Antifouling Photoelectrochemical DNA Sensor Constructed Based on Peptide-Hairpin DNA Conjugates

Abstract

The present invention provides an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate, which relates to the technical field of HIV detection. The detection process of the anti-pollution photoelectrochemical DNA sensor includes the preparation of a PDA / TCPP / COF-V photoanode, the preparation of a conjugate LZP-hDNA, the preparation of a nucleic acid probe pDNA-AgInS2, the construction of an anti-pollution DNA sensor, and photoelectrochemical testing. The present invention uses a click reaction to react a linear zwitterionic peptide LZP modified with an -N3 azide group and a hairpin DNA (hDNA) modified with 3'-dibenzocyclooctyne (DBCO) to form an LZP-hDNA conjugate. This conjugate not only has an anti-pollution effect, but the presence of hDNA also facilitates the progress of the λ-Exo-assisted target recycling amplification reaction. By modifying a composite material of meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and polydopamine (PDA) on a COF-V electrode, a ternary composite photoanode of PDA / TCPP / COF-V is prepared, which serves as a signal transducer for the sensing platform and firmly anchors the LZP-hDNA conjugate.

Description

Technical Field

[0001] The present invention relates to the technical field of HIV detection, and specifically to an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate. Background Art

[0002] Highly sensitive and selective detection of specific DNA sequences is of great significance for early cancer screening, mutation analysis, environmental monitoring, molecular diagnosis, etc. Photoelectrochemical biosensing is an emerging and booming analytical detection technology. Its working principle is based on the photocurrent change triggered by a specific biorecognition event between a capture probe and a target. Due to the complete separation between the detection signal and the excitation source, compared with traditional optical and electrochemical analyses, PEC detection has high sensitivity and low background signals. In order to detect specific DNA sequences at lower content levels, various efficient signal amplification strategies have been developed successively, including hybridization chain reaction, rolling circle amplification, catalytic hairpin assembly, polymerase chain reaction, and exonuclease-assisted target recycling. Among them, exonuclease-assisted target recycling has the characteristics of not requiring additional labeling steps, auxiliary substances, and short incubation time, showing its superiority. Generally, λ-exonuclease (λ-Exo) can recognize double-stranded DNA and catalyze the stepwise hydrolysis of single nucleotides in the 5' to 3' direction. This property makes λ-Exo an effective tool for DNA signal amplification. Nevertheless, there are still some deficiencies in the actual detection of complex biological samples by photoelectrochemical DNA biosensors.

[0003] The following technical problems exist in the prior art during use:

[0004] Problem 1: There are some potential interfering proteins in actual biological samples. These proteins will non-specifically adsorb on the electrode interface, thereby affecting the analytical performance of the DNA biosensor.

[0005] Problem 2: In recent years, more and more biosensors use zwitterionic peptides to achieve anti-pollution effects. As is well known, zwitterionic peptides have the advantages of simple synthesis, non-toxicity, good biocompatibility, etc., which have attracted wide attention. However, currently most biosensors mainly involve the co-anchoring of zwitterionic peptides and capture probes, which leads to competitive immobilization between the two, thereby affecting the anti-pollution ability and sensitivity of the sensor.

[0006] Therefore, an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate is needed to solve the above problems. Summary of the Invention

[0007] Technical Problems to be Solved

[0008] Aiming at the deficiencies of the prior art, the present invention provides an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate. The method includes: by combining zwitterionic peptides, hairpin DNA conjugates, composite organic semiconductor materials, and signal amplification strategies, a novel anti-pollution PEC DNA detection platform is established for accurately and sensitively detecting the target analyte HIV in human body fluids.

[0009] Technical solution

[0010] To achieve the above objectives, the present invention is realized through the following technical solutions: an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate. The detection process of the anti-pollution photoelectrochemical DNA sensor includes the preparation of a PDA / TCPP / COF-V photoelectrode, the preparation of the conjugate LZP-hDNA, the preparation of the nucleic acid probe pDNA-AgInS2, the construction of the anti-pollution DNA sensor, and photoelectrochemical testing. The preparation of the PDA / TCPP / COF-V photoelectrode includes the following steps.

[0011] Sp1. When preparing the PDA / TCPP / COF-V photoelectrode, first dissolve 40 mg of 1,3,5-tris(4-aminophenyl)benzene and 32 mg of 1,4-dialdehyde-2,5-divinylbenzene in 20 mL of acetonitrile.

[0012] Sp2. Add 4 mL of 12 M acetic acid to the solution as a catalyst.

[0013] Sp3. Oscillate the solution obtained in Sp2 for 30 s and let it stand for 72 h. Centrifuge the solution using a centrifuge, collect the precipitate, and then wash it thoroughly with tetrahydrofuran and ethanol. Subsequently, place it in a vacuum dryer at 60 °C to obtain the final product COF-V.

[0014] Sp4. After obtaining COF-V according to the Sp3 step, weigh 5 mg of COF-V powder and disperse it in 10 mL of deionized water. After ultrasonic treatment for 30 min, obtain a uniformly dispersed COF-V suspension with a concentration of 0.5 mg / mL. Disperse 20 μL of the 0.5 mg / mL COF-V suspension uniformly on an ITO substrate electrode with a modified area of 0.25 cm2, and let it dry naturally at room temperature to obtain a COF-V modified electrode.

[0015] Sp5. Dissolve 5 mg of TCPP and 10 mg of DA in 5 mL of Tris-HCl buffer (10 mM, pH 8.5), and fully dissolve TCPP by ultrasonic treatment.

[0016] Sp6. Disperse 20 μL of this solution on the COF-V modified electrode, keep it standing still in the dark environment for 30 min to allow DA to fully self-polymerize, and finally wash the electrode with deionized water to obtain the desired PDA / TCPP / COF-V electrode.

[0017] Preferably, the preparation method of the conjugate LZP-hDNA is to mix 300 μL of 40 μM LZP and 300 μL of 40 μM hDNA solution, place it in a shaker, keep shaking at 37 °C for 5 min, and finally obtain a LZP-hDNA conjugate with a concentration of 20 μM.

[0018] Preferably, before the preparation of the nucleic acid probe pDNA-AgInS2, water-soluble AgInS2 quantum dots need to be prepared. When preparing the water-soluble AgInS2 quantum dots, first place 25 mL of an aqueous solution containing 0.1 mM MPA, 0.4 mM In(NO3)3, and 0.1 mM AgNO3 in a 50 mL three-necked flask.

[0019] Preferably, 1.5 mL of 0.2 M Na2S solution should be quickly added to the aqueous solution placed in the three-necked flask under rapid stirring, heat the solution to 100 °C, reflux and maintain for 2 h to allow the AgInS2 quantum dots to grow sufficiently, and finally store the obtained quantum dots at 4 °C for normal temperature preservation.

[0020] Preferably, the quantum dots are used to synthesize the pDNA-AgInS2 nucleic acid probe through an amide coupling reaction. When synthesizing the nucleic acid probe pDNA-AgInS2, mix 150 μL of AgInS2 quantum dots and 100 μL of 10 mM EDC / NHS solution, keep at room temperature for 30 min to fully activate the carboxyl groups on the surface of the AgInS2 quantum dots, then add 400 μL of 30 μM amino-modified pDNA to the above solution, react at room temperature for 2 h, and finally ultrafilter and purify the obtained solution and dilute it with 600 μL of Tris-HCl buffer, and place it in a 4 °C environment for standby.

[0021] Preferably, during the construction of the anti-pollution DNA sensor, disperse 20 μL of 20 μM LZP-hDNA conjugate on the surface of the PDA / TCPP / COF-V photoanode, incubate overnight in a humid environment at 4 °C, anchor LZP-hDNA to the photoanode surface through the Michael reaction, then wash the electrode with Tris-HCl buffer to remove the unconnected conjugate, subsequently incubate the electrode with 20 μL of tDNA solution containing 10 U λ-Exo at different concentrations at 37 °C for 1 h, after washing with Tris-HCl buffer, incubate the electrode with 20 μL of 20 μM pDNA-AgInS2 nucleic acid probe at 37 °C for another 1 h, and after washing with Tris-HCl buffer, the obtained electrode is used for subsequent PEC testing.

[0022] Preferably, during the photoelectrochemical test, the prepared sensing electrode is used as the working electrode, a platinum wire is used as the counter electrode, a saturated Ag / AgCl electrode is used as the reference electrode, the test buffer solution is a phosphate buffer solution, and the dissolved oxygen in the PBS solution is used as the electron acceptor.

[0023] Preferably, the instruments used in the detection process of the anti-fouling photoelectrochemical DNA sensor include a transmission electron microscope, a scanning electron microscope, an X-ray photoelectron spectrometer, an X-ray diffractometer, a Fourier transform infrared spectrometer, a photoelectrochemical workstation, an electrochemical workstation, a contact angle measuring instrument, a laser confocal microscope, and an ultraviolet-visible diffuse reflectance spectrometer.

[0024] Beneficial effects

[0025] The present invention provides an anti-fouling photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate. It has the following beneficial effects:

[0026] 1. The present invention uses click reaction to react the linear zwitterionic peptide LZP modified with -N3 azide group and the hairpin DNA (hDNA) modified with 3'-dibenzocyclooctyne (DBCO) to form an LZP-hDNA conjugate. This conjugate not only has an anti-fouling effect, but the presence of hDNA is also beneficial to the progress of the λ-Exo-assisted target recycling amplification reaction. By modifying the composite material of meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and polydopamine (PDA) on the COF-V electrode, a ternary composite photoanode PDA / TCPP / COF-V is prepared, which acts as a signal converter of the sensing platform and firmly anchors the LZP-hDNA conjugate. When tDNA and λ-Exo are present, tDNA can hybridize with hDNA to form a double-stranded structure, and λ-Exo will gradually hydrolyze it from the 5' to 3' direction and release tDNA to continue participating in the hybridization reaction, thereby playing a role in target recycling amplification. The uncut part of hDNA will further hybridize with the pDNA-AgInS2 signal probe. At this time, the AgInS2 quantum dots are close to the electrode, and due to the bandgap mismatch, the photocurrent is significantly reduced.

[0027] 2. In the present invention, scanning electron microscopy (SEM) further confirmed that the synthesized covalent organic framework (COF-V) has advantages such as uniform size and good dispersibility. The SEM images provide direct evidence of the morphology of the COF-V material, enabling researchers to visually evaluate the size distribution, uniformity, and dispersibility of the material. This is crucial for ensuring the high-quality materials used in subsequent experiments. As an important component of the photoanode, the morphology and properties of COF-V directly affect the photoelectric conversion efficiency and sensitivity of the sensor. COF-V with uniform size and good dispersibility helps to form a more uniform and stable photoanode surface, thereby improving the overall performance of the sensor. Due to the good morphology and stability of COF-V, it can provide a more stable and anti-pollution interface for the subsequently modified LZP-hDNA conjugate. This is crucial for maintaining the sensitivity and accuracy of the sensor in a complex biological environment.

[0028] 3. The uniformly distributed COF-V in the present invention helps the λ-Exo-assisted target cycling amplification reaction to proceed more effectively on the photoanode surface, which means that more tDNA can be released and participate in the cycling reaction, thereby increasing the number of AgInS2 quantum dots on the photoanode, resulting in a more significant decrease in photocurrent and improving the detection sensitivity. The quality of the COF-V material verified by SEM ensures the reliability and repeatability of the experimental results, which is crucial for the rigor and verifiability of scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a model diagram of the anti-pollution PEC DNA sensor constructed based on the zwitterionic peptide-hairpin DNA conjugate of the present invention;

[0030] Figure 2 It is a table diagram of the reagents and materials used in the present invention;

[0031] Figure 3 It is a table diagram of the test instruments used in the present invention;

[0032] Figure 4 It is a morphology diagram of COF-V of the present invention;

[0033] Figure 5 It is a characterization diagram of AgInS2 quantum dots and TCPP of the present invention;

[0034] Figure 6 It is a spectrogram of TCPP of the present invention;

[0035] Figure 7 It is a characterization diagram of the PDA / TCPP / COF-V photoanode of the present invention;

[0036] Figure 8 It is a PEC performance characterization diagram of the photoanode of the present invention;

[0037] Figure 9 Schematic diagram for the characterization of the LZP-hDNA conjugate and pDNA-AgInS of the present invention;

[0038] Figure 10 Schematic diagram for the characterization of the DNA sensor of the present invention;

[0039] Figure 11 Schematic diagram for the optimization of photocurrent response of the present invention;

[0040] Figure 12 Schematic diagram for the display of fluorescence microscope images of the present invention;

[0041] Figure 13 Performance analysis diagram of the DNA sensor of the present invention;

[0042] Figure 14 Schematic diagram for the anti-interference change of the present invention. Detailed implementation manners

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention. Specific embodiment 1:

[0045] As Figures 1-14 shown, an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate. The detection process of the anti-pollution photoelectrochemical DNA sensor includes the preparation of a PDA / TCPP / COF-V photoanode, the preparation of a conjugate LZP-hDNA, the preparation of a nucleic acid probe pDNA-AgInS2, the construction of an anti-pollution DNA sensor, and photoelectrochemical testing. The preparation of the PDA / TCPP / COF-V photoanode includes the following steps,

[0046] Sp1. When preparing the PDA / TCPP / COF-V photoanode, first dissolve 40 mg of 1,3,5-tris(4-aminophenyl)benzene and 32 mg of 1,4-dialdehyde-2,5-divinylbenzene in 20 mL of acetonitrile;

[0047] Sp2. Add 4 mL of 12 M acetic acid to the solution as a catalyst;

[0048] Sp3. Shake the solution obtained in Sp2 for 30 s and let it stand for 72 h. Centrifuge the solution using a centrifuge and collect the precipitate. Then wash it thoroughly with tetrahydrofuran and ethanol, and subsequently place it in a vacuum dryer at 60 °C to obtain the final product COF-V;

[0049] Sp4. After obtaining COF-V according to the steps of Sp3, weigh 5 mg of COF-V powder and disperse it in 10 mL of deionized water. After ultrasonic treatment for 30 min, a uniformly dispersed COF-V suspension with a concentration of 0.5 mg / mL is obtained. Disperse 20 μL of the 0.5 mg / mL COF-V suspension evenly on an ITO substrate electrode with a modified area of 0.25 cm2 and let it dry naturally at room temperature to obtain a COF-V modified electrode;

[0050] Sp5. Dissolve 5 mg of TCPP and 10 mg of DA in 5 mL of Tris-HCl buffer (10 mM, pH 8.5), and fully dissolve TCPP by ultrasonic treatment;

[0051] Sp6. Disperse 20 μL of this solution on the COF-V modified electrode, keep it standing in a dark environment for 30 min to allow DA to fully self-polymerize, and finally wash the electrode with deionized water to obtain the required PDA / TCPP / COF-V electrode.

[0052] The preparation method of the conjugate LZP-hDNA is to mix 300 μL of 40 μM LZP and 300 μL of 40 μM hDNA solution and place it in a shaker, keep shaking at 37 °C for 5 min, and finally obtain an LZP-hDNA conjugate with a concentration of 20 μM.

[0053] Before the preparation of the nucleic acid probe pDNA-AgInS2, water-soluble AgInS2 quantum dots need to be prepared. When preparing the water-soluble AgInS2 quantum dots, first place 25 mL of an aqueous solution containing 0.1 mM MPA, 0.4 mM In(NO3)3, and 0.1 mM AgNO3 in a 50 mL three-necked flask.

[0054] Quickly add 1.5 mL of 0.2 M Na2S solution to the aqueous solution in the three-necked flask under rapid stirring, heat the solution to 100 °C, and reflux for 2 h to allow the AgInS2 quantum dots to grow sufficiently. Finally, store the obtained quantum dots at 4 °C for normal temperature preservation.

[0055] Quantum dots are synthesized into pDNA-AgInS2 nucleic acid probes through an amide coupling reaction. When synthesizing the nucleic acid probe pDNA-AgInS2, 150 μL of AgInS2 quantum dots and 100 μL of 10 mM EDC / NHS solution are mixed and kept at room temperature for 30 min to fully activate the carboxyl groups on the surface of AgInS2 quantum dots. Then, 400 μL of 30 μM amino-modified pDNA is added to the above solution and reacted at room temperature for 2 h. Finally, the obtained solution is ultrafiltered and purified and diluted with 600 μL of Tris-HCl buffer solution, and stored at 4 °C for later use.

[0056] During the construction of the anti-pollution DNA sensor, 20 μL of 20 μM LZP-hDNA conjugate is dispersed on the surface of the PDA / TCPP / COF-V optoelectrode and incubated overnight in a humid environment at 4 °C. The LZP-hDNA is anchored to the surface of the optoelectrode through a Michael reaction. Then, the electrode is washed with Tris-HCl buffer solution to remove the unconnected conjugate. Subsequently, the electrode is incubated with 20 μL of tDNA solution with different concentrations containing 10 U λ-Exo at 37 °C for 1 h. After washing with Tris-HCl buffer solution, the electrode is further incubated with 20 μL of 20 μM pDNA-AgInS2 nucleic acid probe at 37 °C for 1 h. After washing with Tris-HCl buffer solution, the obtained electrode is used for subsequent PEC testing.

[0057] During photoelectrochemical testing, the prepared sensing electrode is used as the working electrode, a platinum wire is used as the counter electrode, and a saturated Ag / AgCl electrode is used as the reference electrode. The test buffer solution is phosphate buffer solution, and the dissolved oxygen in the PBS solution is used as the electron acceptor.

[0058] The instruments used in the detection process of the anti-pollution photoelectrochemical DNA sensor include a transmission electron microscope, a scanning electron microscope, an X-ray photoelectron spectrometer, an X-ray diffractometer, a Fourier transform infrared spectrometer, a photoelectrochemical workstation, an electrochemical workstation, a contact angle measuring instrument, a laser confocal microscope, and an ultraviolet-visible diffuse reflectance spectrometer.

[0059] It should be noted that the above anti-pollution photoelectrochemical DNA sensor constructed based on the polypeptide-hairpin DNA conjugate is used for the detection of HIV. Specific Example 2:

[0061] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0062] Using the DNA sequence (tDNA) corresponding to human immunodeficiency virus (HIV) as a detection model, HIV is a virus that destroys lymphocytes and causes the body to lose its immune function, which can lead to acquired immunodeficiency syndrome (AIDS). Specifically, the click reaction is used to react the linear zwitterionic peptide LZP modified with -N3 azide group and the hairpin DNA (hDNA) modified with 3'-dibenzocyclooctyne DBCO to form the LZP-hDNA conjugate. This conjugate not only has an anti-pollution effect, but the presence of hDNA also facilitates the λ-Exo-assisted target cycling amplification reaction. By modifying the composite material of meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and polydopamine (PDA) on the COF-V electrode, a ternary composite optoelectrode PDA / TCPP / COF-V is prepared, which acts as a signal converter of the sensing platform and firmly anchors the LZP-hDNA conjugate. When tDNA and λ-Exo are present, tDNA can hybridize with hDNA to form a double-stranded structure, and λ-Exo will gradually hydrolyze it from the 5' to 3' direction and release tDNA to continue participating in the hybridization reaction, thereby playing a role in target cycling amplification. The uncut part of hDNA will further hybridize with the pDNA-AgInS2 signal probe. At this time, the AgInS2 quantum dots are close to the electrode, and the photocurrent is significantly reduced due to the bandgap mismatch. The results show that this PECDNA sensing platform has the potential to be applied in complex environments. Specific Embodiment Three:

[0064] As Figures 1-14 shown, based on the content in Specific Embodiment One, the following content is further disclosed:

[0065] The PDA / TCPP / COF-V optoelectrode, as a signal converter for DNA sensors, has a high photocurrent signal output. Covalent organic frameworks (COFs) are porous crystalline materials formed by strong covalent bonds between organic molecules. Due to their large number of π-conjugated structures, porous structures, good biocompatibility, and structural stability, they exhibit excellent optical and electrical properties. The covalent organic framework COF-V with a 3D structure, due to its extensive π-conjugated structure, high crystallinity, and porous structure, makes it a good optoelectronic material for constructing PEC sensors. To enhance the photoelectric conversion efficiency and the absorption ability of visible light, a composite of meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and polydopamine (PDA) was introduced. The two-dimensional large π-conjugated system of TCPP endows it with excellent optical and electrochemical catalytic properties and has been widely used in the optoelectronic field. TCPP has a Soret band at around 420 nm, which helps it absorb visible light. The electrons on the conduction band of COF-V just flow to the valence band of TCPP, thus increasing the cathodic photocurrent. However, the bandgap of TCPP is about 2.89 eV, indicating its limited absorption of visible light and only improving the photoelectric conversion efficiency to a certain extent. PDA has excellent charge transfer ability and can be used as a high-performance optoelectronic material. At the same time, PDA has a narrow bandgap of 1.6 eV, making it exhibit excellent light absorption ability. Therefore, the composite of TCPP and PDA can increase the light absorption range and improve the photoelectric conversion efficiency, thus enabling the PDA / TCPP / COF-V optoelectrode to generate a large photocurrent output.

[0066] Interfering proteins present in complex biological matrices can affect the sensitivity of the sensor during the detection of the target. Therefore, by introducing a zwitterionic peptide-DNA conjugate to improve the anti-pollution ability of the sensor. Since hairpin DNA is more easily used for signal amplification and has a higher binding efficiency with linear zwitterionic peptides, the linear zwitterionic peptide (LZP) was conjugated with hairpin DNA (hDNA), and the LZP-hDNA conjugate was successfully prepared. An anti-pollution biointerface was constructed by anchoring the LZP-hDNA conjugate on the optoelectrode. Among them, LZP is used to resist the non-specific adsorption of proteins on the biointerface, and hDNA participates in the λ-Exo-assisted target recycling amplification process. LZP consists of two parts, namely the anchoring part CPPPP and the anti-pollution part DKDKDKDK. The anchoring sequence ensures the firm and stable connection of LZP on the optoelectrode. The anti-pollution part selects the DK alternating sequence with similar hydrophilicity and electrical neutrality to the EK alternating sequence. This zwitterionic peptide exhibits excellent anti-pollution ability. Based on this, the zwitterionic peptide LZP with the sequence CPPPPDKDKDKDK-N3 was designed in this work.

[0067] It is very important for PEC sensors to have high sensitivity. Here, pDNA-AgInS2 nucleic acid probe and λ-Exo are used as signal amplification elements to effectively improve the detection sensitivity. In the pDNA-AgInS2 nucleic acid probe, due to the bandgap mismatch between AgInS2 quantum dots and PDA / TCPP / COF-V optoelectrode material, it acts as an electron donor in the sensor, hindering the transfer of electrons from the electrode to the solution. When the target tDNA is present, tDNA hybridizes with hDNA to form double-stranded DNA, exposing the 5'-phosphate group end of hDNA. λ-Exo will gradually hydrolyze hDNA in the 5'-3' direction, releasing tDNA, which continues to react with unreacted hDNA, thus playing a role of cyclic amplification. The remaining part of hDNA after being sheared fixes pDNA-AgInS2 to the electrode surface through DNA hybridization. At this time, AgInS2 quantum dots approach the electrode, causing the photocurrent to decrease. This λ-Exo-assisted target cyclic amplification strategy results in an increase in AgInS2 quantum dots on the optoelectrode, thereby causing a significant decrease in photocurrent. This PEC DNA sensor method successfully realizes the accurate and sensitive detection of HIV. Specific Example Four:

[0069] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0070] The morphology of COF-V was observed by TEM. The TEM image shows that the synthesized COF-V is a nanosphere with a diameter of about 400 - 500 nm. As Figure 4 shown in -b, SEM further shows that the synthesized COF-V has the advantages of uniform size and good dispersibility, which provides convenience for the subsequent preparation of optoelectrodes. At the same time, as Figure 4 shown in the -c elemental distribution image, the results show that this nanomaterial contains C, N, and O elements, and the three elements are evenly distributed in the nanosphere. In addition, XRD characterization was carried out on COF-V. As Figure 5 shown in -d, at 2θ = 5.5°, 7.3°, 9.6° and 25.1°, different diffraction peaks of the pure COF-V phase can be clearly observed, corresponding to the crystal planes (200), (210), (220) and (001), further proving its successful synthesis. It should be noted that in Figure 4 a is the TEM image of COF-V, b is the TEM image, c is the elemental distribution image, and d is the XRD pattern. Specific Example Five:

[0072] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0073] First, the morphology of water-soluble AgInS2 quantum dots was observed by TEM. Their sizes were uniform and the dispersion was good. Figure 5 The inset in -a shows the high-resolution transmission electron microscope (HRTEM) of AgInS2 quantum dots. After that, the sizes of AgInS2 quantum dots were statistically analyzed based on the TEM images, and their particle size distribution diagrams were obtained. It was observed from the diagrams that the average diameter of AgInS2 quantum dots was 2.8 ± 0.6 nm.

[0074] Figure 5 -c shows the UV-vis absorption spectrum of AgInS2 quantum dots. Their light absorption range was as wide as 470 nm, and this result was consistent with the results in the published literature. Figure 5 -d is the fluorescence spectrum of AgInS2 quantum dots. The emission peak was at 593 nm, which also matched the peak position in the published literature. Both the fluorescence spectrum and the ultraviolet-visible absorption spectrum proved that AgInS2 quantum dots were successfully synthesized and the quantum dots had good optical properties, where 5-b was the size distribution diagram. Specific Example Five:

[0076] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0077] From the UV-vis absorption spectrum ( Figure 6 -a), it can be observed that TCPP had a strongest absorption peak at 420 nm and relatively weak absorption in the 500 - 700 nm region, indicating that TCPP had excellent optical properties. In addition, its fluorescence spectrum was used for further characterization (6-b), and the results showed that TCPP had a fluorescence emission peak at 685 nm. Specific Example Six:

[0079] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0080] In Figure 7 a was the characterization diagram of COF-V, b was the SEM image of the PDA / TCPP / COF-V electrode, c and d were the XPS full scan diagrams and UV-vis DRS diagrams of the COF-V, TCPP / COF-V, and PDA / TCPP / COF-V electrodes. The surface morphologies of the COF-V and TCPP / COF-V electrodes were observed by SEM. As Figure 7 -a shown, COF-V nanospheres were uniformly covered on the ITO electrode, and their sizes were uniform. After being modified with the PDA / TCPP composite material, it could be in Figure 7It can be clearly observed from -b that the surface of the COF-V nanospheres changes from the original rough surface to a relatively smooth surface, indicating the presence of the PDA / TCPP composite material and preliminarily proving the successful preparation of the PDA / TCPP / COF-V electrode.

[0081] Subsequently, XPS was used to further explore the construction of the photoanode. As Figure 5 shown in c, since TCPP, PDA, and COF-V contain the same elements, the characteristic peaks of COF-V, TCPP / COF-V, and PDA / TCPP / COF-V are the same, only the peak intensities are different. When only COF-V is present, the characteristic peaks of C1 s, N1 s, and O1 s appear in the XPS spectrum. When TCPP is modified, the contents of N and O elements increase, resulting in an increase in the characteristic peak intensities of N1 s and O1 s. When the PDA / TCPP composite material is modified, the characteristic peak intensities of N1 s and O1 s are the largest. In addition, no characteristic peaks of other elements are detected, indicating the successful construction of the photoanode.

[0082] Figure 7 -d shows the UV-vis DRS spectra of COF-V, TCPP / COF-V, and PDA / TCPP / COF-V. The absorption edge of COF-V is located at 535 nm. After only TCPP is modified, the absorption edge expands to 605 nm, indicating that TCPP can broaden the light absorption range. However, when the PDA / TCPP composite material is co-modified onto the COF-V electrode, the absorption edge increases to 648 nm, indicating that this composite material can significantly enhance light absorption and thus significantly improve the electron transfer ability. Specific Embodiment Seven:

[0084] As Figures 1-14 shown, based on the content in Specific Embodiment 1, the following content is further disclosed:

[0085] The PDA / TCPP / COF-V photoanode, as the signal converter of the PEC DNA biosensor, its optoelectronic performance has an important impact on the analytical performance of the sensor. As Figure 8As shown in Figure -a, we respectively compared the photocurrent outputs of COF-V, TCPP / COF-V, PDA / COF-V, and PDA / TCPP / COF-V photoanodes. The COF-V electrode exhibited relatively poor photocurrent output (curve a) due to its limited optoelectronic properties. However, after modification with TCPP, the photocurrent output of the TCPP / COF-V electrode increased (curve b), which was 4.8 times higher than that of the COF-V electrode. Similarly, after modification with PDA, the photocurrent output of the PDA / COF-V electrode also increased (curve c), reaching 12.5 times that of the COF-V electrode. Notably, when the PDA / TCPP composite was modified on the surface of the COF-V electrode, the photocurrent output of the PDA / TCPP / COF-V electrode increased significantly (curve d), reaching 35.5 times that of the COF-V electrode. This enhancement was attributed to the superior electron transfer ability of TCPP and PDA and their light absorption ability in the visible range. In addition, we investigated the stability of the photoanode by continuously turning the light on and off for a long time. As Figure 8 shown in Figure -b, during the entire illumination process, the PEC signal did not decrease significantly, demonstrating the good stability of the proposed PDA / TCPP / COF-V electrode. Figure 8-a shows the photocurrent output curves of COF-V, TCPP / COF-V, PDA / COF-V, and PDA / TCPP / COF-V electrodes, and Figure 8-b shows the photocurrent intensity of the PDA / TCPP / COF-V photoanode as a function of time. Specific Example VIII:

[0087] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0088] In Figure 9-a, the PAGE images of hDNA, LZP, and the LZP-hDNA conjugate are shown, and in Figure 9-b, the UV-vis absorption spectral image of pDNA-AgInS2 is shown. According to the previously reported reagent-free click reaction, the LZP-hDNA conjugate was prepared by mixing azide-modified LZP and DBCO-modified hDNA at a molar ratio of 1:1 and reacting them at 37 °C for 5 min, 30 min, 2 h, 4 h, and 12 h, respectively. As Figure 9As shown in Figure -a, the synthesized LZP-hDNA conjugate was characterized by polyacrylamide gel electrophoresis (PAGE). Lane 2 was the hDNA control, while lanes 3, 4, 5, 6, and 7 were the LZP-hDNA conjugates synthesized at 5 min, 30 min, 2 h, 4 h, and 12 h, respectively. Obviously, the positions of the PAGE bands of the LZP-hDNA conjugates (lanes 3, 4, 5, 6, 7) were significantly higher than those of the hDNA bands (lane 2), indicating the successful binding of LZP and hDNA, and the binding was complete at 5 minutes of the reaction, suggesting that the optimal reaction time for the conjugation of LZP and hDNA was 5 min, demonstrating the high efficiency of the click reaction. In addition, the formation of the pDNA-AgInS2 probe was verified by UV-vis absorption spectroscopy ( Figure 9 -b). When pDNA was linked to AgInS2 quantum dots, an absorption peak appeared at 242 nm for pDNA, showing a blue shift compared to that at 260 nm, indicating the successful formation of the pDNA-AgInS2 nucleic acid probe. Specific Example Nine:

[0090] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0091] The prepared DNA sensor was characterized by the photocurrent output signal, as Figure 10 shown in Figure -a. After modifying the COF-V electrode with the PDA / TCPP composite material, the light absorption range of COF-V could be broadened and the charge transfer ability could be enhanced, thus obtaining a relatively high initial photocurrent (curve a). After immobilizing the LZP-hDNA conjugate, the output of the photocurrent decreased (curve b), which was related to the relatively weak charge transfer ability of the LZP-hDNA conjugate. After incubating tDNA and λ-Exo together, the output of the photocurrent increased moderately (curve c). This was because after hDNA formed a double-stranded structure with tDNA, hDNA could be cleaved by λ-Exo, and the generated single-stranded DNA fragments had better charge transfer ability than hDNA. Then, the pDNA-AgInS2 probe was bound to the electrode through base complementary pairing between pDNA and the single-stranded DNA, and the photocurrent intensity decreased significantly (curve d). This was because the conduction band position of AgInS2 was higher than that of PDA, thus hindering the charge transfer from the electrode to the solution. The changing trend of the photocurrent signal proved the successful construction of the DNA sensor, and the successful construction of the DNA sensor was further confirmed by electrochemical impedance, as Figure 10As shown in Figure -b, the diameter of the semi - circle in the figure represents the charge transfer resistance Rct of the modified electrode. After the COF - V electrode is loaded with the PDA / TCPP composite material, Rct is relatively small (curve a). Further anchoring the LZP - hDNA conjugate, due to the relatively low conductivity of the zwitterionic peptide and DNA, Rct increases (curve b). After incubating the electrode with tDNA and λ - Exo together, Rct decreases (curve c), indicating that λ - Exo successfully cleaves the double - stranded DNA structure and generates a large number of single - stranded DNA fragments on the electrode surface. With the anchoring of the pDNA - AgInS2 probe, Rct increases again (curve d), which is attributed to the low electron transfer ability of DNA and AgInS2. Specific Example Ten:

[0093] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0094] To obtain better detection performance, some important parameters and conditions were optimized. Figure 11 Figure -a shows the photocurrent output of COF - V electrodes prepared using COF - V dispersions with different concentrations. It can be found that when the concentration of COF - V is 0.5 mg / mL, the current output presents the best value. Initially, as the concentration of COF - V increases, COF - V accumulates appropriately on the electrode, and the current output signal gradually increases. When the concentration of COF - V exceeds 0.5 mg / mL, the excessive COF - V leads to a significant increase in the diffusion resistance of electron movement, and the output photocurrent gradually decreases. Therefore, the concentration of the COF - V dispersion is selected as 0.5 mg / mL.

[0095] Figure 11 Figure -b describes the change diagram of the photocurrent response of PDA / TCPP / COF - V electrodes prepared using TCPP solutions with different concentrations. As the concentration of TCPP increases, the ability of photoelectron transport continuously enhances, so the photocurrent intensity increases accordingly. When the concentration of TCPP exceeds 1.0 mg / mL, due to the increase in the resistance of electron movement, the photocurrent signal decreases. Therefore, the concentration of the TCPP solution for preparing the PDA / TCPP / COF - V photo - electrode is selected as 1.0 mg / mL. Figure 11 - c is the modified electrode with different concentrations of LZP - hDNA conjugate solutions, Figure 11 - d is the DNA sensor with different incubation times in the presence of 10 pM tDNA and 10 U λ - Exo, and Figure 11 - a is the COF - V electrode with different concentrations of COF - V dispersions.

[0096] Figure 11-c describes the photocurrent output of the LZP-hDNA modified electrode prepared using different concentrations of the LZP-hDNA conjugate. As the concentration of LZP-hDNA increases, the photocurrent intensity gradually decreases. When the concentration of the LZP-hDNA conjugate reaches 20 μM, the photocurrent begins to level off, indicating that sufficient LZP-hDNA conjugate is anchored on the electrode at this time. Therefore, 20 μM is used as the optimal incubation concentration of the LZP-hDNA conjugate.

[0097] Figure 11 -d shows the photocurrent output of the DNA sensor at different reaction times in the presence of 10 pM tDNA and 10 U λ-Exo. It can be seen from the figure that as the reaction time increases, the photocurrent intensity gradually increases and finally remains basically stable at 1.0 h, indicating that the cleavage of λ-Exo triggered by tDNA is completed, thus determining 1.0 h as the optimal reaction time. Specific Example XI:

[0099] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0100] The anti-pollution performance of the designed DNA sensor was further verified using a fluorescence microscope, and green fluorescent protein BSA-FITC was used as a fluorescence indicator. It can be observed from Figure 12 -a that many green fluorescent proteins were non-specifically adsorbed on the surface of the COF-V electrode. After PDA / TCPP modification, as Figure 12 shown in -b, the amount of green fluorescent protein adsorbed on the surface of the PDA / TCPP / COF-V electrode was significantly reduced. This is because PDA has certain anti-pollution ability. After LZP-hDNA or LZP was anchored, as Figure 12 shown in -c and Figure 12 -d, almost no green fluorescent protein was adsorbed on the electrode, indicating that both LZP-hDNA and LZP have good ability to resist protein adsorption. Therefore, the above results show that the DNA sensor has good anti-pollution performance. Specific Example XII:

[0102] As Figures 1-14 shown, based on the content in Specific Example 1, the following content is further disclosed:

[0103] Figure 13 -a shows the photocurrent response of the DNA sensor to different concentrations of tDNA, indicating that the photocurrent response gradually decreases as the concentration of tDNA increases. Figure 13-b shows good linear correlation in a wide concentration range from 0.1 fM to 100 pM. The linear regression equation is ΔI = -185.78 - 64.39 log C (fM), the correlation coefficient is 0.9974, and the limit of detection (LOD, S / N = 3) is 0.04 fM, which is lower than or comparable to many sensitive DNA biosensors reported previously.

[0104] To evaluate the anti-pollution ability of the constructed DNA sensor in complex biological matrices, the signal changes of the optoelectrode anchored with LZP-hDNA and hDNA were detected during incubation in human serum samples, as Figure 13 shown in -c. The optoelectrode anchored with the LZP-hDNA conjugate had the smallest photocurrent change and showed a significant anti-pollution effect (4.89%) even when exposed to 15% diluted human serum. However, the biosensor modified with hDNA had an obvious photocurrent change and could only resist the interference of 1% human serum, indicating that hDNA alone was significantly insufficient in anti-pollution performance. The stability of the PEC DNA biosensor was initially verified by monitoring the photocurrent signal. The biosensor showed no significant change in photocurrent intensity during dozens of repeated light irradiations ( Figure 13 -d), demonstrating that the proposed DNA sensor has high stability.

[0105] The selectivity of the DNA sensor was evaluated by comparing the photocurrent responses to different DNA sequences at the same concentration (10 pM). These sequences included the target tDNA (HIV), DNA with a single-base mismatch at the 5'-end, DNA with a single-base mismatch at the 3'-end, and non-complementary DNA (HCV, HPV, HBV), as Figure 14 shown in -a. The change in photocurrent response to HIV was obvious. Smaller photocurrent changes were observed for DNA with a single-base mismatch at different positions, which were almost negligible compared to the signal response of tDNA. In addition, the photocurrent response to non-complementary DNA was very small. The above results indicate that HIV specifically reduced the photocurrent signal and further amplified the signal change through target cycling reactions. Therefore, the DNA biosensor we constructed has excellent selectivity.

[0106] To achieve accurate detection of DNA sensors in complex biological samples, good anti-interference ability is of great significance. Since there are usually interfering substances such as reducing agents in biological samples, we selected some of these substances such as ascorbic acid (AA), glucose (Glu), glutathione (GSH), dopamine (DA), and cysteine (Cys) for anti-interference experiments, as Figure 14As shown in Figure -b, these interfering substances that may exist in real biological samples have no significant effect on the photocurrent signal, thus verifying that the PECDNA biosensor has a satisfactory anti-interference ability.

[0107] In summary:

[0108] By combining zwitterionic peptide-hairpin DNA conjugates, composite organic semiconductor materials, and signal amplification strategies, a novel anti-fouling PECDNA detection platform was established for the accurate and sensitive detection of the target analyte HIV in human body fluids. The ternary composite photoanode based on PDA / TCPP / COF-V provides a relatively high initial photocurrent, making it an ideal signal transducer for PEC sensors and laying a foundation for improving the detection performance of DNA sensors. Introducing the LZP-hDNA conjugate into the electrode established an anti-fouling biological interface and paved the way for signal amplification at the same time. Subsequently, through the hydrolysis of double-stranded DNA by λ-Exo, the pDNA-AgInS2 signal probe was further introduced to significantly reduce the photocurrent, improving the sensitivity of the sensor. Based on the PEC strategy of composite organic semiconductor materials and combined with signal amplification, ultrasensitive and robust detection of HIV was achieved. This innovative strategy of combining organic composite materials, anti-biofouling, and signal amplification provides a promising approach for the development of more advanced and feasible biological detection platforms.

[0109] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a reference structure" does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0110] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made in these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A detection method for an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate, the detection method for the anti-pollution photoelectrochemical DNA sensor is for non-disease diagnosis purposes, and the detection method for the anti-pollution photoelectrochemical DNA sensor includes the preparation of a PDA / TCPP / COF-V photoanode, the preparation of a conjugate of a linear zwitterionic peptide LZP and hairpin DNA (hDNA) LZP-hDNA, the preparation of a nucleic acid probe modified with AgInS2 quantum dots, the construction of an anti-pollution DNA sensor, and photoelectrochemical testing, characterized in that: The preparation of the PDA / TCPP / COF-V photoanode includes the following steps: Sp1. When preparing the PDA / TCPP / COF-V photoanode, first dissolve 40 mg of 1,3,5-tris(4-aminophenyl)benzene and 32 mg of 1,4-dialdehyde-2,5-divinylbenzene in 20 mL of acetonitrile. Sp2. Add 4 mL of 12 M acetic acid to the solution as a catalyst. Sp3. Oscillate the solution obtained in Sp2 for 30 s and let it stand for 72 h. Centrifuge the solution using a centrifuge, collect the precipitate, then wash it thoroughly with tetrahydrofuran and ethanol. Subsequently, place it in a vacuum dryer at 60 °C to obtain the final product, covalent organic framework COF-V. Sp4. After obtaining COF-V according to step Sp3, weigh 5 mg of COF-V powder and disperse it in 10 mL of deionized water. After ultrasonic treatment for 30 min, obtain a uniformly dispersed COF-V suspension with a concentration of 0.5 mg / mL. Disperse 20 μL of the 0.5 mg / mL COF-V suspension evenly on an ITO substrate electrode with a modified area of 0.25 cm², and let it air dry at room temperature to obtain a COF-V modified electrode. Sp5. Dissolve 5 mg of meso-tetra(4-carboxyphenyl)porphyrin TCPP and 10 mg of dopamine DA in 5 mL of 10 mM pH 8.5 Tris-HCl buffer solution, and ultrasonically dissolve TCPP thoroughly. Sp6. Disperse 20 μL of this solution on the COF-V modified electrode, keep it in a dark environment and let it stand for 30 min to allow DA to fully self-polymerize. Finally, wash the electrode with deionized water to obtain the required PDA / TCPP / COF-V electrode.

2. The detection method of an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate according to claim 1, characterized in that: The preparation method of the conjugate LZP-hDNA is to mix 300 μL of 40 μM LZP solution and 300 μL of 40 μM hDNA solution, then place it in a shaker and keep shaking at 37 °C for 5 min to finally obtain an LZP-hDNA conjugate with a concentration of 20 μM.

3. The detection method of the anti-pollution photoelectrochemical DNA sensor constructed based on the polypeptide-hairpin DNA conjugate according to claim 2, wherein: Before preparing the nucleic acid probe pDNA-AgInS2, it is necessary to prepare water-soluble AgInS2 quantum dots. When preparing the water-soluble AgInS2 quantum dots, first place 25 mL of an aqueous solution containing 0.1 mM 3-mercaptopropionic acid, 0.4 mM indium nitrate, and 0.1 mM silver nitrate in a 50 mL three-necked flask.

4. The detection method of the anti-pollution photoelectrochemical DNA sensor constructed based on the polypeptide-hairpin DNA conjugate according to claim 3, characterized in that: Quickly add 1.5 mL of 0.2 M Na2S solution to the aqueous solution placed in the three-necked flask under rapid stirring, heat the solution to 100 °C, and reflux for 2 h to allow the AgInS2 quantum dots to grow sufficiently. Finally, store the obtained quantum dots at 4 °C at room temperature.

5. The detection method of the anti-pollution photoelectrochemical DNA sensor constructed based on the polypeptide-hairpin DNA conjugate according to claim 4, characterized in that: The quantum dots are synthesized into pDNA-AgInS2 nucleic acid probes through an amide coupling reaction. When synthesizing the nucleic acid probe pDNA-AgInS2, 150 μL of AgInS2 quantum dots and 100 μL of 10 mM EDC / NHS solution are mixed and kept at room temperature for 30 min to fully activate the carboxyl groups on the surface of the AgInS2 quantum dots. Then, 400 μL of 30 μM amino-modified pDNA is added to the above solution, and the reaction is carried out at room temperature for 2 h. Finally, the obtained solution is ultrafiltered and purified and diluted with 600 μL of Tris-HCl buffer solution, and placed in an environment at 4°C for standby.

6. The detection method of an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate according to claim 5, wherein: During the construction of the anti-pollution DNA sensor, 20 μL of 20 μM LZP-hDNA conjugate is dispersed on the surface of the PDA / TCPP / COF-V optoelectrode and incubated overnight in a humid environment at 4°C. The LZP-hDNA is anchored to the surface of the optoelectrode through a Michael reaction. Then, the electrode is washed with Tris-HCl buffer solution to remove the unconnected conjugate. Subsequently, the electrode is incubated with 20 μL of tDNA solution containing 10 U λ-Exo at different concentrations at 37°C for 1 h. After washing with Tris-HCl buffer solution, the electrode is further incubated with 20 μL of 20 μM pDNA-AgInS2 nucleic acid probe at 37°C for 1 h. After washing with Tris-HCl buffer solution, the obtained electrode is used for subsequent PEC testing. The DNA sequence tDNA corresponding to the human immunodeficiency virus is used as a detection model, and the -N3 azide group is modified by click reaction The linear zwitterionic peptide LZP modified with -N3 azide group and the hairpin DNA modified with 3'-dibenzocyclooctyne DBCO react to form the LZP-hDNA conjugate. By modifying the composite material of meso-tetraporphyrin and polydopamine on the COF-V electrode, a PDA / TCPP / COF-V ternary composite optoelectrode is prepared, which acts as a signal transducer for the sensing platform and firmly anchors the LZP-hDNA conjugate. When tDNA and λ-Exo are present, tDNA can hybridize with hDNA to form a double-stranded structure, and λ-Exo will gradually hydrolyze it from the 5' to 3' direction and release tDNA to continue to participate in the hybridization reaction.

7. The detection method of an anti-pollution photoelectrochemical DNA sensor constructed based on a polypeptide-hairpin DNA conjugate according to claim 6, characterized in that: During the photoelectrochemical test, the prepared sensing electrode is used as the working electrode, a platinum wire is used as the counter electrode, and a saturated Ag / AgCl electrode is used as the reference electrode. The test buffer solution is a phosphate buffer solution, and the dissolved oxygen in the PBS solution is used as the electron acceptor.

8. The detection method of the anti-pollution photoelectrochemical DNA sensor constructed based on the polypeptide-hairpin DNA conjugate according to claim 7, characterized in that: The instruments used in the detection process of the anti-pollution photoelectrochemical DNA sensor include a transmission electron microscope, a scanning electron microscope, an X-ray photoelectron spectrometer, an X-ray diffractometer, a Fourier transform infrared spectrometer, a photoelectrochemical workstation, an electrochemical workstation, a contact angle measuring instrument, a laser confocal microscope, and an ultraviolet-visible diffuse reflectance spectrometer.

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