Self-energized photoelectrochemical biosensor based on AgIn5S8-coated Cu-Por-MOF heterojunction and application of self-energized photoelectrochemical biosensor in streptomycin detection

Abstract

The invention belongs to the technical field of photoelectrochemical biosensors, and particularly relates to a self-energized photoelectrochemical biosensor based on an AgIn5S8-coated Cu-Por-MOF heterojunction and application of the self-energized photoelectrochemical biosensor in streptomycin detection. The self-powered photoelectrochemical biosensor based on the AgIn5S8 (at) Cu-Por-MOF heterojunction comprises a photoelectric cathode, wherein the photoelectric cathode comprises a conductive substrate and an AgIn5S8 (at) Cu-Por-MOF composite material layer compounded on the surface of the conductive substrate; the AgIn5S8-coated Cu-Por-MOF composite material is obtained by carrying out hydrothermal reaction on AgIn5S8, a copper source and imidazole phenyl porphyrin in a solvent. A built-in electric field formed at the interface of AgIn5S8 and Cu-Por-MOF drives directional migration of photo-induced electrons-holes, the photoelectric conversion efficiency and the charge mobility are improved, PEC response signals are amplified, and further the streptomycin detection sensitivity is improved.

Description

Technical Field

[0001] This invention relates to the field of photoelectrochemical biosensor technology, specifically to a self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction and its application in streptomycin detection. Background Technology

[0002] Streptomycin (STR), an aminoglycoside antibiotic, is derived from *Streptomyces griseus*. STRs are highly effective at inhibiting Gram-negative bacteria by interfering with protein synthesis, leading to bacterial death. Therefore, they are widely used as antibacterial agents in the pharmaceutical, livestock, and agricultural fields. However, the misuse of STRs can lead to their residues in animals, causing bacterial mutations and antibiotic resistance, thus impacting human health. Several methods for detecting STRs have been developed, including enzyme-linked immunosorbent assay (ELISA), fluorescence detection (FL), and enzyme-linked immunosorbent assay (ELISA). However, these methods typically have drawbacks, such as requiring extensive equipment, complex operation, time consumption, and specialized personnel.

[0003] Chinese patent application CN118604080A, published on September 6, 2024, discloses a photoelectrochemical aptamer sensor based on CuO-CuInS2 and ferrocene and its application. The composite material CuO-CuInS2 is used as the photocathode active material, providing a stable initial photocurrent. Ferrocene (Fe) is used as the signal amplifier to construct an on-off PEC aptamer sensor for sensitive detection of streptomycin. The photoelectric signal decreases with increasing streptomycin concentration and has a linear relationship with the logarithm of streptomycin concentration, with a linear range of 500 fM-100 nM and a detection limit of 5.26 fM (3.06 fg / mL).

[0004] However, the detection limits of the aforementioned photoelectrochemical sensors for streptomycin detection remain relatively high. Self-powered photoelectrochemical biosensing analysis, as a promising analytical method, has attracted widespread research attention due to its advantages such as ease of operation, simple equipment, and extremely low background noise. Therefore, it is essential to develop highly sensitive and selective self-powered photoelectrochemical (PEC) biosensors for the analysis of streptomycin. Summary of the Invention

[0005] The purpose of this invention is to provide a self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction, which solves the problem of low sensitivity of existing photoelectrochemical sensors in streptomycin detection.

[0006] The second objective of this invention is to provide an application of a self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction in streptomycin detection, thereby solving the problem of low sensitivity of existing photoelectrochemical sensors in streptomycin detection.

[0007] To address the aforementioned technical problems, the technical solution of the self-powered photoelectrochemical biosensor based on the AgIn5S8@Cu-Por-MOF heterojunction of the present invention is as follows:

[0008] A self-powered photoelectrochemical biosensor based on an AgIn5S8@Cu-Por-MOF heterojunction includes a photocathode comprising a first conductive substrate and an AgIn5S8@Cu-Por-MOF composite material layer composited on the surface of the first conductive substrate; the AgIn5S8@Cu-Por-MOF composite material is obtained by hydrothermal reaction of AgIn5S8, a copper source, and imidazophenylporphyrin in a solvent.

[0009] This invention improves upon existing technologies by providing a self-powered photoelectrochemical biosensor based on an AgIn5S8@Cu-Por-MOF heterojunction. Cu-Por-MOF is synthesized via a solvothermal method, and an AgIn5S8@Cu-Por-MOF pn-type heterojunction is prepared through in-situ growth. This heterojunction exhibits excellent light absorption and electrochemical activity. A built-in electric field is formed at the AgIn5S8 and Cu-Por-MOF interface, causing photogenerated electrons and holes to separate under the influence of this field. This drives the directional migration of photogenerated electrons and holes, improving photoelectric conversion efficiency and charge mobility, and effectively amplifying the PEC response signal. Using AgIn5S8@Cu-Por-MOF as the photocathode and photoanode, a self-powered PEC biosensor driven by a dual-photoelectrode photofuel cell is constructed for the efficient detection of trace STRs in food.

[0010] The self-powered PEC aptamer sensor provided by this invention operates at 1.0 fg / mL. -1 -1 μg mL -1 It showed an ultra-low limit of detection (LOD) for STR detection within the concentration range of 0.81 fg / mL. -1 Furthermore, this sensor also exhibits high selectivity, good reproducibility, and stability, demonstrating significant practical value in actual testing of pig liver, sheep kidney, and milk samples.

[0011] To further improve photoelectric conversion efficiency and charge mobility, the preferred mass ratio of AgIn5S8, copper source and imidazophenylporphyrin is (30~40):(25~30):(35~40).

[0012] To further improve the performance of the heterojunction, preferably, the hydrothermal reaction temperature is 120~160℃ and the hydrothermal reaction time is 24~36h.

[0013] To further improve the target recognition capability, preferably, a bio-modification layer is provided on the surface of the AgIn5S8@Cu-Por-MOF composite material layer. The bio-modification layer is obtained by sequentially incubating aptamers and non-specific site blockers on the surface of the AgIn5S8@Cu-Por-MOF composite material layer.

[0014] To further improve the open-circuit voltage and enhance detection stability, preferably, 0.1-1 nmol of aptamer and 1-2 μmol of nonspecific site blocking agent are incubated for every 2.5-10 mg of AgIn5S8@Cu-Por-MOF composite material.

[0015] To further improve the photocurrent response, preferably, the aptamer is incubated by coating the surface of the AgIn5S8@Cu-Por-MOF composite material layer with an aptamer solution and incubating at 4~8℃, with the concentration of the aptamer solution being 100nM~1μM and the incubation time being 10~60min.

[0016] To further improve photocurrent response, preferably, the self-powered photoelectrochemical biosensor includes a photoanode, which comprises a second conductive substrate and a CuInS2 layer composited on the second conductive substrate. Using AgIn5S8@Cu-Por-MOF as the photocathode working electrode and photoactive CuInS2 as the photoanode, a self-powered PEC biosensor driven by a dual-photoelectrode photofuel cell was constructed for the efficient detection of trace amounts of STRs in food.

[0017] To further improve the photocurrent response, preferably, for every 2.5~10mg of AgIn5S8@Cu-Por-MOF composite material in the photocathode, the corresponding mass of CuInS2 in the photoanode is 2.5~10mg.

[0018] The technical solution for the application of the self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction in streptomycin detection is as follows:

[0019] Application of the self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction in streptomycin detection.

[0020] The application of the self-powered photoelectrochemical biosensor of this invention in streptomycin detection utilizes AgIn5S8@Cu-Por-MOF as the photocathode working electrode. The built-in electric field formed at the interface of AgIn5S8 and Cu-Por-MOF drives the directional migration of photogenerated electrons and holes, improving photoelectric conversion efficiency and charge mobility, effectively amplifying the PEC response signal. The resulting biosensor exhibits an extremely low detection limit of 0.81 fg / mL when detecting streptomycin. -1 This significantly improves the sensitivity of the sensor, which also features high selectivity, good reproducibility, and stability.

[0021] Preferably, the method includes the following steps: before detection, streptomycin solutions of different concentrations are incubated on the surface of a photocathode for 10 to 50 minutes. Attached Figure Description

[0022] Figure 1 Surface morphology images of Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF;

[0023] Figure 2 TEM image and elemental distribution map of AgIn5S8@Cu-Por-MOF;

[0024] Figure 3 XRD and XPS full spectra of Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF;

[0025] Figure 4 High-resolution XPS spectra of elements in Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF;

[0026] Figure 5 Figures showing the photoelectric response test results of each step in the construction process of the AgIn5S8@Cu-Por-MOF self-powered PEC aptamer sensor and the STR detection.

[0027] Figure 6 Optimization test results for the construction conditions of AgIn5S8@Cu-Por-MOF self-powered PEC aptamer sensor;

[0028] Figure 7 The graph shows the sensitivity test results of the AgIn5S8@Cu-Por-MOF self-powered PEC aptamer sensor for STR detection.

[0029] Figure 8 Figure 1 shows the test results of selectivity, reproducibility, and stability of AgIn5S8@Cu-Por-MOF-based self-powered PEC aptamer sensor for detecting STR. Detailed Implementation

[0030] The technical concept of the self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction provided by this invention is as follows:

[0031] Existing photoelectrochemical aptamer sensors based on CuO-CuInS2 and ferrocene have a detection limit of 5.26 fM (3.06 fg / mL) when detecting STRs, indicating low sensor sensitivity.

[0032] Porphyrins are macroscopic cyclic organic compounds, typically containing four nitrogen atoms, used as catalysts, photosensitizers, and electron transport materials. When porphyrins and their derivatives are used as organic compounds, the linkers combine with metal nodes to form porphyrin MOFs (Por-MOFs). Porphyrin MOF molecules are periodically arranged, preventing self-aggregation and exhibiting excellent chemical stability. Simultaneously, porphyrin MOFs exhibit relatively small band gaps, promoting photoinduced electron transfer processes and demonstrating good photoelectric activity and catalytic performance. Furthermore, combining porphyrin MOF composites with functional components (such as nanomaterials or biomolecules) offers dual advantages: introducing nanomaterials significantly enhances catalytic activity, accelerating charge transport rates and thus improving signal response and detection sensitivity; while combining with biomolecules provides highly selective specific recognition functions for the target analytes.

[0033] Based on this, the present invention provides a self-powered photoelectrochemical biosensor. Combining the advantages of high porosity and large specific surface area of ​​Por-MOF, an AgIn5S8@Cu-Por-MOF heterojunction is synthesized and used as the photocathode material to modify the working electrode. Simultaneously, a photoactive material is used as the photoanode to form a dual-photoelectrode driven photofuel cell (PFC), thereby constructing a novel self-powered PEC biosensor for sensitive detection of STRs in various foods. The built-in electric field formed at the AgIn5S8 and Cu-Por-MOF interface drives the directional migration of photogenerated electrons and holes, thereby improving photoelectric conversion efficiency and charge mobility, amplifying the response signal, and ultimately enhancing the sensor's sensitivity.

[0034] The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction provided by the present invention includes a photocathode and a photoanode; the photocathode includes a conductive substrate and an AgIn5S8@Cu-Por-MOF composite material layer composited on the surface of the conductive substrate.

[0035] The AgIn5S8@Cu-Por-MOF composite material is obtained by subjecting AgIn5S8, a copper source, and imidazole phenyl porphyrin in a solvent at a mass ratio of (30~40):(25~30):(35~40) to a first hydrothermal reaction at 120~160℃ for 24~36 h. The solvent is a mixture of methanol and chloroform in a volume ratio of 1~2:1~2. The copper source is copper sulfate. 16~20 mL of the mixed solvent is added for every 30~40 mg of AgIn5S8. After adding AgIn5S8, the copper source, and imidazole phenyl porphyrin to the solvent, the mixture is first sonicated for 30~45 min, then stirred for 30~40 min.

[0036] AgIn5S8 is obtained by a second hydrothermal reaction in water with a silver source, an indium source, and a sulfur source in a molar ratio of 1~1.5:1.95~2.5:4.5~5. The temperature of the second hydrothermal reaction is 160~200℃, and the reaction time is 90~120 min. The silver source is silver nitrate; the indium source is indium nitrate; and the sulfur source is C2H5NS. 20~40 mL of deionized water is added for every 1~1.5 mmol of silver source. After adding the silver, indium, and sulfur sources to the water, the mixture is first sonicated for 15~30 min, and then stirred at room temperature for 20~30 min.

[0037] The AgIn5S8@Cu-Por-MOF composite layer is obtained by coating a first conductive substrate with an AgIn5S8@Cu-Por-MOF solution and then drying it. The concentration of the AgIn5S8@Cu-Por-MOF solution is 2.5~10 mg / mL, and the volume is 10~20 µL. More preferably, the concentration of the AgIn5S8@Cu-Por-MOF solution is 6 mg / mL. The first conductive substrate is conductive glass ITO.

[0038] A bio-modified layer is deposited on the surface of the AgIn5S8@Cu-Por-MOF composite material layer. The bio-modified layer is obtained by sequentially incubating an aptamer and a non-specific site blocker on the surface of the AgIn5S8@Cu-Por-MOF composite material layer. Each 2.5~10 mg of AgIn5S8@Cu-Por-MOF composite material corresponds to the incubation of 0.1~1 nmol of aptamer and 1~2 μmol of non-specific site blocker.

[0039] Specifically, the aptamer incubation involves coating the AgIn5S8@Cu-Por-MOF composite layer with an aptamer solution and incubating at 4–8°C. The concentration of the aptamer solution is 100 nM–1 μM, the incubation time is 10–60 min, and the volume is 10–20 µL. After aptamer incubation, a nonspecific site blocking agent solution is coated and incubated at 4–8°C to block nonspecific sites. The concentration of the nonspecific site blocking agent solution is 1–2 mM, and the volume is 10–20 µL. The nonspecific site blocking agent is MCH.

[0040] The photoanode comprises a second conductive substrate and a CuInS2 layer composited on the second conductive substrate. For every 2.5–10 mg of AgIn5S8@Cu-Por-MOF composite material in the photocathode, the corresponding mass of CuInS2 in the photoanode is 2.5–10 mg. Specifically, the CuInS2 layer is obtained by coating a CuInS2 solution onto the conductive substrate and then drying it; the concentration of the CuInS2 solution is 2.5–10 mg / mL.

[0041] CuInS2 is obtained by a third hydrothermal reaction in water using copper, indium, and sulfur sources in a molar ratio of 1–1.5:1.95–2.5:4.5–5. The temperature of the third hydrothermal reaction is 160–200 °C, and the time of the second hydrothermal reaction is 90–120 min. The copper source is copper nitrate; the indium source is indium nitrate; and the sulfur source is C2H5NS. 20–40 mL of deionized water is added for every 1–1.5 mmol of silver source. After adding the copper, indium, and sulfur sources to the water, the mixture is first sonicated for 15–30 min, and then stirred at room temperature for 20–30 min.

[0042] The embodiments of the present invention will be further described below with reference to specific examples. Unless otherwise specified, the chemical reagents involved in the following examples are all commercially available conventional products.

[0043] I. Specific Embodiments of the Self-Powered Photoelectric, Chemical, and Biosensor Based on AgIn5S8@Cu-Por-MOF Heterojunction of the Present Invention

[0044] Example 1

[0045] The self-powered photoelectrochemical biosensor based on the AgIn5S8@Cu-Por-MOF heterojunction in this embodiment includes a photocathode and a photoanode. The photocathode includes ITO and an AgIn5S8@Cu-Por-MOF composite material layer on the surface of ITO, and the photocathode serves as the working electrode. The photoanode includes ITO and a CuInS2 layer on the surface of ITO.

[0046] A bio-modified layer is formed on the surface of the AgIn5S8@Cu-Por-MOF composite material layer. The bio-modified layer is formed by sequentially incubating aptamers and non-specific site blockers on the surface of the AgIn5S8@Cu-Por-MOF composite material layer.

[0047] The base sequence of the aptamer Apt is as follows: STR Aptamer: 5'-NH2-GCT TCC AGC TTATTG AAT TAC ACG CAG AGGGTA GCG GCT CTG CGC ATT CAA TTG CTG CGC GCT GAA GCGCGG AAG C-3'. The nonspecific site blocking agent is 6-mercapto-1-hexanol (MCH).

[0048] The method for preparing the working electrode is as follows:

[0049] 1) Preparation of AgIn5S8

[0050] The basic steps for preparing AgIn5S8 by hydrothermal method are as follows: 1 mmol AgNO3, 1.95 mmol In(NO3)3 and 4.5 mmol C2H5NS are added to a beaker, followed by 20 mL of deionized water. The mixture is sonicated for 15 min to ensure uniform dispersion, stirred at room temperature for 20 min, and then transferred to a 40 mL reaction vessel. After reacting in a 160℃ oven for 90 min, the mixture is washed three times with water and ethanol, and then dried in a vacuum oven to obtain AgIn5S8.

[0051] 2) Preparation of AgIn5S8@Cu-Por-MOF composite material

[0052] The AgIn5S8@Cu-Por-MOF composite material was prepared by a hydrothermal method. The basic steps were as follows: 30 mg of AgIn5S8, 25 mg of CuSO4 and 35 mg of imidazole phenyl porphyrin (CAS: 1311998-62-5,5,10,15,20-tetrakis(4-(1H-imidazol-1-yl)phenyl)porphyrin) synthesized above were added to a mixed solvent of 8 mL methanol and 8 mL chloroform. After sonication for 30 min and stirring for 30 min, the mixture was loaded into a reaction vessel and placed in a high-temperature oven at 120 °C for 24 h. After cooling to room temperature, the mixture was washed three times with methanol and DMF and dried overnight in a vacuum oven at 60 °C to obtain the AgIn5S8@Cu-Por-MOF composite material.

[0053] 3) Working electrode

[0054] 10 µL of AgIn5S8@Cu-Por-MOF solution (concentration 6 mg / mL) was added. -1The electrode was added dropwise to the ITO surface and dried at room temperature to obtain the working electrode, denoted as AgIn5S8@Cu-Por-MOF / ITO.

[0055] Preparation of the biomodified layer: 10 µL of 500 nM aptamer solution was added to the AgIn5S8@Cu-Por-MOF / ITO surface and incubated at 4 °C for 50 min, denoted as Apt / AgIn5S8@Cu-Por-MOF / ITO; then Apt / AgIn5S8@Cu-Por-MOF / ITO was incubated with 10 µL of 1 mM MCH at 4 °C to block non-specific sites, denoted as MCH / Apt / AgIn5S8@Cu-Por-MOF / ITO.

[0056] Preparation of photoanodes:

[0057] 1) Preparation of CuInS2

[0058] The basic steps for preparing CuInS2 by hydrothermal method are as follows: 1 mmol Cu(NO3)2, 1.95 mmol In(NO3)3 and 4.5 mmol C2H5NS are added to a beaker, followed by 20 mL of deionized water. The mixture is sonicated for 15 min to ensure uniform dispersion, stirred at room temperature for 20 min, and then transferred to a 40 mL reaction vessel. After reacting in a 160℃ oven for 90 min, the mixture is washed three times with water and ethanol, and then dried in a vacuum oven to obtain CuInS2.

[0059] 2) Photoanode

[0060] 30 µL of a solution with a concentration of 5 mg / mL -1 CuInS2 solution was added dropwise to the ITO surface and dried at room temperature.

[0061] II. Application of the self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction in streptomycin detection.

[0062] Example 2

[0063] The application of the self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction in streptomycin detection in this embodiment is as follows: The self-powered photoelectrochemical biosensor constructed in Example 1 is used. ITO is modified with AgIn5S8@Cu-Por-MOF as the sensitive membrane, and aptamer molecules are further immobilized. Non-specific active sites are then blocked using MCH for the identification and detection of streptomycin (STR). The photoanode is CuInS2 / ITO, and the photocathode is AgIn5S8@Cu-Por-MOF / ITO (working electrode), forming a dual-photoelectrode driven photofuel cell system for constructing the self-powered PEC aptamer sensor. Using the photocathode as the working electrode to construct the sensing platform avoids false positives. Before detection, different concentrations of STR are incubated on the surface of the working electrode.

[0064] PEC measurements were performed using a CHI 760E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China) and a 500 W xenon lamp light source with a 420 nm filter (Beijing Pofilai Technology Co., Ltd.). The electrode was 3 cm away from the light source. A three-electrode system was used for testing: the working electrode was AgIn5S8@Cu-Por-MOF / ITO, the counter electrode was CuInS2 / ITO, and the reference electrode was an Ag / AgCl electrode. No additional electron donors were added to the electrolyte, and the bias voltage was 0 V. The photocurrent response was measured at 30 s intervals, and the It curves were recorded.

[0065] The electrolyte was a 0.01 M phosphate buffer solution, prepared by dissolving 0.242 g potassium dihydrogen phosphate, 2.951 g disodium hydrogen phosphate, 0.2 g KCl and 8.003 g sodium chloride in deionized water, bringing the volume to 1.0 L, and adjusting the pH of the solution to 7.4 with 0.1 M hydrochloric acid solution.

[0066] III. Experimental Examples

[0067] 1) SEM

[0068] The surface morphology of Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF was characterized using SEM, and the obtained surface morphology images are shown below. Figure 1 As shown. Figure 1 (a) and Figure 1 (b) shows that the surface of Cu-Por-MOF has a nanosheet structure, which is assembled from small nanocrystals and has a rough surface. AgIn5S8 exhibits an irregular stacked nanobulk structure with an average diameter of 100 nm. Figure 1 (c) and Figure 1 (d)). For example Figure 1 (e) and Figure 1 As shown in (f), the surface morphology of the AgIn5S8@Cu-Por-MOF composite material exhibits a nanobulk agglomerate structure with a diameter of approximately 600 nm–1µm.

[0069] TEM images and elemental distribution maps of AgIn5S8@Cu-Por-MOF are as follows: Figure 2 As shown. Figure 2 (a) and Figure 2 (b) TEM images at different magnifications. Figure 2 (a) and Figure 2 (b) shows that AgIn5S8@Cu-Por-MOF exhibits agglomerates formed by the aggregation of multiple circular nanosheets, with irregularly serrated edges. In the high-resolution TEM image of AgIn5S8@Cu-Por-MOF ( Figure 2 (c) The lattice stripes with a spacing of 0.31 nm belong to the (311) plane of AgIn5S8. Figure 2 (d) is the EDS element mapping image.

[0070] 2) XRD and XPS

[0071] The XRD and XPS full spectra of Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF are as follows: Figure 3 As shown, Figure 3 (a) and (b) are the XRD and XPS spectra, respectively, where (i) represents AgIn5S8, (ii) represents Cu-Por-MOF, and (iii) represents AgIn5S8@Cu-Por-MOF. The XRD pattern of CuCo-MOF shows two diffraction peaks at 2θ, at 6.8° and 9.5°, indicating that Cu(II) forms a complex with the imidazole ligand. In the XRD pattern of AgIn5S8, diffraction peaks at 27.3°, 33.1°, 43.2°, and 47.4° correspond to the (311), (400), (511), and (440) crystal planes of AgIn5S8 (JCPDS No. 25-1329). The TEM spectrum of AgIn5S8@Cu-Por-MOF (…) Figure 2 The (311) crystal plane in the XRD pattern of AgIn5S8 can also be observed in c).

[0072] XPS energy dispersive spectroscopy analysis was performed on the AgIn5S8@Cu-Por-MOF sample. Figure 3The results in (b) indicate that the AgIn5S8@Cu-Por-MOF composite material contains six elements, of which Cu (1.66%), C (66.3%), N (15.72%), and O (12.39%) are derived from Cu-Por-MOF, while In (0.62%), Ag (0.61%), and S (2.69%) are mainly derived from AgIn5S8 material. Clear In 3d (447.7 eV), Ag 3d (368.2 eV), and S 2p (161.6 eV) signals were observed in AgIn5S8 (curve i, Figure 3(b)). The XPS scan spectrum of Cu-Por-MOF (curve ii, Fig. 3(b)) includes C 1s (283.9 eV), N 1s (398.9 eV), O 1s (531.1 eV), and Cu 2p (935.2 eV) signals. Furthermore, AgIn5S8@Cu-Por-MOF includes C 1s, N 1s, O 1s, Cu 2p, In 3d, Ag 3d, and S 2p signals.

[0073] The electronic structure and chemical composition of AgIn5S8, Cu-Por-MOF, and AgIn5S8@Cu-Por-MOF samples were further investigated using high-resolution XPS spectra. High-resolution XPS spectra of elements in Cu-Por-MOF, AgIn5S8, and AgIn5S8@Cu-Por-MOF are shown below. Figure 4 As shown. Figure 4 (a)-(c) are high-resolution XPS spectra of C 1s, N 1s, and Cu 2p for Cu-Por-MOF and AgIn5S8@Cu-Por-MOF, respectively. Figure 4 In (a)-(c), (i) represents Cu-Por-MOF, and (ii) represents AgIn5S8@Cu-Por-MOF; Figure 4 (d)-(f) are high-resolution XPS spectra of Ag 3d, In 3d, and S 2p in AgIn5S8 and AgIn5S8@Cu-Por-MOF, respectively. Figure 4 In (d)-(f), (i) represents AgIn5S8 and (ii) represents AgIn5S8@Cu-Por-MOF.

[0074] The C 1s XPS spectra of Cu-Por-MOF and AgIn5S8@Cu-Por-MOF (Figure 4(a)) include two main groups: C–C / C=C (284.6 eV) and C–N (285.9 eV). Furthermore, the N 1s XPS spectra of both samples (Figure 4(b)) can be separated into C–N (398.6 eV), porphyrin N (399.7 eV), N–H (401.1 eV), and N–O (403.8 eV). Additionally, the peaks at BE of 932.7 eV and 934.9 eV in the Cu 2p XPS spectrum of Cu-Por-MOF (Figure 4(c)) are attributed to Cu2p. 3 / 2 The orbitals belong to Cu. + and Cu 2+ The peaks at 952.8 eV and 954.8 eV are attributed to Cu 2p. 1 / 2 The orbitals are Cu + and Cu 2+ The peaks are also observed in the Cu 2p XPS spectra of AgIn5S8@Cu-Por-MOF. However, the peaks at BE of 961.7 eV and 944.1 eV are attributed to Cu 2p. 3 / 2 The values ​​at 947.6 eV and 963.4 eV belong to Cu 2p. 1 / 2 The satellite peaks in the orbit disappeared. Furthermore, the Ag 3d XPS spectra of the AgIn5S8 and AgIn5S8@Cu-Por-MOF samples (Figure 4(d)) can be divided into Ag3d... 5 / 2 (368.1 eV) and Ag 3d 3 / 2 (374.1 eV). The In 3d XPS spectrum shown in Figure 4(e) reveals that the In 3d region... 5 / 2 (452.5 eV) orbital and In 3d 3 / 2 The diffraction peak of the (445.0 eV) orbital. The S 2p XPS spectrum of AgIn5S8@Cu-Por-MOF (Figure 4(f)) is attributed to S 2- 2p 3 / 2 (162.7 eV) and S 2- 2p 1 / 2 (161.6 eV).

[0075] 3) EIS and PEC responses of photocathodes at different modification stages

[0076] To verify the successful construction of the self-powered sensor, the EIS and PEC behaviors of the photocathode were studied in each modification stage. Figure 5The figures show the photoelectric response test results of each step in the construction process of the AgIn5S8@Cu-Por-MOF self-powered PEC aptamer sensor and the STR detection. Figure 5 (a)-(c) represent the photocurrent response diagram, EIS diagram, and open-circuit voltage diagram, respectively. (i) represents ITO, (ii) represents AgIn5S8@Cu-Por-MOF / ITO, (iii) represents Apt / AgIn5S8@Cu-Por-MOF / ITO, (iv) represents MCH / Apt / AgIn5S8@Cu-Por-MOF / ITO, and (v) represents STR / MCH / Apt / AgIn5S8@Cu-Por-MOF / ITO. Figure 5 (d) CuInS2 / ITO photoanode and AgIn5S8@Cu-Por-MOF / ITO photocathode in 0.1 M phosphate buffer solution (PBS, pH 7.4) under illumination at 2 mV s -1 Polarization curves obtained from the scan rate.

[0077] like Figure 5 As shown in figure a, the photocurrent of bare ITO is negligible. AgIn5S8@Cu-Por-MOF exhibits an enhanced photocurrent (8.24 μA), which is due to the high light absorption performance and photoelectric conversion efficiency of the heterojunction. However, after aptamer molecule adsorption, the photocurrent decreases to 6.22 μA. This is because a large number of aptamers are immobilized in the porous AgIn5S8@Cu-Por-MOF network, and the insulating molecular layer hinders the transfer of photogenerated carriers / electrolytes at the photocathode / electrolyte interface, leading to a decrease in PEC response. Modifying Apt / AgIn5S8@Cu-Por-MOF / ITO with MCH to eliminate nonspecific adsorption leads to a further decrease in photocurrent (4.53 μA). When STRs are present, the aptamers specifically recognize STRs, and the signal further decreases (4.14 μA).

[0078] Furthermore, the sensor construction and STR detection process were further validated using electrochemical techniques (EIS). Figure 5 (b) In this case, the ITO substrate was replaced with a gold electrode AE ​​for testing. The results showed that ITO has a smaller charge transfer resistance (R0). ct The Ω value is 75.2, indicating good electrochemical activity. Furthermore, the Ro of AgIn5S8@Cu-Por-MOF / ITO is... ct The value is 197 Ω, slightly greater than that of ITO. Furthermore, the Ro of the Apt / AgIn5S8@Cu-Por-MOF / ITO electrode... ctThe resistance increases to 233 Ω, which is clearly caused by the fixed Apt, which hinders electrons from the electrolyte to the electrode surface, thus increasing the resistance. Subsequently, for MCH / Apt / AgIn5S8@Cu-Por-MOF / ITO, R... ct A slight increase (335 Ω). Subsequently, the constructed sensor was used to detect STR, further increasing R. ct The Ω was increased to 451 Ω. The CV curve showed results similar to those obtained by EIS measurements.

[0079] To investigate the output energy performance of the PFC system, its output voltage-current (VI) was tested. For example... Figure 5 As shown in (c), the open-circuit voltage of the AgIn5S8@Cu-Por-MOF-based PFC sensing system is 0.91 V. Subsequently, due to the strong biocompatibility of AgIn5S8@Cu-Por-MOF, aptamers adsorb onto the material surface, forming an insulating layer covering the ITO surface, hindering electron transport in the photocathode, with an open-circuit voltage of 0.83 V. Further, after sealing with MCH, the open-circuit voltage of Apt / AgIn5S8@Cu-Por-MOF / ITO is 0.78 V. Finally, with the introduction of STR, the open-circuit voltage drops to 0.74 V, indicating that the aptamer and the target STR recognize each other and specifically bind, and the formed Aptamer-STR complex further inhibits electron transfer.

[0080] The thermodynamic feasibility of the constructed photofuel cell was analyzed by studying the polarization curves of the photoanode and photocathode. Figure 5 (d) On the one hand, the onset potential for photoelectrochemical oxidation of water on the CuInS2 photoanode is approximately -0.97 V. On the other hand, the onset potential for photoreduction of oxygen on the AgIn5S8@Cu-Por-MOF photocathode is -0.06 V. The onset potential of photoanode oxidation is lower than that of photocathode reduction, which proves that the photofuel cell based on AgIn5S8@Cu-Por-MOF / ITO photocathode and CuInS2 / ITO photoanode is thermodynamically feasible. The theoretical open-circuit potential (OCP) of the cell, estimated from the difference between these two onset potentials, is 0.91 V, which is consistent with the actual open-circuit potential value (0.91 V) shown in the open-circuit potential curve.

[0081] 4) Condition Optimization

[0082] Before using the AgIn5S8@Cu-Por-MOF-based PEC aptamer sensor for quantitative analysis of STRs, the sensor construction conditions were optimized, and the effects of AgIn5S8@Cu-Por-MOF dispersion concentration, CuInS2 dispersion concentration, aptamer concentration, aptamer binding time, and target analyte binding time on the PEC response signal were investigated. Figure 6 Optimize test results by constructing conditions for the sensor. Figure 6 (a)-(e) are the results of the condition optimization test for AgIn5S8@Cu-Por-MOF concentration, CuInS2 dispersion concentration, aptamer concentration, aptamer binding time and target binding time, respectively.

[0083] Figure 6 (a) shows the photocurrent response induced by coating ITO with different concentrations of AgIn5S8@Cu-Por-MOF. The results show that the photocurrent response is significantly higher in the 2.5–6 mg / mL range. -1 Within the concentration range, the open-circuit voltage increases with increasing material concentration, while when the material concentration increases from 6 mg / mL... -1 Increase to 7 mg / mL -1 At that time, the open-circuit voltage did not change much, indicating that the material concentration that could be coated on ITO had reached saturation. Coating a thicker layer of material (10 mg / mL) would be more feasible. -1 This makes it easier for the AgIn5S8@Cu-Por-MOF dispersion to peel off from the electrode, leading to decreased measurement stability. Therefore, the concentration of the AgIn5S8@Cu-Por-MOF dispersion used to construct the PEC aptamer sensor was set to 6.0 mg / mL. -1 . Figure 6 (b) shows that as the CuInS2 concentration increases, ΔI first increases and then gradually remains constant, especially at a CuInS2 concentration of 8 mg / mL. -1 The signal reaches its maximum at this point, so this concentration is chosen as the concentration of the photoanode material during testing. Figure 6 (c) shows that the variation in the photocurrent response of the constructed aptamer sensor originates from the fixation of aptamers at different concentrations. Within the range of 100-500 nM, ΔI gradually increases with increasing aptamer concentration. When the aptamer concentration exceeds this value, it reaches saturation, and the photocurrent response signal plateaus. Therefore, a 500 nM aptamer solution was selected to develop the PEC aptamer sensor. At this level, the adsorption behavior of the aptamer is as follows: Figure 6 As shown in (d), the change in photocurrent signal response caused by aptamer anchoring reaches equilibrium after 50 min; therefore, the aptamer adsorption time is set to 50 min. Finally, the effect of the binding time of the target STR on detection is as follows: Figure 6As shown in (e), equilibrium was reached after 40 min, therefore the optimal binding time for the target and aptamer to bind is 40 min.

[0084] Therefore, the optimal conditions for sensor fabrication were determined as follows: the concentration of the ITO-modified AgIn5S8@Cu-Por-MOF solution was 6.0 mg / mL. -1 The concentration of CuInS2 was 8.0 mg / mL. -1 The aptamer concentration was 500 nM; the adsorption time of the aptamer on AgIn5S8@Cu-Por-MOF / ITO was 50 min; and the binding time of STR to the aptamer was 40 min.

[0085] 5) Detection sensitivity

[0086] Under the optimal conditions described in section 4) of the condition optimization section, a self-powered PEC aptamer sensor was constructed, and its sensitivity for STR detection was studied. The sensitivity test results are as follows: Figure 7 As shown, where Figure 7 (a)-(b) are the photocurrent response diagrams and calibration curves of the photocurrent response change (ΔI) versus STR concentration detected by the AgIn5S8@Cu-Por-MOF based PEC sensor, respectively. Figure 7 (b) shows the linear fit of ΔI as the logarithmic function of STR concentration; Figure 7 (c)-(d) are calibration curves showing the open-circuit voltage and the change in open-circuit voltage (ΔE) versus STR concentration for different concentrations of STR detected by the AgIn5S8@Cu-Por-MOF based PEC sensor, respectively. Figure 7 The inset in (d) is a linear fit plot of ΔE as a logarithmic function of STR concentration.

[0087] like Figure 7 As shown in (a), the photocurrent response of STR detection decreases with increasing STR concentration. As previously mentioned, the binding of more STR molecules to the aptamer chain leads to a thickening of the insulating layer on the electrode surface, resulting in a decrease in photocurrent response. Figure 7 (b) shows △I (△I = I0 – I) STR The relationship between STR levels and STR concentration. As STR levels increased from 1.0 fg / mL... -1 Increase to 10 pg mL -1 The resulting sharp increase in ΔI gradually stabilizes. ΔI is used as the STR concentration (lgC). STR The logarithmic function of ) in 1.0 fg mL -1 Up to 1 µg mL -1A good linear relationship can be obtained within the range of y = 0.61 + 0.42x, with a correlation coefficient R. 2 The value is 0.98, therefore, the calculated LOD is 0.81 fg mL. -1 (S / N = 3).

[0088] In addition, such as Figure 7 As shown in (c), the open-circuit voltage was also tested as a function of STR concentration. The open-circuit voltage gradually decreased as the STR concentration increased. This is because when the STR concentration is low, the active sites on the photocathode surface are relatively sufficient, and the specific recognition reaction between STR and Apt is more complete, resulting in a higher electrode potential. However, as the STR concentration increases, the active sites on the electrode surface are occupied, which leads to a decrease in the open-circuit voltage. Figure 7 (d) shows △E (△E = E0 – E STR The relationship between ΔE and STR concentration. Let ΔE be the STR concentration (lgC). STR The logarithmic function of ) in 1.0 fg mL -1 Up to 1 µg mL -1 A good linear relationship can be obtained within the range of y = 0.028 + 0.043x, with a correlation coefficient R. 2 The value is 0.99. Therefore, the calculated value of LOD is 0.82 fg mL. -1 (S / N = 3), which is close to the LOD obtained under the PEC mode.

[0089] 6) Selectivity, reproducibility, and stability

[0090] To test the selectivity of this biosensor, different antibiotics were used as interfering agents, including gentamicin, erythromycin, tetracycline, ampicillin, clindamycin, lincomycin, and kanamycin. A mixed solution of STR and the above interfering agents was also used as an interfering agent. The concentration of all these interfering agents was set at 100 fg / mL. -1 , is STR (1 fgmL) -1 The concentration was 100 times that of the target substance. The selectivity of the biosensor was analyzed by comparing the photocurrent response induced by STR and interfering substance detection.

[0091] To investigate the stability of the biosensor, short-term stability tests were conducted (the photocurrent response of the PEC aptamer sensor was recorded every 30 seconds using an electrochemical workstation for 500 seconds) and long-term stability tests were conducted (the same electrode pair with the same STR concentration (1 fg / mL) was recorded for 15 consecutive days). -1 (Photocurrent response under)

[0092] Five independent biosensors were prepared for STR (1 fg mL) -1 They are tested to ensure that they are as consistent as possible in terms of preparation process, material composition, and testing conditions, in order to assess their reproducibility.

[0093] The test results of the selectivity, reproducibility, and stability of the AgIn5S8@Cu-Por-MOF-based self-powered PEC aptamer sensor for detecting STRs are as follows: Figure 8 As shown, where Figure 8 (a)-(d) show the results of the selectivity, reproducibility, short-term stability and long-term stability tests, respectively.

[0094] Figure 8 (a) The results show that the photocurrent response change caused by the detection of interfering substances using the aptamer sensor of the present invention is very small. The photocurrent signal generated by the STR is significantly higher than that of the interfering substances, and comparable to the photocurrent signals of all mixtures of interfering substances and STR. This indicates that the constructed PEC aptamer sensor has excellent selectivity in detecting STR in complex environments. Furthermore, reproducibility was evaluated using five identical PEC aptamer sensors. Figure 8 (b) It can be seen that the photocurrent response of the five aptamer sensors caused by STR detection is similar, with a relative standard deviation (RSD) of 0.82%.

[0095] Figure 8 (c) The photocurrent remains almost constant throughout the 10 repeated on / off light cycles. The final photocurrent response of the detected STR is close to the initial value. Figure 8 As shown in (d), the long-term stability of the given PEC aptamer sensor was investigated by recording the photocurrent response of STR analysis daily for 15 days. The results showed a low RSD of 0.89%. These results indicate that the constructed PEC aptamer sensor has good short-term and long-term stability.

[0096] 7) Actual sample testing

[0097] To verify the practicality of this biosensor, a recovery experiment was conducted using a spiking method, with milk, sheep kidney, and pig liver selected as actual samples. For milk pretreatment, the milk sample was thoroughly shaken, and 10 mL was measured into a centrifuge tube. 20 mL of acetonitrile was added, and the mixture was thoroughly shaken to denature and precipitate the proteins. The sample was then centrifuged at 8000 r / min for 5 min, and the supernatant was collected. This centrifugation process was repeated 2-3 times. After protein removal, the milk samples were filtered through 0.45 μm and 0.22 μm filters to remove any remaining small particulate impurities. The resulting filtrate was diluted 100 times with deionized water and thoroughly mixed. Sheep kidneys and pig liver samples were purchased from a local supermarket. 5 g of the minced meat was weighed and added to 15 mL of 2% lead acetate solution. The mixture was sonicated for 20 min, stirred for 30 min, and centrifuged at 14000 r / min for 5 min. The supernatant was diluted 500 times with pure water and filtered multiple times through a 0.22 µm filter to serve as a blank sample. Different standard concentrations of STR were added to the blank sample, and the recovery rate was calculated by comparing it with a linear standard curve to verify the practicality of the biosensor.

[0098] After detection using the developed PEC aptamer sensor, data was obtained from the calibration curve (see...). Figure 7 (b) The true concentration of STR was derived and compared with the spiked value. The spiked recoveries obtained from the tests are shown in Table 1. The detection results of STR in pig liver, sheep kidney, and milk showed that the recoveries of the sensor were 92.5%~95.8%, 95.2%~99.2%, and 88.3%~95.9%, respectively, with RSDs of 1.25%~3.42%, 1.67%~5.29%, and 2.04%~4.76%, respectively. These results demonstrate that the constructed AgIn5S8@Cu-Por-MOF-based self-powered PEC aptamer sensor has acceptable applicability in the detection of real samples.

[0099] Table 1. Self-powered PEC sensor used for STR content determination in different samples (n = 3)

[0100]

[0101] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction, characterized in that, The invention includes a photocathode, which comprises a first conductive substrate and an AgIn5S8@Cu-Por-MOF composite material layer composited on the surface of the first conductive substrate; the AgIn5S8@Cu-Por-MOF composite material is obtained by hydrothermal reaction of AgIn5S8, a copper source and imidazophenyl porphyrin in a solvent.

2. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 1, characterized in that, The mass ratio of AgIn5S8, copper source and imidazophenylporphyrin is (30~40):(25~30):(35~40).

3. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 1 or 2, characterized in that, The hydrothermal reaction temperature is 120~160℃, and the hydrothermal reaction time is 24~36h.

4. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 1, characterized in that, A bio-modified layer is disposed on the surface of the AgIn5S8@Cu-Por-MOF composite material layer. The bio-modified layer is obtained by sequentially incubating aptamers and non-specific site blockers on the surface of the AgIn5S8@Cu-Por-MOF composite material layer.

5. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 4, characterized in that, For every 2.5~10 mg of AgIn5S8@Cu-Por-MOF composite material, 0.1~1 nmol of aptamer and 1~2 μmol of nonspecific site blocking agent are incubated.

6. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 5, characterized in that, The aptamer incubation method involves coating the surface of the AgIn5S8@Cu-Por-MOF composite material layer with an aptamer solution and incubating it at 4~8℃. The concentration of the aptamer solution is 100nM~1μM, and the incubation time is 10~60min.

7. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 1, characterized in that, The self-powered photoelectrochemical biosensor includes a photoanode, which comprises a second conductive substrate and a CuInS2 layer composited on the second conductive substrate.

8. The self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in claim 7, characterized in that, For every 2.5~10 mg AgIn5S8@Cu-Por-MOF composite material in the photocathode, the corresponding mass of CuInS2 in the photoanode is 2.5~10 mg.

9. The application of a self-powered photoelectrochemical biosensor based on AgIn5S8@Cu-Por-MOF heterojunction as described in any one of claims 1-8 in streptomycin detection.

10. The application as described in claim 9, characterized in that, Includes the following steps: Before detection, streptomycin solutions of different concentrations were incubated on the surface of the photocathode for 10-50 minutes.

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