A method for photoelectrochemical detection of hAAG based on sulfur vacancy engineering

Abstract

The present application relates to a kind of hAAG photoelectrochemical detection method based on sulfur vacancy engineering, belong to analytical detection technical field.The present application will prepare hairpin structure DNA to single-stranded DNA, sequentially join different concentrations of human alkyladenine DNA glycosylase (hAAG), endonuclease IV, exonuclease I and its reaction buffer;Dopamine solution is added to reaction, and the enzymatic reaction system of hAAG is obtained;CdZnS modified electrode is immersed in enzymatic reaction product solution and reacts, so that dopamine and zinc atom on the surface of CdZnS are combined by ortho-hydroxy coordination, and sulfur vacancy is induced to generate;Photoelectrochemical test is carried out to CdZnS modified electrode, the photocurrent signal is detected, the linear relationship between photocurrent signal and hAAG concentration is established, and the quantitative detection of hAAG is realized.The present application not only provides efficient strategy for hAAG detection, but also provides new ideas for performance optimization of sulfide-based photoelectrochemical device.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering. Background Technology

[0002] Human alkyladenine DNA glycosylase (hAAG) is a key enzyme in the base excision repair pathway, responsible for recognizing and excising alkylated adenine, hypoxanthine, and other abnormal bases in damaged DNA, which is crucial for maintaining genome integrity [L. Milano, C. Charlier, R. Andreguetti, et al., "A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response," Proceedings of the National Academy of Sciences, 119 (2022):e2111404119]. Abnormal hAAG expression is closely related to the occurrence and development of various diseases such as cancer, neurodegenerative diseases, and chronic inflammation, and is an important biomarker for early disease diagnosis, disease monitoring, and treatment efficacy evaluation. Therefore, establishing a highly sensitive and specific hAAG detection method has significant clinical significance and application value.

[0003] Currently, the detection methods for hAAG are mainly based on fluorescence methods. Despite continuous efforts, these methods still suffer from inherent drawbacks such as high background noise, a single detection mechanism, and complex operation, making it difficult to meet the precise and efficient requirements of clinical testing. Photoelectrochemical (PEC) sensing technology has attracted much attention in the field of biological detection due to its advantages of simple operation, high sensitivity, and low cost [Y. Zhou, H. Yin, S. Ai., "Applications of two-dimensional layered nanomaterials in photoelectrochemical sensors: a comprehensive review," Coordination Chemistry Reviews, 447 (2021): 214156]. The core performance of this technology depends on the photoelectric conversion efficiency of photoactive materials. Among them, metal sulfides, due to their shallow valence band position, wide light absorption range, and fast charge migration rate, have become ideal candidates for photoactive materials.

[0004] However, traditional metal sulfide-based PEC sensors primarily rely on photoinduced electron transfer mechanisms for signal conversion, often using signal quenching as the output method. Furthermore, they cannot address the inherent photocorrosion problem of sulfide materials, leading to unsatisfactory detection selectivity and stability. Sulfur vacancies, as an important defect type in metal sulfides, can enhance photoelectric performance by trapping charge carriers, reducing band gaps, and inducing polarization electric fields. Existing methods for introducing sulfur vacancies (such as high-temperature hydrothermal methods, plasma etching, and heteroatom doping) suffer from high energy consumption, long reaction times, and complex operations, limiting their practical applications. Therefore, developing simple and efficient sulfur vacancy engineering strategies and constructing novel sulfide-based PEC sensing platforms is of great significance for overcoming the bottlenecks in hAAG detection technology. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a photoelectrochemical detection method for human alkyladenine DNA glycosylation enzyme (hAAG) based on sulfur vacancy engineering. Specifically, it is a method for the photoelectrochemical detection of hAAG based on sulfur vacancy engineering. By coordinating dopamine with the CdZnS surface, the sulfur vacancy formation energy is reduced, enabling rapid and efficient generation of sulfur vacancies. Combined with the enzymatically specific recognition reaction mediated by hAAG, a highly sensitive PEC biosensor is constructed. This invention, based on a novel PEC sensing strategy using sulfur vacancy engineering driven by surface reactions to reduce formation energy, achieves highly selective and sensitive hAAG PEC detection, overcoming the limitations of traditional sulfide-based PEC sensors, such as severe photocorrosion and a single detection mechanism, significantly improving detection performance.

[0006] This invention is achieved through the following technical solution:

[0007] The purpose of this invention is to provide a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, comprising the following steps:

[0008] S1: CdZnS nanomaterials are modified onto the surface of a conductive electrode to obtain a CdZnS modified electrode;

[0009] S2: Hairpin DNA structure was prepared from single-stranded DNA. Different concentrations of hAAG and hAAG reaction buffer, endonuclease IV and endonuclease IV reaction buffer, and exonuclease I and exonuclease I buffer were added sequentially for incubation. Then, dopamine solution was added to react and obtain the enzymatic reaction system of hAAG. The specific enzymatic reaction occurred depending on the presence or absence of hAAG, releasing or consuming dopamine (DA) in the system.

[0010] S3: Immerse the CdZnS modified electrode obtained in step S1 into the enzyme-catalyzed reaction product solution in step S2 to react, so that dopamine and zinc atoms on the CdZnS surface are coordinated and bound through ortho-hydroxyl groups, inducing the generation of sulfur vacancies.

[0011] S4: Perform photoelectrochemical testing on the CdZnS modified electrode obtained in step S3, detect the photocurrent signal, establish a linear relationship between the photocurrent signal and the hAAG concentration, and realize the quantitative detection of hAAG.

[0012] In one embodiment of the present invention, in step S1, the CdZnS nanomaterial is prepared by a hydrothermal method; the CdZnS nanomaterial is obtained by dispersing zinc salt, cadmium salt and sulfide precursor in water, reacting by heating, centrifuging, washing and drying.

[0013] And / or, the CdZnS nanomaterials have a particle size of 15~25 nm and exhibit a quasi-spherical morphology, such as... Figure 1 The TEM image is shown below;

[0014] In one embodiment of the present invention, the zinc salt is one or more selected from Zn(Ac)2·2H2O, ZnCl2, and Zn(NO3)2·6H2O;

[0015] And / or, the cadmium salt is one or more of Cd(Ac)2·2H2O, CdCl2·2.5H2O, and Cd(NO3)2·4H2O;

[0016] And / or, the sulfide is one or more of Na2S·9H2O, thiourea, and thioacetamide;

[0017] And / or, the molar ratio of the zinc salt, cadmium salt and sulfide precursor is 1.5~2.5:0.3~0.7:2.0~3.0;

[0018] And / or, the conditions for the heating reaction are: 150~170℃ for 10~14 h.

[0019] In one embodiment of the present invention, 2.0-3.0 mmol Na2S·9H2O, 1.5-2.5 mmol Zn(Ac)2·2H2O and 0.3-0.7 mmol Cd(Ac)2·2H2O were dissolved in 20 mL of deionized water, and after magnetic stirring for 0.5-1.5 h, the solution was transferred to a 50 mL polytetrafluoroethylene-lined reactor and reacted at 150-170 °C for 10-14 h. The precipitate was collected by centrifugation, washed 3-5 times alternately with deionized water and ethanol, and dried at 60-70 °C for 8-12 h to obtain quasi-spherical CdZnS nanoparticles with an average particle size of 15-25 nm.

[0020] In one embodiment of the present invention, in step S1, the CdZnS modified electrode is prepared by the following method: dispersing CdZnS nanomaterials in water to obtain a CdZnS suspension; coating the CdZnS suspension onto the surface of a pretreated conductive electrode, and drying it to obtain the CdZnS modified electrode.

[0021] In one embodiment of the present invention, the concentration of the CdZnS suspension is 0.5~1.5 mg / mL;

[0022] And / or, the coating amount of the CdZnS suspension is 1.5~2.2 μL / mm. 2 ;

[0023] And / or, the conductive electrode is an indium tin oxide (ITO) electrode. The pretreatment method is as follows: the ITO electrode is ultrasonically cleaned with isopropanol and deionized water for 10-20 min in sequence, and then dried.

[0024] In one embodiment of the present invention, in step S2, the enzyme-catalyzed reaction product solution is prepared by the following method:

[0025] (1) Dissolve single-stranded DNA in a buffer solution containing magnesium ions, heat, and cool to form hairpin DNA;

[0026] (2) Hairpin structure DNA, different concentrations of hAAG and hAAG reaction buffer and water were mixed and incubated. hAAG recognized and removed hypoxanthine sites on hairpin structure DNA to form purine-free / pyrimidine-free sites.

[0027] (3) Add endonuclease IV and endonuclease IV reaction buffer and incubate to cleave the purine-free / pyrimidine-free sites to release single-chain dopamine aptamers;

[0028] (4) Add exonuclease I and exonuclease I buffer and incubate to degrade single-chain dopamine aptamers;

[0029] (5) Heating terminates the enzymatic reaction, and after cooling, dopamine solution is added to react and the enzymatic reaction product solution is obtained.

[0030] In one embodiment of the present invention, in step S2, the single-stranded DNA is a DNA capable of forming a stem-loop structure, one complementary strand of the stem structure is modified with hypoxanthine, and the hypoxanthine to terminal sequence in the stem structure is a dopamine aptamer sequence.

[0031] The sequence of the single-stranded DNA is shown in SEQ ID NO. 1; the sequence of the dopamine aptamer is shown in SEQ ID NO. 2;

[0032] SEQ ID NO. 1: 5'-HO-AT GTC TCT GTG TGC GCC AGA GAC ACT GGG GCA GAT ATGGGC CAG CAC AGA ATG AGG CCC TG CCN CCA CCG GTG TTT CGG TGG TGG C-OH-3'; where N represents I, and I represents the hypoxanthine site;

[0033] SEQ ID NO.2: 5'-HO- GTC TCT GTG TGC GCC AGA GAC ACT GGG GCA GAT ATGGGC CAG CAC AGA ATG AGG CCC-3';

[0034] The single-stranded DNA needs to be pretreated: dissolved in 10 mM Tris-HCl buffer (pH=8.0) containing 0.1 mM MgCl2, heated at 95 ℃ for 4~6 min, and then naturally cooled to room temperature;

[0035] And / or, the concentration of hAAG is 5 × 10⁻⁶. -4 ~1.0 U / mL; the hAAG reaction temperature is 36~38 ℃, and the reaction time is 60~80 min;

[0036] And / or, the concentration of Endo IV is 400~600 U / mL; the reaction time is 60~80 min;

[0037] And / or, the concentration of Exo I is 8~12 U / μL; the reaction time is 30~50 min;

[0038] And / or, the concentration of the dopamine solution is 40-60 μM, and the reaction time with the enzyme-catalyzed reaction product solution is 25-35 min.

[0039] In one embodiment of the present invention, in S3, the reaction conditions are: 25-30°C for 6-10 min.

[0040] In one embodiment of the present invention, in step S4, the photoelectrochemical test adopts a three-electrode system; the CdZnS modified electrode is the working electrode, the saturated Ag / AgCl electrode is the reference electrode, and the platinum electrode is the counter electrode.

[0041] In one embodiment of the present invention, in step S4, the buffer solution for the photoelectrochemical test is a Tris-HCl buffer solution with a pH of 7.0-9.0; the voltage is +0.2 to +0.4 V; and the excitation wavelength is 400 to 410 nm.

[0042] This invention proposes a sulfur vacancy engineering strategy based on surface reaction-driven reduction of formation energy to achieve highly sensitive PEC detection of hAAG. The core mechanism is as follows: DA coordinates with Zn atoms on the CdZnS surface through ortho-hydroxyl groups, significantly reducing the sulfur vacancy formation energy. Sulfur vacancies can be rapidly generated without harsh conditions such as high temperature and high pressure. Sulfur vacancies introduce defect energy levels, trapping photogenerated electrons and suppressing carrier recombination. On the other hand, they induce LPEF, accelerating charge separation. This dual effect amplifies the photocurrent response.

[0043] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0044] This invention provides a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering. It utilizes the coordination effect between DA and the CdZnS surface to reduce the sulfur vacancy formation energy, thereby achieving rapid and efficient generation of sulfur vacancies. The sulfur vacancy introduces defect energy levels and induces a localized surface polarization electric field (LPEF), which synergistically suppresses carrier recombination and improves charge separation efficiency, significantly amplifying the photocurrent response, and ultimately achieving highly sensitive and selective detection of hAAG.

[0045] This invention achieves selective detection of hAAG by combining sulfur vacancy engineering with enzyme-catalyzed specific recognition reactions. It solves the drawbacks of existing sulfur vacancy introduction methods, such as high energy consumption and complex operation, improves the sensitivity and specificity of detection, reduces the detection limit, opens up a new method for selective detection of hAAG, and provides new ideas for the application of sulfide semiconductors in the field of photoelectrochemical sensing. Attached Figure Description

[0046] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Figure 1 This is a TEM characterization image of CdZnS in this invention;

[0048] Figure 2 This is a comparison diagram of the photocurrent of CdZnS and CdZnS / DA in this invention;

[0049] Figure 3 These are the S 2p high-resolution XPS spectra of CdZnS and CdZnS / DA in this invention;

[0050] Figure 4 These are the ESR spectra of CdZnS and CdZnS / DA in this invention;

[0051] Figure 5 This is the calculated result of the sulfur vacancy formation energy of CdZnS and CdZnS / DA in this invention;

[0052] Figure 6 The photocurrent response (A) and standard curve (B) for different hAAG concentrations in this invention are shown.

[0053] Figure 7 This is a selective test diagram of the method in this invention. Detailed Implementation

[0054] The detection of human alkyladenine DNA glycosylation enzyme (hAAG) currently relies heavily on fluorescence sensing platforms, which suffer from high background noise, a single detection mechanism, and complex operation. While traditional metal sulfide-based photoelectrochemical (PEC) sensors offer advantages, they are susceptible to photocorrosion, resulting in poor selectivity and stability. Furthermore, sulfur vacancy introduction methods are limited by high energy consumption and operational complexity. This invention provides a highly sensitive and selective PEC detection method for hAAG based on surface reaction-driven sulfur vacancy engineering.

[0055] The first aspect of this invention protects a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, comprising the following steps:

[0056] S1: CdZnS nanomaterials were prepared by hydrothermal method and dispersed in deionized water to obtain a CdZnS suspension;

[0057] S2: CdZnS suspension was coated onto the surface of the pretreated conductive electrode and dried at room temperature to obtain a CdZnS modified electrode.

[0058] S3: Construct an hAAG enzymatic reaction system, which includes hairpin DNA (HP), hAAG, endonuclease IV (Endo Ⅳ), exonuclease I (Exo Ⅰ) and corresponding reaction buffers. Specific enzymatic reactions occur based on the presence or absence of hAAG, achieving selective release or consumption of dopamine (DA).

[0059] S4: The CdZnS modified electrode prepared in S2 is immersed in the enzyme reaction product solution of S3. DA binds to zinc atoms on the CdZnS surface through ortho-hydroxyl groups, inducing the generation of sulfur vacancies.

[0060] S5: Perform photoelectrochemical tests on the treated CdZnS modified electrode, record the photocurrent intensity, and realize the quantitative detection of hAAG based on the linear relationship between the photocurrent signal and the hAAG concentration.

[0061] Understandably, this invention significantly reduces the sulfur vacancy formation energy through the coordination of DA with zinc atoms on the CdZnS surface, achieving rapid and efficient sulfur vacancy generation. Sulfur vacancies introduce defect energy levels, suppressing photogenerated carrier recombination, and induce a localized surface polarization electric field (LPEF), accelerating charge separation; this dual effect amplifies the photocurrent response. Combined with the hAAG-mediated enzymatic specific recognition reaction, selective release or consumption of DA is achieved, allowing for quantitative detection of hAAG through changes in photocurrent signal. This solves the problems of photocorrosion and a single detection mechanism in traditional sulfide sensors.

[0062] In some embodiments, in S1, the CdZnS nanomaterial is prepared by a hydrothermal method, specifically by dispersing zinc salt, cadmium salt and sulfide precursor in deionized water, reacting at high temperature, and then centrifuging, washing and drying to obtain the nanomaterial.

[0063] In some embodiments, the zinc salt comprises one or more of Zn(Ac)2·2H2O, ZnCl2, and Zn(NO3)2·6H2O; the cadmium salt comprises one or more of Cd(Ac)2·2H2O, CdCl2·2.5H2O, and Cd(NO3)2·4H2O; and the sulfide precursor comprises one or more of Na2S·9H2O, thiourea, and thioacetamide.

[0064] In some embodiments, the molar ratio of the zinc salt, cadmium salt and sulfide precursor is 1.5~2.5:0.3~0.7:2.0~3.0, including but not limited to 2.0:0.5:2.5, 1.5:0.3:2.0, and 2.5:0.7:3.0.

[0065] In some embodiments, the temperature of the hydrothermal reaction is 150~170 ℃, including but not limited to 150 ℃, 160 ℃, and 170 ℃; the reaction time is 10~14 h, including but not limited to 10 h, 12 h, and 14 h.

[0066] In some embodiments, the concentration of the CdZnS suspension is 0.5~1.5 mg / mL, including but not limited to 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, and 1.5 mg / mL, preferably 1.0 mg / mL, at which concentration the material dispersibility is optimal and the photoelectric response is strongest.

[0067] In some embodiments, in S2, the conductive electrode is an indium tin oxide (ITO) conductive electrode, and the pretreatment method includes: ultrasonically cleaning the ITO electrode with isopropanol and deionized water for 10-20 min in sequence, and then drying it for later use.

[0068] In some embodiments, the coating amount of the CdZnS suspension is 25~35 μL, including but not limited to 25 μL, 30 μL, and 35 μL; the drying method is natural drying at room temperature for 12~24 h to ensure uniform loading of the material and the formation of a stable modified layer.

[0069] In some embodiments, the HP needs to be pretreated: dissolved in 10 mM Tris-HCl buffer (pH=8.0) containing 0.1 mM MgCl2, heated at 95 °C for 4~6 min, and naturally cooled to room temperature to form a stable hairpin structure.

[0070] In some embodiments, the hAAG reaction is carried out at a temperature of 36-38 °C and for a reaction time of 60-80 min, including but not limited to 60 min, 70 min, and 80 min, preferably 70 min, to ensure that hAAG fully recognizes and removes hypoxanthine sites on HP.

[0071] In some embodiments, the concentration of Endo Ⅳ is 400~600 U / mL, and the reaction time is 60~80 min; the concentration of Exo Ⅰ is 8~12 U / μL, and the reaction time is 30~50 min, to ensure complete degradation of the single-chain aptamer.

[0072] In some embodiments, the concentration of the DA solution is 40-60 μM, and the reaction time with the enzyme-catalyzed reaction product solution is 25-35 min to ensure that the DA is fully released or consumed.

[0073] In some embodiments, in S4, the reaction temperature of the CdZnS modified electrode and the enzyme-catalyzed reaction product solution is room temperature, and the reaction time is 6 to 10 min, including but not limited to 6 min, 8 min, and 10 min, preferably 8 min, to ensure that DA is fully coordinated with the CdZnS surface and induces the generation of sulfur vacancies.

[0074] In some embodiments, in S5, the photoelectrochemical test uses a three-electrode system: a CdZnS modified electrode as the working electrode, a saturated Ag / AgCl electrode as the reference electrode, and a platinum electrode as the counter electrode.

[0075] In some embodiments, the test buffer is 0.1 mol / L Tris-HCl (pH=8.0), the test voltage is +0.2~+0.4 V (vs. Ag / AgCl), preferably +0.3 V; and the excitation wavelength is 400~410 nm, under which the photocurrent response is strongest.

[0076] In some embodiments, the specific process of the photoelectrochemical detection is as follows: insert the three electrodes into the buffer solution, turn on the electrochemical workstation and excitation light source, record the photocurrent intensity, and calculate the photocurrent difference (ΔI) when hAAG is present and when hAAG is not present.

[0077] In the specific sample testing of this invention, the actual sample to be tested (such as diluted human serum) can replace the standard hAAG solution. After processing according to the enzymatic reaction procedure in S3, it is immersed in a CdZnS modified electrode for photoelectrochemical testing. The concentration of hAAG in the actual sample is obtained by comparing the measured photocurrent intensity with the standard curve. The specific detection conditions are the same as those in S3~S5 above, and will not be repeated here.

[0078] It is understood that the detection method provided by this invention is applicable to the detection of hAAG in a variety of real samples, including but not limited to human serum, cell lysate, etc., providing reliable technical support for early disease diagnosis and disease monitoring.

[0079] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0081] Hairpin DNA (HP) was purchased from Shanghai Sangon Biotech, Endo IV was purchased from Shanghai Sangon Biotech, and Exo I was purchased from Shanghai Sangon Biotech.

[0082] Example 1:

[0083] This embodiment provides a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, including the following steps:

[0084] (1) Preparation of CdZnS nanomaterials: 2.5 mmol Na2S·9H2O, 2.0 mmol Zn(Ac)2·2H2O and 0.5 mmol Cd(Ac)2·2H2O were dissolved in 20 mL of deionized water and magnetically stirred at room temperature for 1 h until completely dispersed. The suspension was transferred to a 50 mL polytetrafluoroethylene-lined reactor and reacted at 160 ℃ for 12 h. After the reaction was completed, the precipitate was collected by centrifugation at 8000 r / min for 10 min, washed three times alternately with deionized water and ethanol, and dried for 10 h to obtain CdZnS nanopowder.

[0085] (2) Preparation of ITO / CdZnS electrode: CdZnS nanoparticles were dispersed in ultrapure water to prepare a CdZnS suspension of 1.0 mg / mL. 30 μL of the suspension was drop-coated onto the surface of the pretreated ITO electrode and allowed to air dry at room temperature for 12 h. The pretreatment method for the ITO electrode was as follows: it was boiled in a 2.0 mol / L KOH isopropanol solution for 25 min, rinsed with deionized water, and dried at 125℃.

[0086] (3) Construction of the enzymatic reaction system for hAAG: 10 μL of 5 μM single-stranded DNA (HP) was dissolved in 10 mM Tris-HCl buffer (pH=8.0) containing 0.1 mM MgCl2, heated at 95 ℃ for 5 min, and then naturally cooled to form a stable hairpin DNA structure. 10 μL of different concentrations of hAAG (0, 5 × 10⁻⁶) were added to the system. -4 U / mL, 1×10 -3 U / mL, 5×10 -3 U / mL, 1×10 -2 U / mL, 5×10 -2 The reaction mixture consisted of 5 μL of 500 U / mL Endo IV and its corresponding buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, pH=8.8), 0.1 U / mL Endo IV, 0.1 U / mL Endo IV and its corresponding buffer (500 mM Tris-acetate, 500 mM KCl, 10 mM EDTA, 0.5% (v / v) Triton X-100, pH=7.5), and deionized water was added to a final volume of 50 μL. The mixture was incubated at 37 °C for 70 min and then heated at 80 °C for 20 min to terminate the reaction. After cooling, add 3 μL of 10 U / μL Exo I and the corresponding buffer (670 mM glycine-KOH, 67 mM MgCl2, 10 mM DTT, pH=9.5), add deionized water to 100 μL, incubate at 37 ℃ for 40 min, heat at 80 ℃ for 20 min to terminate the reaction, and finally add 25 μL of 50 μM dopamine (DA) solution and react at room temperature for 30 min.

[0087] (4) Sulfur vacancy induction and PEC detection: The ITO / CdZnS electrode was immersed in the above-mentioned enzymatic reaction product solution for 8 min, and then rinsed with 0.1 mol / L Tris-HCl buffer (pH=8.0). A three-electrode system was constructed with the ITO / CdZnS electrode as the working electrode, saturated Ag / AgCl as the reference electrode, and a platinum electrode as the counter electrode. PEC was tested in 0.1 mol / L Tris-HCl buffer (pH=8.0) with a test voltage of +0.3 V and an excitation light source of 400~410 nm LED. The photocurrent intensity was recorded.

[0088] (5) Construction of standard curve: such as Figure 6 As shown in Figures A and B, with the logarithm of hAAG concentration on the x-axis and the photocurrent difference (ΔI = photocurrent with hAAG present - photocurrent without hAAG) on the y-axis, the linear equation ΔI = 0.518 × Log[hAAG] + 1.756 is obtained, with a correlation coefficient R² = 0.984 and a detection range of 5 × 10⁻⁶. -4 ~1.0 U / mL, detection limit is 1.7 × 10⁻⁶ -4 U / mL (S / N=3).

[0089] To verify the accuracy of the method, 0.02 U / mL, 0.05 U / mL, 0.1 U / mL, 0.2 U / mL, and 0.5 U / mL hAAG were added to healthy human serum samples (centrifuged at 4500 r / min for 5 min, and the supernatant was diluted 20 times with 10 mM Tris-HCl buffer (pH=7.4)) for spike recovery experiments. The results showed that the spike recovery rate was 95.5%~108.4%, and the relative standard deviation was ≤3.5%, indicating that the method is suitable for the detection of actual samples.

[0090] Table 1. Test results of actual samples with different spiking concentrations

[0091]

[0092] Figure 1 The image shows the TEM characterization of CdZnS prepared in Example 1 of this invention; it can be seen that CdZnS consists of nanoparticles of about 20 nm.

[0093] Figure 2 The image shows a comparison of the photocurrent of CdZnS and CdZnS / DA in this invention. It can be seen that the photocurrent is significantly enhanced after DA treatment, which confirms that the reaction between DA and the material causes a surface effect, inducing an enhancement of the photocurrent.

[0094] Figure 3The images show the high-resolution XPS spectra of S 2p for CdZnS and CdZnS / DA; it can be seen that S 2p 1 / 2 The area ratio of characteristic peaks (attributed to sulfur vacancies) increased from 12.65% to 28.60%, indicating that a large number of sulfur vacancies were generated.

[0095] Figure 4 The ESR spectra of CdZnS and CdZnS / DA are shown. It can be seen that CdZnS / DA has a characteristic signal peak at 3364 G, and the calculated g=2.003 further verifies the presence of sulfur vacancies.

[0096] Figure 5 The results show the sulfur vacancy formation energy calculations for CdZnS and CdZnS / DA. DFT calculations show that the sulfur vacancy formation energy of CdZnS / DA (1.132 eV) is much lower than that of pure CdZnS (1.881 eV), a decrease of 40%.

[0097] Example 2:

[0098] This embodiment provides a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, including the following steps:

[0099] (1) Preparation of CdZnS nanomaterials: 3.0 mmol Na2S·9H2O, 2.5 mmol Zn(Ac)2·2H2O and 0.5 mmol Cd(Ac)2·2H2O were dissolved in 24 mL of deionized water, stirred at room temperature for 1.5 h, and then transferred to a reaction vessel. The reaction was carried out at 170 °C for 10 h. After centrifugation, washing and drying, CdZnS solid powder was obtained.

[0100] (2) Preparation of ITO / CdZnS electrode: CdZnS was prepared into a CdZnS suspension of 1.0 mg / mL. 30 μL was dropped onto the pretreated ITO electrode and air-dried to obtain the ITO / CdZnS electrode.

[0101] (3) Enzymatic reaction and PEC detection of hAAG: Following the method for constructing the enzymatic reaction system in Example 1, the hAAG reaction time was adjusted to 60 min, the Endo IV concentration to 600 U / mL, and the Exo I reaction time to 30 min, while keeping other conditions unchanged. The ITO / CdZnS electrode was immersed in the enzymatic reaction product solution for 6 min, and the PEC test was performed according to the conditions in Example 1. The photocurrent intensity was recorded, and a standard curve was constructed to obtain the detection model.

[0102] Example 3:

[0103] This embodiment presents a photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, comprising the following steps:

[0104] (1) Preparation of CdZnS nanomaterials: 2.0 mmol Na2S·9H2O, 1.5 mmol Zn(Ac)2·2H2O and 0.5 mmol Cd(Ac)2·2H2O were dissolved in 16 mL of deionized water, stirred at room temperature for 0.5 h, and then transferred to a reaction vessel. The reaction was carried out at 150 ℃ for 14 h. After centrifugation, washing and drying, CdZnS solid powder was obtained.

[0105] (2) Preparation of ITO / CdZnS electrode: CdZnS was prepared into a CdZnS suspension of 1.0 mg / mL. 30 μL was dropped onto the pretreated ITO electrode and air-dried to obtain the ITO / CdZnS electrode.

[0106] (3) Enzymatic reaction and PEC detection of hAAG: Following the method for constructing the enzymatic reaction system in Example 1, the hAAG reaction time was adjusted to 80 min, the Endo IV concentration to 400 U / mL, and the Exo I concentration to 12 U / μL, while keeping other conditions unchanged. The ITO / CdZnS electrode was immersed in the enzymatic reaction product solution for 10 min, and the detection was performed according to the PEC test conditions in Example 1. The photocurrent intensity was recorded, and a standard curve was constructed to obtain the detection model.

[0107] This invention also investigated the effect of CdZnS suspension concentration on detection. The suspension concentration in step (2) of Example 1 was independently modified to 0.5 mg / mL and 1.5 mg / mL, respectively. The photocurrent enhancement effect was not as significant as at 1.0 mg / mL. At 1.0 mg / mL, the material dispersibility was optimal, the coordination with DA was most complete, the sulfur vacancy generation was the highest, the charge separation efficiency was optimal, and the photocurrent response was the strongest, with an improvement of over 30% compared to other concentrations.

[0108] This invention also investigated the selectivity of the method in Example 1, detecting interfering substances such as 5.0 mg / mL bovine serum albumin (BSA), human serum albumin (HSA), 8-oxoguanine DNA glycosyltransferase (hOGG1), uracil DNA glycosyltransferase (UDG), alkaline phosphatase (ALP), T4 ligase, thymine DNA glycosyltransferase (TDG), and immunoglobulin G (IgG). The results are as follows. Figure 7 As shown, only hAAG elicited a significant photocurrent response, while the photocurrent signals of other interfering substances were close to those of the blank group, indicating excellent method selectivity.

[0109] Compared to traditional fluorescence detection methods, this method offers a lower detection limit, simpler operation, and significantly reduced background noise. Compared to existing sulfide-based PEC sensors, it effectively solves the stability problem caused by photocorrosion, while overcoming the drawbacks of high energy consumption and complex operation of traditional sulfur vacancy introduction methods, resulting in a significant improvement in detection sensitivity. Furthermore, this sulfur vacancy engineering strategy is not only applicable to hAAG detection but can also be extended to PEC sensing of other biomarkers. It also provides new insights for performance optimization of sulfide-based photocatalysts, electrocatalysts, solar cells, and other devices, demonstrating broad application prospects.

[0110] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A photoelectrochemical detection method for hAAG based on sulfur vacancy engineering, characterized in that, Includes the following steps: S1: CdZnS nanomaterials are modified onto the surface of a conductive electrode to obtain a CdZnS modified electrode; S2: Prepare hairpin DNA from single-stranded DNA, and incubate with different concentrations of hAAG and hAAG reaction buffer, endonuclease IV and endonuclease IV reaction buffer, and exonuclease I and exonuclease I buffer; then add dopamine solution to react and obtain the enzymatic reaction system of hAAG; S3: Immerse the CdZnS modified electrode obtained in step S1 into the enzyme-catalyzed reaction product solution in step S2 to react, so that dopamine and zinc atoms on the CdZnS surface are coordinated and bound through ortho-hydroxyl groups, inducing the generation of sulfur vacancies. S4: Perform photoelectrochemical testing on the CdZnS modified electrode obtained in step S3, detect the photocurrent signal, establish a linear relationship between the photocurrent signal and the hAAG concentration, and realize the quantitative detection of hAAG.

2. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In step S1, the CdZnS nanomaterials are prepared by a hydrothermal method; the CdZnS nanomaterials are obtained by dispersing zinc salt, cadmium salt and sulfide precursor in water, reacting them by heating, centrifuging, washing and drying. And / or, the particle size of the CdZnS nanomaterial is 15~25 nm.

3. The hAAG photoelectrochemical detection method according to claim 2, characterized in that, The zinc salt is one or more of Zn(Ac)2·2H2O, ZnCl2, and Zn(NO3)2·6H2O; And / or, the cadmium salt is one or more of Cd(Ac)2·2H2O, CdCl2·2.5H2O, and Cd(NO3)2·4H2O; And / or, the sulfide is one or more of Na2S·9H2O, thiourea, and thioacetamide; And / or, the molar ratio of the zinc salt, cadmium salt and sulfide precursor is 1.5~2.5:0.3~0.7:2.0~3.0; And / or, the conditions for the heating reaction are: 150~170℃ for 10~14 h.

4. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In step S1, the CdZnS modified electrode is prepared by the following method: dispersing CdZnS nanomaterials in water to obtain a CdZnS suspension; coating the CdZnS suspension onto the surface of a pretreated conductive electrode, and drying it to obtain the CdZnS modified electrode.

5. The hAAG photoelectrochemical detection method according to claim 4, characterized in that, The concentration of the CdZnS suspension is 0.5~1.5 mg / mL; And / or, the coating amount of the CdZnS suspension is 1.5~2.2 μL / mm. 2 ; And / or, the conductive electrode is an indium tin oxide electrode.

6. The hAAG photoelectrochemical detection method according to claim 1, characterized in that, In step S2, the enzyme-catalyzed reaction product solution is prepared by the following method: (1) Dissolve single-stranded DNA in a buffer solution containing magnesium ions, heat, and cool to form hairpin DNA; (2) Hairpin structure DNA, different concentrations of hAAG and hAAG reaction buffer and water were mixed and incubated. hAAG recognized and removed hypoxanthine sites on hairpin structure DNA to form purine-free / pyrimidine-free sites. (3) Add endonuclease IV and endonuclease IV reaction buffer and incubate to cleave the purine-free / pyrimidine-free sites to release single-chain dopamine aptamers; (4) Add exonuclease I and exonuclease I buffer and incubate to degrade single-chain dopamine aptamers; (5) Heating terminates the enzymatic reaction, and after cooling, dopamine solution is added to react and the enzymatic reaction product solution is obtained.

7. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In step S2, the single-stranded DNA is a stem-loop structured single-stranded DNA, one complementary strand of the stem structure is modified with hypoxanthine, and the hypoxanthine to terminal sequence in the stem structure is a dopamine aptamer sequence. The sequence of the single-stranded DNA is shown in SEQ ID NO. 1; the sequence of the dopamine aptamer is shown in SEQ ID NO.

2.

8. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In step S2, the concentration of hAAG is 5 × 10⁻⁶. -4 ~1.0 U / mL; And / or, the concentration of the dopamine solution is 40~60 μM.

9. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In S3, the reaction conditions are: 25-30℃ for 6-10 min.

10. The photoelectrochemical detection method for hAAG according to claim 1, characterized in that, In step S4, the buffer solution for the photoelectrochemical test is a Tris-HCl buffer solution with a pH of 7.0-9.0; the voltage is +0.2~+0.4 V; The excitation wavelength is 400~410 nm.

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