene (H4TBAPy), stirred evenly and allowed to stand, and the precipitate is collected by centrifugation and washing to obtain a HOF-101 product; a HOF-101 dispersion is prepared by pure water, and dripped onto the ZnO nanoarray electrode, and dried at room temperature to form a HOF-101 / ZnO electrode; a Tris-HCl buffer solution (pH 8.5, 10 mM) containing dopamine is dripped onto the HOF-101 / ZnO electrode, and dopamine self-polymerizes at room temperature to form PDA, thereby obtaining the desired PDA / HOF-101 / ZnO photoelectrode.

[0020] It should be noted that the electrode material used in the PDA / HOF-101 / ZnO photoelectrode is environmentally friendly, has strong biocompatibility, a simple preparation process, and has a very obvious photocurrent signal response and good photochemical stability.

[0021] Further preferably, the wet chemical method for preparing the ZnO nanoarray electrode has a treatment time of 2 to 5 hours; when preparing the HOF-101 / ZnO electrode, the concentration of the HOF-101 dispersion is 0.5 to 2.0 mg / mL; when preparing the PDA / HOF-101 / ZnO photoelectrode, the concentration of the dopamine solution is 5 to 20 mM.

[0022] Preferably, in step (2), the click chemistry reaction successfully occurs between the exposed azide group (-N3) on BZP and the dibenzocyclooctyne (DBCO) modified at the 3' end of pDNA, and after stirring on a shaker at room temperature, a BZP-pDNA conjugate is obtained.

[0023] It should be noted that the process of obtaining the BZP-pDNA conjugate by click chemistry reaction is fast, efficient, simple and convenient, and the reaction degree is thorough.

[0024] Further preferably, the concentrations of BZP and pDNA used in the preparation of the BZP-pDNA conjugate are the same and are 20-100 μM, and the stirring is carried out on a shaker at room temperature for 5 minutes to 2 hours.

[0025] Preferably, in the step (3), a BZP-pDNA conjugate is added dropwise to the PDA / HOF-101 / ZnO photoelectrode, incubated at low temperature, and then rinsed with Tris-HCl buffer to finally obtain the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

[0026] It should be noted that the above-mentioned photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugates not only has mild construction conditions, simple and fast process steps, and low consumption of biological samples, but also has simple detection operation for the target MUC1, no purification is required, and it has high sensitivity, strong resistance to biological contamination, and is accurate and fast.

[0027] Further preferably, the concentration of the BZP-pDNA conjugate during low temperature incubation is 10-50 μM.

[0028] Exemplary, the preferred preparation scheme of the present invention is:

[0029] (1) Using a UV-absorbing ZnO nanoarray electrode as a substrate, two sensitizers, a visible-light-absorbing hydrogen-bonded organic framework material (HOF-101) and polydopamine (PDA), were modified on the substrate surface to prepare a PDA / HOF-101 / ZnO photoelectrode:

[0030] 1) A ZnO nanoarray was prepared by a wet chemical method. A ZnO seed layer was deposited on an original indium tin oxide electrode using a 5 mM Zn(AC)2 ethanol solution. The ZnO seed layer electrode was immersed in an aqueous solution containing 25 mM hexamethylenetetramine and Zn(NO3)2 for 3 hours at 95°C to obtain a ZnO nanoarray electrode.

[0031] 2) Prepare 1 mL of 1 mg / mL 1,3,6,8-tetrakis(p-benzoyl)pyrene (H4TBAPy) anhydrous DMF solution, quickly add 8 mL of methanol, stir for 10 minutes, let stand for 30 minutes, collect the precipitate by centrifugation, and then wash it thoroughly with deionized water several times to obtain HOF-101 product;

[0032] 3) using pure water to prepare a HOF-101 dispersion with a concentration of 1 mg / mL, dropping 20 μL of the HOF-101 dispersion on the ZnO nanoarray electrode, and drying at room temperature to form a HOF-101 / ZnO electrode;

[0033] 4) A 10 mM dopamine solution was prepared using Tris-HCl buffer (pH 8.5, 10 mM), and 10 μL of the dopamine solution was dropped onto the HOF-101 / ZnO electrode. After self-polymerization at room temperature for 30 minutes, a PDA / HOF-101 / ZnO photoelectrode was obtained.

[0034] (2) The branched zwitterionic peptide (BZP) with anti-biological contamination and the nucleic acid aptamer (pDNA) that specifically recognizes the target MUC1 are coupled in one step through a fast and efficient click chemistry reaction to form a peptide-nucleic acid (BZP-pDNA) conjugate:

[0035] 1 mL of 40 μM BZP solution and 1 mL of 40 μM pDNA solution were prepared using Tris-HCl buffer (pH 7.4, 10 mM), 500 μL of the BZP solution and 500 μL of the pDNA were mixed at room temperature, and stirred on a shaker for 5 minutes to obtain a BZP-pDNA conjugate with a final concentration of 20 μM.

[0036] (3) The BZP-pDNA conjugate synthesized in step (2) is directly anchored to the PDA / HOF-101 / ZnO photoelectrode prepared in step (1), thereby obtaining the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

[0037] 20 μL of 20 μM BZP-pDNA conjugate was added to the PDA / HOF-101 / ZnO photoelectrode, incubated at low temperature for 6 hours, and the electrode was gently rinsed with Tris-HCl buffer (pH 7.4, 10 mM) to complete the construction of the sensing electrode, and finally the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate was obtained.

[0038] The third technical purpose of the present invention is to provide the application of the above-mentioned photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate in in vitro detection products.

[0039] In some application scenarios, the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate is also used to detect MUC1 in blood.

[0040] Furthermore, the test environment of the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate is a buffer solution with a pH of 6.5 to 7.5, and ascorbic acid is used as an electron donor.

[0041] It can be seen from the above technical solutions that, compared with the prior art, the present invention provides a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate and a preparation method and application thereof, which has the following excellent effects:

[0042] 1) The aptamer sensor prepared by the present invention utilizes for the first time a conjugate formed by a branched zwitterionic peptide with anti-biofouling properties and a nucleic acid aptamer with a specific target to construct a photoelectrochemical aptamer sensor, which effectively solves the problem of competition between the modification of zwitterionic peptides and capture probes on electrodes in traditional anti-biofouling sensors, and further improves the sensitivity and anti-biofouling ability of the sensor.

[0043] 2) The aptamer sensor prepared by the present invention has the remarkable characteristics of simple device, convenient operation, fast signal response, low background interference, and self-powered system. At the same time, the excellent ability of polypeptide-nucleic acid conjugates to resist biomacromolecule contamination of the sensing interface and the obvious steric hindrance effect of the target MUC1 on the sensor charge transfer are utilized to realize photocurrent signal detection.

[0044] 3) The preparation process of the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate provided by the present invention has simple and efficient steps, mild conditions, simple operation and low cost. The detection of the target MUC1 does not require actual sample purification, is convenient and rapid, and the biosensor has the potential to accurately and sensitively detect MUC1 in actual biological samples. It is suitable for market promotion and application, and has far-reaching significance for in vitro diagnosis of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the embodiment will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0046] Figure 1 This is the photocurrent signal response diagram of the HOF-101 / ZnO electrode corresponding to different concentrations of HOF-101 dispersions in Example 1.

[0047] Figure 2 This is the photocurrent signal response diagram of the PDA / HOF-101 / ZnO photoelectrode corresponding to dopamine solutions of different concentrations in Example 1.

[0048] Figure 3 This is the photocurrent signal response diagram of the modified photoelectrode corresponding to different incubation concentrations of BZP-pDNA conjugates in Example 2.

[0049] Figure 4This is a scanning electron microscope photograph of the PDA / HOF-101 / ZnO photoelectrode preparation process in Example 3.

[0050] Figure 5 The X-ray diffraction diagram and X-ray photoelectron spectrum diagram of the preparation process of the PDA / HOF-101 / ZnO photoelectrode in Example 3.

[0051] Figure 6 This is the UV-visible diffuse reflectance spectrum of the PDA / HOF-101 / ZnO photoelectrode preparation process in Example 4.

[0052] Figure 7 This is the polyacrylamide gel electrophoresis diagram of the BZP-pDNA conjugate synthesis process in Example 5.

[0053] Figure 8 This is a photocurrent signal response diagram of the photoelectrochemical aptamer sensor preparation process described in Example 6.

[0054] Fig. 9 This is a standard curve diagram of the photoelectrochemical aptamer sensor described in Example 7 for detecting the target MUC1.

[0055] Fig.10 This is a graph showing the anti-biological contamination performance test data of the photoelectrochemical aptamer sensor described in Example 8.

[0056] Fig.11 This is a graph showing the specific recognition performance test data of the photoelectrochemical aptamer sensor described in Example 9.

[0057] Fig.12 This is a data analysis diagram of the photoelectrochemical aptamer sensor described in Example 10 detecting the target in a serum sample. DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0060] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0061] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0062] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0063] First of all, it should be noted that the current signal response in the experimental process of the following examples was tested on a photoelectrochemical system, as follows:

[0064] Use white light emission, wavelength 200nm ~ 2500nm, light intensity 150W / m 2 A xenon lamp was used as the excitation light source, which was turned on and off every 10 seconds. The photocurrent was recorded by an electrochemical workstation. When testing the current signal response, a three-electrode system was used: a photoelectrode with a modified area of ​​0.5 cm × 0.5 cm was used as the working electrode, a platinum wire electrode was used as the counter electrode, and an Ag / AgCl electrode was used as the reference electrode; and there was no external voltage applied to the system, the system was self-powered, and the test solution was Tris-HCl buffer (pH 7.4, 10 mM) containing 0.1 M ascorbic acid.

[0065] Example 1

[0066] Since the photocurrent signal response size of the PDA / HOF-101 / ZnO photoelectrode has an important influence on the detection sensitivity of the final prepared photoelectrochemical aptamer sensor based on peptide-nucleic acid conjugate, the main preparation process parameters of the PDA / HOF-101 / ZnO photoelectrode were optimized as follows:

[0067] 1) Since the modification amount of HOF-101 on the electrode can be adjusted by the concentration of the HOF-101 dispersion, the concentration of the HOF-101 dispersion is optimized as follows:

[0068] HOF-101 dispersions with concentrations of 0 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL were prepared with pure water, and 20 μL of the HOF-101 dispersion was dripped onto the ZnO nanoarray electrode. After drying at room temperature, a HOF-101 / ZnO electrode was obtained.

[0069] By performing photocurrent characterization tests, the following Figure 1 As shown in the figure, when the concentration of HOF-101 dispersion is 1 mg / mL, the photocurrent signal response of HOF-101 / ZnO electrode reaches the best. This is because HOF-101 with appropriate thickness can effectively improve the photoelectric conversion efficiency of the photoelectrode, while HOF-101 with excessive thickness will significantly increase the probability of charge recombination, resulting in electron annihilation. Therefore, the concentration of HOF-101 dispersion 1 mg / mL is selected as the optimal preparation process parameter.

[0070] 2) Since the modification thickness of dopamine (DA) on the electrode can be adjusted by the concentration of DA solution, the concentration of DA solution is optimized as follows:

[0071] DA solutions with concentrations of 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM were prepared using Tris-HCl buffer (pH 8.5, 10 mM), and 10 μL of the DA solution was dropped onto the HOF-101 / ZnO electrode. After self-polymerization at room temperature for 30 minutes, a PDA / HOF-101 / ZnO photoelectrode was obtained.

[0072] By performing photocurrent characterization tests, it can be obtained, as shown in the attached Figure 2 As shown in the figure, when the DA solution concentration is 10mM, the photocurrent signal response of the PDA / HOF-101 / ZnO photoelectrode reaches the best. This is because the appropriate thickness of PDA can further effectively improve the photoelectric conversion efficiency of the photoelectrode, while the excessive thickness of HOF-101 will significantly increase the probability of charge recombination of the photoelectrode, resulting in electron annihilation. Therefore, the DA solution concentration of 10mM is selected as the optimal preparation process parameter.

[0073] Example 2

[0074] Since the modification amount of BZP-pDNA conjugate on the sensing electrode has a significant impact on the quantitative detection range of the final prepared photoelectrochemical aptamer sensor based on peptide-nucleic acid conjugate, the preparation process parameters of the modification amount of BZP-pDNA conjugate on the sensing electrode were optimized:

[0075] Since the modification amount on the sensing electrode can be reflected by its incubation concentration on the electrode, the incubation concentration of the BZP-pDNA conjugate was optimized.

[0076] 20 μL of BZP-pDNA conjugates with concentrations of 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM were added to the PDA / HOF-101 / ZnO photoelectrode, incubated at low temperature for 6 hours, and the electrode was gently rinsed with Tris-HCl buffer (pH 7.4, 10 mM) to complete the construction of the sensing electrode, and finally the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate was obtained.

[0077] By performing photocurrent characterization tests, it can be obtained, as shown in the attached Figure 3 As shown, the incubation concentration of the BZP-pDNA conjugate needs to be greater than or equal to 20 μM, so as to ensure that the BZP-pDNA conjugate is fully anchored on the sensing electrode to obtain the best quantitative detection range. Therefore, a BZP-pDNA conjugate greater than or equal to 20 μM is selected as the optimal incubation concentration.

[0078] Example 3

[0079] Preparation of PDA / HOF-101 / ZnO photoelectrode

[0080] The ZnO nanoarray was prepared by a wet chemical method. A ZnO seed layer was deposited on the original indium tin oxide electrode using a 5 mM Zn(AC)2 ethanol solution. The ZnO seed layer electrode was immersed in an aqueous solution containing 25 mM hexamethylenetetramine and Zn(NO3)2 and treated at 95°C for 3 hours to obtain a ZnO nanoarray electrode. 1 mL of anhydrous DMF solution of 1 mg / mL 1,3,6,8-tetra[p-benzoic acid]pyrene (H4TBAPy) was prepared, and 8 mL of methanol was quickly added. After stirring for 10 minutes, the solution was allowed to stand for 30 minutes, and the precipitate was collected by centrifugation. The precipitate was then washed thoroughly with deionized water for multiple times to obtain a HOF-101 product. A HOF-101 dispersion with a concentration of 1 mg / mL was prepared using pure water, and 20 μL of the HOF-101 dispersion was dripped onto the ZnO nanoarray electrode. After drying at room temperature, a HOF-101 / ZnO electrode was formed. A Tris-HCl buffer (pH 8.5, 10 mM) was prepared with a dopamine solution of 10 mM concentration, and 10 μL of the dopamine solution was dropped onto the HOF-101 / ZnO electrode. Dopamine self-polymerized at room temperature for 30 minutes to form PDA, thereby obtaining a PDA / HOF-101 / ZnO photoelectrode.

[0081] Scanning electron microscopy images are attached. Figure 4 As shown in a, it can be seen from the figure that the ZnO nanoarray electrode is composed of highly ordered, tightly and vertically arranged ZnO nanorods with a rod diameter of 60 to 90 nm. The scanning electron microscope image is shown in the attached figure. Figure 4As shown in Figure b, it can be seen that the surface of the ZnO nanoarray electrode is covered with many large-sized rod-shaped HOF-101 with a diameter of 300-400nm and a length of 8-12μm, proving that the HOF-101 / ZnO electrode was successfully prepared. The scanning electron microscope image is shown in the attached figure. Figure 4 As shown in c, it can be seen from the figure that the surface of the HOF-101 rod-like structure is covered with a compact PDA film; the characteristic N element mapping analysis in the illustration further illustrates the successful modification of PDA, proving that the PDA / HOF-101 / ZnO photoelectrode was successfully prepared.

[0082] The X-ray diffraction of the HOF-101 / ZnO electrode is shown in the attached Figure 5 As shown in a, the diffraction peaks at 31.8°, 34.4°, 36.3°, 47.6°, 56.6°, 62.8°, 66.4°, 67.9° and 69.1° correspond to the (100), (002), (101), (102), (110), (103), (200), (112) and (201) crystal planes of pure hexagonal ZnO (PDFNo.36-1451), respectively. After modification of HOF-101, characteristic diffraction peaks of HOF-101 appeared in the XRD spectrum, which were located at 2θ=8.7°, 9.8° and 13.2°, respectively, which confirmed the successful preparation of HOF-101 / ZnO electrode. X-ray photoelectron spectroscopy of PDA / HOF-101 / ZnO photoelectrode Figure 5 As shown in Figure b, the characteristic peaks of Zn 3d, Zn 3p, Zn 3s, Zn 2p, O 1s and C1s are derived from the HOF-101 / ZnO electrode, while the typical N1s characteristic peak is attributed to the presence of PDA. This further confirms the successful preparation of the PDA / HOF-101 / ZnO photoelectrode.

[0083] Example 4

[0084] Spectral Absorption Characterization of PDA / HOF-101 / ZnO Photoelectrode

[0085] UV-Vis diffuse reflectance spectra of the step-by-step formation process of the PDA / HOF-101 / ZnO photoelectrode, as shown in the attached figure. Figure 6As shown. It can be observed that the spectral absorption edge of the ZnO nanoarray electrode is roughly located at 425nm, and can only absorb ultraviolet light; after modifying HOF-101, the spectral absorption edge of the HOF-101 / ZnO electrode is significantly extended to 532nm, the light absorption range is significantly expanded, and the electron transfer ability is improved; after further modification of the PDA film, the spectral absorption edge of the PDA / HOF-101 / ZnO photoelectrode is extended to 605nm, indicating further enhanced visible light absorption. Therefore, it is proved that the PDA / HOF-101 / ZnO photoelectrode has a wide range of spectral absorption bands, which is conducive to increasing light absorption efficiency and improving photocurrent intensity.

[0086] Example 5

[0087] Synthesis of BZP-pDNA Conjugate

[0088] The coupling reaction between BZP and pDNA was completed in one step by a fast and efficient click chemistry reaction to form a polypeptide-nucleic acid BZP-pDNA conjugate. 1 mL of 40 μM BZP solution and 1 mL of 40 μM pDNA solution were prepared using Tris-HCl buffer (pH 7.4, 10 mM), and 500 μL of the BZP solution and 500 μL of the pDNA were mixed at room temperature. After stirring on a shaker for 5 minutes, a BZP-pDNA conjugate with a final concentration of 20 μM was obtained.

[0089] Polyacrylamide gel electrophoresis (PAGE) is shown in the attached figure. Figure 7 As shown in the figure, DBCO-modified pDNA and -N3 group-modified BZP were mixed in a molar ratio of 1:1 and reacted at different reaction times of 5 minutes, 10 minutes, 1 hour, 2 hours and 4 hours to obtain BZP-pDNA conjugates. Lane 2 represents the pDNA control, while lanes 3, 4, 5, 6 and 7 correspond to the BZP-pDNA conjugates synthesized at 5 minutes, 10 minutes, 1 hour, 2 hours and 4 hours, respectively. It is clearly observed from the figure that the PAGE band of the BZP-pDNA conjugate has a significant upward shift relative to the pDNA control band, indicating the successful coupling of BZP and pDNA. In addition, at the 5-minute coupling time corresponding to lane 3, the pDNA control band almost completely disappeared, confirming the remarkable characteristics of the rapid and efficient synthesis of BZP-pDNA conjugates by click chemistry.

[0090] Example 6

[0091] Preparation of photoelectrochemical aptamer sensors based on peptide-nucleic acid conjugates

[0092] 20 μL of 20 μM BZP-pDNA conjugate was added to the PDA / HOF-101 / ZnO photoelectrode, incubated at low temperature for 6 hours, and the electrode was gently rinsed with Tris-HCl buffer (pH 7.4, 10 mM) to complete the construction of the sensing electrode, and finally the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate was obtained.

[0093] The successful preparation of the photoelectrochemical aptamer sensor based on peptide-nucleic acid conjugates was verified by photocurrent signal response test. Figure 8 As shown. The nanoarray electrode has a relatively small photocurrent signal response (curve a), which is due to its limited ultraviolet light absorption ability; after modification of HOF-101, the photocurrent signal response of the HOF-101 / ZnO electrode increased significantly (curve b), because HOF-101 has excellent electron transfer ability and a significant expansion of the visible light absorption range; after further modification of PDA, the photocurrent signal response of the PDA / HOF-101 / ZnO photoelectrode is further enhanced (curve c), which is attributed to the additional expansion of the visible light absorption range of the PDA film. The PDA film can also provide a large number of binding sites for the subsequent anchoring of the BZP-pDNA conjugate; after the BZP-pDNA conjugate is anchored to the photoelectrode, the photocurrent signal response decreases (curve d), which is due to the weak charge conduction ability of the BZP-pDNA conjugate. Therefore, the photocurrent signal response test proves the successful preparation of the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

[0094] Example 7

[0095] Detection of the target MUC1 by the prepared photoelectrochemical aptamer sensor

[0096] The photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate prepared in Example 6 was incubated with 20 μL of different concentrations of the target MUC1 at room temperature for 1.5 hours to allow the target MUC1 to undergo a specific recognition reaction with the MUC1 nucleic acid aptamer in the conjugate. After washing with Tris-HCl buffer (pH 7.4, 10 mM), the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate incubated with the target MUC1 was finally tested for photocurrent signal in Tris-HCl buffer (pH 7.4, 0.1 M) containing an electron donor 0.1 M ascorbic acid, and photocurrent signal detection was achieved by utilizing the obvious steric hindrance effect of the target MUC1 to hinder the charge transfer of the sensor.

[0097] The detection results show that as the concentration of the target MUC1 increases, the corresponding photocurrent detection signal gradually weakens, and in the range of MUC1 concentration from 1pg / mL to 10ng / mL, the photocurrent detection signal and the logarithm of the target MUC1 concentration show a good linear relationship. Fig. 9 As shown, the linear fitting equation is I=14.02-2.41logC (ng / mL), the linear correlation coefficient is 0.9989, and the experimental minimum detection limit is 0.36pg / mL, which indicates that the photoelectrochemical aptamer sensor of the polypeptide-nucleic acid conjugate prepared by the present invention has high sensitivity in detecting the target MUC1.

[0098] Example 8

[0099] Anti-biological contamination performance test of the prepared photoelectrochemical aptasensor for detecting MUC1

[0100] The photoelectrode anchored by the BZP-pDNA conjugate prepared in Example 5 was incubated with serum samples of different dilution concentrations at room temperature for 1.5 hours, and the electrode was washed with Tris-HCl buffer (pH 7.4, 10mM), and then the photocurrent signal response test was performed. For comparison, the photoelectrode anchored by pDNA alone was also incubated with serum samples of different dilution concentrations under the same conditions and the electrode was washed.

[0101] The photocurrent signal changes as shown in the attached Fig.10 As shown, even when incubated with 30% diluted human serum, the photoelectrode anchored by the BZP-pDNA conjugate showed a very small signal change (less than 5%), indicating its significant anti-pollution ability. In contrast, the photoelectrode anchored by pDNA alone also showed a significant signal change in 1% diluted human serum, indicating that it has obvious deficiencies in anti-pollution performance. The above comparison results fully demonstrate the excellent anti-biological contamination ability of the BZP-pDNA conjugate in the design of the aptamer sensor.

[0102] Example 9

[0103] Specific recognition performance test of the prepared photoelectrochemical aptamer sensor for detecting MUC1

[0104] In order to prove that the above-mentioned photoelectrochemical aptasensor for detecting MUC1 has excellent specific recognition ability, protein macromolecules commonly found in blood: human immunoglobulin G (HIgG), carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and neuron-specific enolase (NSE) were selected as typical interferents for specificity verification experiments. The specific operations are as follows:

[0105] 1 ng / mL of the target substance MUC1 and 10 ng / mL of interfering proteins HIgG, CA125, CEA, AFP, and NSE were added separately or mixed into 10% diluted serum, and the photoelectrochemical aptamer sensor for detecting MUC1 prepared by the present invention was used for detection according to the above method. The changes in the photocurrent signal were as shown in the attached figure. Fig.11 As shown. The results show that the photocurrent changes caused by the interfering proteins HIgG, CA125, CEA, AFP, NSE and their mixtures are negligible relative to the detection of the target MUC1. This proves that the photoelectrochemical aptamer sensor for detecting MUC1 prepared by the present invention has excellent specific recognition ability for the target MUC1 and is not affected by interfering proteins.

[0106] Example 10

[0107] The prepared photoelectrochemical aptasensor for detecting MUC1 was used for detection in serum

[0108] The practical application capability of the high-performance photoelectrochemical aptamer sensor for detecting MUC1 prepared by the present invention was evaluated by a spike recovery experiment in serum samples, and the specific operation is as follows:

[0109] The 10% diluted serum was divided into three groups, and MUC1 standard samples with concentrations of 100 pg / mL, 1 ng / mL, and 10 ng / mL were added respectively. The photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate prepared in Example 6 was used to incubate 20 μL of the above serum spiked samples at different concentrations at room temperature for 1.5 hours. After cleaning the electrode, the photocurrent signal was tested 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 7.

[0110] The comparison results of spike recovery experiments in serum samples are shown in the attached Fig.12 As shown in the figure, Serum and PBS represent serum and buffer solution respectively. The recovery rate of the spiked samples is in the range of 96.5% to 104.8%, and the relative standard deviation of the test results is less than 4.5%. This proves that the prepared photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate has excellent application potential in accurately detecting the target MUC1 in actual complex biological samples, and can achieve rapid, sensitive, accurate and efficient detection of the target.

[0111] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate, characterized in that: The sensor is prepared by directly modifying a polypeptide-nucleic acid conjugate on a photoelectrode, and photocurrent signal detection is achieved by utilizing the obvious steric hindrance effect of the target biomarker to hinder the charge transfer of the sensor; In the polypeptide-nucleic acid conjugate, the polypeptide is a branched zwitterionic polypeptide BZP, and the amino acid sequence is (CESKSESKS)2KSEPPPPEK-N3; the nucleic acid is a mucin 1 nucleic acid aptamer pDNA, and the base sequence is 5'-GCA GTT GAT CCT TTG GATACC CTG G-DBCO -3', and BZP and pDNA are formed by a fast and efficient click chemistry reaction.

2. A method for preparing a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate as claimed in claim 1, characterized in that: The specific steps include: (1) Preparation of PDA / HOF-101 / ZnO photoelectrode: Using a UV-absorbing ZnO nanoarray electrode as a substrate, two sensitizers, HOF-101 and polydopamine PDA, which absorb visible light, are sequentially modified on the surface of the substrate to prepare a PDA / HOF-101 / ZnO photoelectrode; (2) Synthetic peptide-nucleic acid conjugate: The branched zwitterionic peptide BZP with anti-biological contamination and the nucleic acid aptamer pDNA that specifically recognizes mucin 1 are coupled in one step through a fast and efficient click chemistry reaction to form a BZP-pDNA conjugate: (3) The BZP-pDNA conjugate synthesized in step (2) is directly anchored to the PDA / HOF-101 / ZnO photoelectrode prepared in step (1), thereby obtaining the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

3. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 2, characterized in that: In the step (1), a ZnO nanoarray is prepared by a wet chemical method, and a ZnO nanoarray electrode is obtained by treating cyclohexamethylenetetramine and a Zn(NO3)2 aqueous solution at 95°C; methanol is quickly added to an anhydrous DMF solution of 1,3,6,8-tetra[p-benzoyl]pyrene H4TBAPy, the mixture is stirred evenly and allowed to stand, and a precipitate is collected by centrifugation and washing to obtain a HOF-101 product; a HOF-101 dispersion is prepared by pure water, and the HOF-101 dispersion is dripped onto the ZnO nanoarray electrode, and the HOF-101 / ZnO electrode is formed after drying at room temperature; a dopamine solution is prepared by Tris-HCl buffer, and the dopamine solution is dripped onto the HOF-101 / ZnO electrode, and the desired PDA / HOF-101 / ZnO photoelectrode is obtained after self-polymerization at room temperature for 30 minutes.

4. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 3, characterized in that: The wet chemical method for preparing the ZnO nanoarray electrode has a treatment time of 2 to 5 hours; when preparing the HOF-101 / ZnO electrode, the concentration of the HOF-101 dispersion is 0.5 to 2.0 mg / mL; when preparing the PDA / HOF-101 / ZnO photoelectrode, the concentration of the dopamine solution is 5 to 20 mM.

5. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 2, characterized in that: In the step (2), the BZP-pDNA conjugate is coupled in one step by a fast and efficient click chemistry reaction, and the click chemistry reaction successfully occurs between the exposed azide group -N3 on the BZP and the dibenzocyclooctyne DBCO modified at the 3' end of the pDNA; the BZP modified with the azide group and the pDNA are mixed at room temperature and stirred on a shaker to obtain a BZP-pDNA conjugate with a concentration of 10-50 μM.

6. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 5, characterized in that: The concentrations of BZP and pDNA used in the preparation of the BZP-pDNA conjugate are the same, both 20-100 μM, and the stirring is carried out on a shaker at room temperature for 5 minutes to 2 hours.

7. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 2, characterized in that: In the step (3), a BZP-pDNA conjugate is added dropwise to the PDA / HOF-101 / ZnO photoelectrode, and after low-temperature incubation, the electrode is gently rinsed with Tris-HCl buffer to complete the construction of the sensing electrode, thereby obtaining the photoelectrochemical aptamer sensor based on the polypeptide-nucleic acid conjugate.

8. The method for preparing the photoelectrochemical aptamer sensor based on polypeptide-nucleic acid conjugate according to claim 7, characterized in that: The concentration of the BZP-pDNA conjugate during low temperature incubation is 10-50 μM.

9. A method for preparing a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate as claimed in claim 1 or a use of a photoelectrochemical aptamer sensor based on a polypeptide-nucleic acid conjugate prepared by any of the methods described in claims 2 to 8 in an in vitro diagnostic product.

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