Photoelectrochemical aptamer sensor based on triple helix molecular switch probe and its application in detection of amoxicillin

Abstract

The application belongs to the field of biological analysis, and relates to detection of amoxicillin, in particular to a photoelectrochemical aptamer sensor based on a triple helix molecular switch probe and application thereof in detection of amoxicillin. The application constructs a signal enhanced PEC aptamer sensor for detection of amoxicillin based on a ferrocene-labeled triple helix molecular probe (THMS). CulnS2 is used as a photoelectrode active material to provide stable initial photocurrent. The ferrocene-labeled T2 probe is replaced by specific recognition of amoxicillin and the ring part of the triple helix molecular probe, and is introduced into the electrode surface through base complementary pairing. Fc can accept electrons from the CulnS2 nanoflower and adjust the catalytic reduction of dissolved oxygen in water, thereby enhancing the cathode photocurrent. The photoelectrochemical aptamer sensing strategy constructed by triggering the conformational change of THMS through a target provides better detection performance for amoxicillin.

Description

Technical Field

[0001] This invention belongs to the field of bioanalysis and relates to the detection of amoxicillin, specifically to a photoelectrochemical aptamer sensor based on a triple-helix molecular switch probe and its application in the detection of amoxicillin. Background Technology

[0002] Amoxicillin (AMOX) is a semi-synthetic penicillin antibiotic with strong antibacterial activity and a broad antibacterial spectrum. It is commonly used to treat bacterial infections caused by Gram-negative and Gram-positive bacteria and is one of the most widely used antibiotics. However, due to improper or excessive use in daily life, AMOX can accumulate in large quantities, leading to bacterial resistance and posing a potential threat to human health. Therefore, developing a simple, sensitive, and specific method is crucial for the effective detection of AMOX. The publications "Eco-friendly copper nanomaterials-based dual-mode optical nanosensors for ultrasensitive trace determination of amoxicillin antibiotics residue in tap water samples" and "Ultrasensitive colorimetric detection of amoxicillin based on Tris-HCl-induced aggregation of gold nanoparticles" disclose colorimetric methods for the detection of amoxicillin, with detection limits of 1.71 × 10⁻⁶ and 1.71 × 10⁻⁶, respectively. 3 nM and 6.70×10 -2 The methods for detecting amoxicillin using fluorescence methods are disclosed in "Hydrogen-bond induced enhanced emission ratiometric fluorescent handy needle for visualization assay of amoxicillin by smartphone sensing platform" and "The rapid synthesis of intrinsic green-fluorescent poly(pyrogallol)-derived carbon dots for amoxicillin drug sensing in clinical samples," with detection limits of 2.39 × 10⁻⁶ and 2.39 × 10⁻⁶, respectively. 0 nM and 9.20×100 The papers "An Electrochemical Sensor Based on Reduced Graphene Oxide, Gold Nanoparticles and Molecular Imprinted Over‐oxidized Polypyrrole for Amoxicillin Determination" and "A New Electrochemical Platform Based on Low-Cost Nanomaterials for Sensitive Detection of the Amoxicillin Antibiotic in Different Matrices" disclose methods for the electrochemical detection of amoxicillin, with detection limits of 1.22 × 10⁻⁶ mM. 3 nM and 5.00×10 1 The method for detecting amoxicillin using Raman spectroscopy with a detection limit of 1.00 × 10⁻⁶ mM is disclosed. 0 The novel molecularly imprinted amoxicillin sensor based on a dual recognition and dual detection strategy discloses a method for detecting amoxicillin using electrochemiluminescence immunoassay, with detection limits of 8.30 × 10⁻⁶. -3 nM.

[0003] Compared with other detection methods, photoelectrochemical (PEC) aptamer sensors offer advantages such as simple equipment, rapid response, low background current, and high sensitivity, demonstrating significant potential in the field of antibiotic detection. As signal converters in PEC aptamer sensors, photoelectroactive materials play a crucial role in the PEC sensing system, and selecting photoelectroactive materials with excellent performance is key to developing high-performance PEC aptamer sensors. Furthermore, in practical applications of aptamer sensors, analytes such as antibiotics and nucleic acids are often present in very low concentrations, typically requiring the involvement of signal enhancement techniques to improve detection sensitivity. Therefore, the preparation of ideal photoelectroactive materials, further improvement of sensitivity, and the appropriate amplification strategies are of significant research importance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a photoelectrochemical aptamer sensor based on a triple-helix molecular switch probe and its application in the detection of amoxicillin.

[0005] The technical solution of this invention is implemented as follows: The preparation steps of a photoelectric active material CulnS2-GR are as follows: (1) Disperse GO in ethylene glycol and sonicate to form a uniform GO dispersion with a mass concentration of 4%; (2) Under stirring conditions, CuCl, InCl3·4H2O and H2NCSNH2 were added to the GO dispersion in sequence, and the mixture was stirred until completely dissolved; the final concentrations of CuCl, InCl3·4H2O and H2NCSNH2 were 0.03 M, 0.03 M and 0.12 M, respectively.

[0006] (3) The mixture was transferred to a reaction vessel and reacted at 200°C for 24 h. After cooling to room temperature, the reaction product was centrifuged, washed and dried to obtain CulnS2-GR, an optoelectronic active material for detecting amoxicillin based on a ferrocene-labeled triple helix molecular probe.

[0007] A modified electrode, the method for preparing the modified electrode is as follows: 1) The CulnS2-GR composite material was dispersed in ultrapure water and ultrasonically mixed to form 6 mg / mL. -1 CulnS2-GR dispersion; 2) Use a pipette to drop 20 μL CulnS2-GR dispersion onto the surface of the ITO electrode and dry it under an infrared lamp. Then place it in an oven at 170°C and heat for 2 h to obtain a stable CulnS2-GR / ITO electrode.

[0008] 3) Drop 10 μL of 0.1 mg / mL solution onto the electrode prepared in step 2). -1 After drying the chitosan solution at 60°C, 10 μL of 2.5% glutaraldehyde solution was added dropwise to the electrode surface, and the electrode was left to stand at room temperature for 1 h.

[0009] 4) Prepare the purchased DNA nucleic acid strand S1 (the base sequence of the S1 strand is shown in SEQ ID No. 1, 5′-NH2-C6-TTTCTGAGACTTCCTTCTATGAATAGTCTCAG-3′) to the required concentration of 1.0 μM using Tris-HCl buffer (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, 5 mM KCl), anneal at 95°C for 5 min, and cool to room temperature.

[0010] 5) Add 10 μL of S1 to the surface of the electrode prepared in step 3), incubate at 37°C for 1 h, and then wash the electrode three times with PBS buffer solution (10 mM, pH 7.4, containing 2.5 mM MgCl2) to remove excess S1.

[0011] 6) Drop 10 μL of 1 mM 6-mercapto-1-hexanol (MCH) solution (prepare 100 mM MCH with ethanol as solvent, then dilute to 1 mM with PBS solution) onto the electrode surface prepared in step 5), and incubate at 25°C for 30 min to eliminate non-specific active sites. After incubation, wash three times in PBS buffer solution (10 mM, pH 7.4, containing 2.5 mM MgCl2) to complete the preparation of the sensing interface.

[0012] Before preparing the modified electrode, the ITO electrode was ultrasonically cleaned with acetone, ethanol and ultrapure water for 15 min each, and then dried in an oven at 60°C.

[0013] A triple-helix molecular switch probe, wherein the triple-helix molecular switch is constructed as follows: ① Prepare the purchased DNA nucleic acid chains T1 (the sequence of T1 chain is shown in SEQ ID No. 2, the base sequence is 5′-CTTCCTTCATTAGTTGGGGTTCAGTTGGACTTCCTTC-3′) and T2 (the sequence of T2 chain is shown in SEQ ID No. 3, the base sequence is 5′-TTCATA GAAGGAAGTCTCAG-Fc-3′) to the required concentration of 10 μM using Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, 5 mM KCl), anneal at 95°C for 5 min, and cool to room temperature.

[0014] ② Mix 50 μL of T1 and 50 μL of T2 in 400 μL of Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, and 5 mM KCl), incubate at 30°C for 1 h to assemble into a triple helix molecular switch probe (THMS), and store at 4°C for later use.

[0015] An optochemical aptamer sensor comprising the modified electrode described above and the triple-helix molecular switch probe described above.

[0016] The application of the photoelectrochemical aptamer sensor in the detection of amoxicillin according to this application involves the following steps: a. Add 10 μL of the triple helical molecular switch probe (THMS) and 10 μL of amoxicillin standard solutions of different concentrations sequentially to 80 μL of Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, 5 mM KCl), and shake in a constant temperature shaker at 37°C for 2 h.

[0017] b. Add the mixed solution prepared in step a to the finally prepared electrode and incubate for 80 min. Clean the electrode three times with PBS buffer solution (10 mM, pH 7.4, containing 2.5 mM MgCl2) and then measure the photoelectric signal.

[0018] c. Substituting the measured photoelectric signal into the linear regression equation, we get ΔI = 0.6478 log(C AMOX The value of amoxicillin in the test solution is calculated as 3.0253, with R being 0.9903.

[0019] The working principle of this invention is as follows: This invention is a PEC aptamer sensor constructed based on a ferrocene-labeled triple-helix molecular probe. First, a photocathode active material, CulnS2-GR, with good photoelectric conversion efficiency and stability, is modified onto an ITO electrode. Then, a hairpin probe (HP) is covalently bound to the electrode surface via chitosan and glutaraldehyde. The triple-helix molecular probe (THMS) is used as a molecular switch. In the presence of amoxicillin, the ferrocene-labeled signal probe (Fc-T2) is released through the opening of the THMS probe, undergoes base complementary pairing with the hairpin probe, and is fixed to the electrode surface. Under visible light irradiation, the photocathode active material CulnS2 can be excited, and electrons transition from VB to CB, generating photogenerated electron-hole pairs. Electrons on the ITO electrode are captured by holes on VB, generating a photocathode current. The band gap (CB) of CulnS2 is -0.61 V, while the reduction potential of ferrocene (Fc) is 0.31 V, both higher than the CB band edge potential of CulnS2. This indicates a potential gradient between the two, prompting electrons on the ITO electrode to rapidly transfer to Fc. Therefore, when the Fc-T2 probe is fixed to the electrode surface, the good energy level matching between CulnS2 and Fc allows electrons to flow more quickly to dissolved oxygen in the water, enhancing the photocurrent signal.

[0020] In summary, the hypersensitivity of the PEC aptamer sensor is attributed to the following factors: ① The CulnS2-GR, with its excellent photoelectric conversion efficiency, provides an ideal initial current; ② The ferrocene-labeled signal probe is released from the triple-helix molecular probe switch after target recognition; ③ The good energy level matching between ferrocene and CulnS2 can accelerate the electron transport rate and enhance the photocurrent signal.

[0021] The present invention has the following beneficial effects: 1. This invention constructs a signal-enhanced PEC aptamer sensor for amoxicillin detection based on a ferrocene-labeled triple-helix molecular probe. CulnS2 is used as the photocathode active material, providing a stable initial photocurrent. The ferrocene-labeled T2 probe is substituted by the specific recognition interaction between amoxicillin and the ring of the triple-helix molecular probe, and introduced onto the electrode surface through base complementary pairing. The Fc can accept electrons from CulnS2 nanoflowers and modulate the catalytic reduction of dissolved oxygen in water, enhancing the cathode photocurrent. This photoelectrochemical aptamer sensing strategy, constructed through target-induced THMS conformational changes, provides superior detection performance for amoxicillin and offers new insights for the rational design of efficient PEC aptamer sensors for detecting other analytes.

[0022] 2. This application constructs a sensitive amoxicillin detection ferrocene-CulnS2 photoelectrochemical aptamer sensor based on the energy level matching between ferrocene and CulnS2. A photocathode active material with good photoelectric conversion efficiency and stability was obtained by compositing p-type semiconductor CulnS2 nanoflowers with reduced graphene oxide (CulnS2 / GR). Then, a hairpin probe (HP) was covalently bound to the electrode surface via chitosan and glutaraldehyde. A triple-helix molecular probe (THMS) was used as a molecular switch. Amoxicillin can open the triple-helix molecular switch, causing the ferrocene-labeled signal probe (Fc-T2) to be released. Subsequently, Fc-T2 undergoes base complementary pairing with the hairpin probe modified on the electrode and is fixed to the electrode surface. The good energy level matching between ferrocene and CulnS2 can accelerate the electron transport rate and effectively enhance the photocurrent output. The increase in photocurrent signal is proportional to the concentration of amoxicillin (AMOX), with a linear range of 100 fM–50 nM and a detection limit of 22.41 fM. This method has also been successfully applied to the detection of milk and lake water samples. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This describes the construction process of the PEC aptamer sensor in this application.

[0025] Figure 2 (A) is the SEM image of CulnS2 nanoflowers; (B, C) are the SEM images of CulnS2-GR; (D) is the TEM image of CulnS2-GR; (EH) is the surface distribution map of C, Cu, In and S elements.

[0026] Figure 3 (A) shows the XRD patterns of CulnS2-GR (curve a) and CulnS2 (curve b); (B) shows the full XPS spectrum of CulnS2-GR; (CF) shows the XPS spectra of C 1s, Cu 2p, In 3d and S 2p, respectively.

[0027] Figure 4 This is a 12% polyacrylamide gel electrophoresis image, where lane 1: Marker, lane 2: T1, lane 3: T2, lane 4: S1, lane 5: T2+S1, lane 6: THMS, and lane 7: THMS+AMOX.

[0028] Figure 5 In the middle (A, B), the photocurrent response curves and electrochemical impedance spectra of CulnS2-GR / ITO (a), CS / CulnS2-GR / ITO (b), S1 / CS / CulnS2-GR / ITO (c), MCH / S1 / CS / CulnS2-GR / ITO (d), and T2 / MCH / S1 / CS / CulnS2-GR / ITO (e) are respectively.

[0029] Figure 6 The CV curves were measured in 0.1 M PBS buffer (pH=7.4): (a) bare ITO electrode, (b) Fc-modified ITO electrode.

[0030] Figure 7 This is a diagram illustrating the mechanism of the constructed PEC sensing strategy.

[0031] Figure 8 The linear potential scan of the cathode and anode measures the conduction band and valence band of CulnS2: (A) CB; (B) VB.

[0032] Figure 9 In the diagram, (A) represents the optimization of the graphene ratio; (B) represents the optimization of the S1 concentration; and (C) represents the optimization of the hybridization time between T2 and S1.

[0033] Figure 10(A) shows the PEC response curves of the aptamer sensor to different concentrations of AMOX (concentration range: 100 fM-300 nM); (B) shows the linear relationship between the photocurrent response and the logarithm of the AMOX concentration; (C) shows the photocurrent response curves of CulnS2-GR / ITO (a) and T2 / MCH / S1 / CS / CulnS2-GR / ITO (b) under 10 on / off illumination cycles; (D) shows the photocurrent change (ΔI) of 100 pM AMOX and other interfering substances (10 nM). Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Experimental instruments and materials Xenon lamp (CEL-HXF300-T3, Beijing Zhongjiao Jinyuan Technology Co., Ltd.); Cryo-dual-beam electron microscopy (Thermoscientific / Aquilos); 120 kV transmission electron microscope (Thermoscientific, Talos L120CG2). Focused ion beam scanning electron microscope (SEM, ZEISS / auriga-bu); Transmission electron microscope (TEM, FEI TalosF200S); CT computed tomography X-ray diffraction system (XRD, Empyrean); X-ray photoelectron spectroscopy (XPS, ESCALB MK-II); Ultraviolet-Vis-NearInfrared Spectrophotometer (Carry 5000, Agilent); PEAC 200A photoelectric reactor (Tianjin Aida Hengsheng Technology Development Co., Ltd.); RST5000 Electrochemical Workstation (Zhengzhou Ruister Instrument Co., Ltd.); High-power CNC ultrasonic cleaner (KQ-600KDE, Kunshan Ultrasonic Instrument Co., Ltd.); pH meter (LE438 Mettler-Toledo); Lambda 35 UV-Vis spectrophotometer (Perkin Elmer); Fluorescence spectrophotometer (F-4600, Hitachi High Technology Corporation).

[0036] CuCl was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. InCl3·4H2O was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Thiourea (H2NCSNH2), potassium dihydrogen phosphate (KH2PO4), potassium chloride (KCl), and trichloroacetic acid (Cl3CCOOH) were purchased from Tianjin Kemeo Chemical Reagent Co., Ltd. Dipotassium hydrogen phosphate (K2HPO4) was purchased from Tianjin Fuchen Chemical Reagent Co., Ltd.

[0037] Graphene oxide (GO) was purchased from Nanjing Xianfeng Nanotechnology Co., Ltd. NaCl was purchased from Tianjin Kemei Chemical Reagent Co., Ltd. Magnesium chloride hexahydrate (MgCl2·6H2O) was purchased from Sigma-Aldrich (USA); The 6×Loading buffer was purchased from Beijing Qingke Biotechnology Co., Ltd. Marker and Sybr gold were purchased from Beijing Baori Biotechnology Co., Ltd. Acrylamide, ammonium persulfate (APS), 5×TBE buffer and DNA oligonucleotides were purchased from Shanghai Sangon Biotech Co., Ltd. 6-Mercapto-1-hexanol (MCH) was purchased from Sigma-Aldrich, USA. Amoxicillin, tobramycin, oxytetracycline, ampicillin, chloramphenicol, tetracycline, and kanamycin were all purchased from Aladdin Reagent Co., Ltd.

[0038] The nucleotide sequences used in this application are as follows: S1 chain (SEQ ID No. 1): 5′-GGGACTTGGTTTAGGTAATGAGTCCCTTTTT-NH2-3′; T1 chain (SEQ ID No. 2): 5′-CTTCCTTCATTAGTTGGGGTTCAGTTGGACTTCCTTC-3′; T2 chain (SEQ ID No. 3): 5′-TTCATA GAAGGAAGTCTCAG-Fc-3′.

[0039] Example: Fabrication of a photoelectrochemical aptamer sensor based on a triple-helix molecular switch probe 1. The preparation method of the photoelectric active material CulnS2-GR is as follows: 4% GO was dispersed in 30 mL of ethylene glycol and sonicated to form a homogeneous GO dispersion. Then, under stirring, 0.03 M CuCl, 0.03 M InCl3·4H2O, and 0.12 M H2NCSNH2 were added sequentially to the GO dispersion, and the mixture was stirred until completely dissolved. The mixture was transferred to a reaction vessel and reacted at 200°C for 24 h. After cooling to room temperature, the resulting reaction product was centrifuged, washed, and dried to obtain the photoelectric active material CulnS2-GR.

[0040] Characterization of CulnS2-GR composite materials as follows: Figure 2 As shown, we used SEM to characterize the microstructure of CulnS2 and CulnS2-GR composites. It can be seen that the synthesized CulnS2 exhibits a flower-like structure. Figure 2 A). When graphene is incorporated, the large number of flower-like CulnS2 structures are not destroyed, but are instead loaded onto the graphene surface with a large specific surface area. Figure 2 B, C). Figure 2 (D) is the TEM image of the CulnS2-GR composite material. Figure 2 (EH) is the EDS spectrum of CulnS2-GR. The figure clearly shows that the synthesized nanomaterial contains all the elements of CulnS2-GR, proving the successful preparation of this composite material.

[0041] The prepared materials were characterized by XRD and XPS, and the crystal structure and elemental valence distribution were analyzed. Figure 3 (A) XRD patterns of CulnS2 and CulnS2-GR are depicted. As can be seen from curve a, CulnS2 has 5 peaks: (112), (004), (204 / 220), (312), and (332). Comparing curve a and curve b, curve a does not have the characteristic peak of GO, but has a clear characteristic peak of GR, corresponding to the (002) crystal plane of GR, indicating that the GO added to the material has been successfully reduced to GR. Figure 3 (B) is the full spectrum of CulnS2-GR, which confirms the presence of elements such as Cu, In, C and S. Figure 3 (CF) are high-resolution XPS spectra of C 1s, Cu 2p, In 3d, and S 2p, respectively. For example... Figure 3 As shown in (C), the C 1s XPS spectrum of the CulnS2-GR composite material exhibits three main peaks, decomposing at 288.5, 286.2, and 284.8 eV, corresponding to OC=O, COC, and CC bonds, respectively. Figure 3 In (D), the Cu 2p plot shows two peaks at 931.8 eV and 951.5 eV, respectively, corresponding to Cu...+ Cu 2p state 3 / 2 and Cu 2p 1 / 2 The binding energy. There is a very weak peak at 945.0 eV, which is related to Cu. + The characteristic peaks correspond to these peaks. The 3p plot of In shows two characteristic peaks at 452.0 eV and 444.5 eV (...). Figure 3 E), respectively corresponding to In 3+ In 3d 3 / 2 and In 3d 5 / 2 The binding energy. Finally, in the S 2p XPS spectrum ( Figure 3 F), S 2p 3 / 2 and S 2p 1 / 2 The binding energy peaks are located at 161.4 eV and 162.7 eV, and are associated with S. 2- The bonding energy is consistent. This further verifies the successful synthesis of the CulnS2-GR composite material.

[0042] 2. The preparation method of the modified electrode is as follows: 1) The CulnS2-GR composite material was dispersed in ultrapure water and ultrasonically mixed to form 6 mg / mL. -1 CulnS2-GR dispersion; 2) Before modifying the electrode, the ITO electrode was ultrasonically cleaned with acetone, ethanol and ultrapure water for 15 min each, and then dried in an oven at 60°C. Then, 20 μL CulnS2-GR dispersion was drop-coated onto the surface of the ITO electrode using a pipette and dried under an infrared lamp. After drying, the electrode was placed in an oven at 170°C for 2 h to obtain a stable CulnS2-GR / ITO electrode.

[0043] 3) Drop 10 μL of 0.1 mg / mL solution onto the electrode prepared in step 2). -1 After drying the chitosan solution at 60°C, 10 μL of 2.5% glutaraldehyde solution was added dropwise to the electrode surface, and the electrode was left to stand at room temperature for 1 h.

[0044] 4) Prepare the purchased DNA nucleic acid strand S1 (the base sequence of S1 is 5′-NH2-C6-TTTCTGAGACTTCCTTCTATGAATAGTCTCAG-3′) to the required concentration of 1.0 μM using Tris-HCl buffer (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, 5 mM KCl), anneal at 95°C for 5 min, and cool to room temperature.

[0045] 5) Add 10 μL of S1 to the surface of the electrode prepared in step 3), incubate at 37°C for 1 h, and then wash the electrode three times with PBS buffer solution (10 mM, pH 7.4, containing 2.5 mM MgCl2) to remove excess S1.

[0046] 6) Drop 10 μL of 1 mM 6-mercapto-1-hexanol (MCH) (preparation method: first prepare 100 mM MCH with ethanol as solvent, then dilute with PBS solution to 1 mM) onto the electrode surface prepared in step 5), and incubate at 25°C for 30 min to eliminate non-specific active sites. After incubation, wash three times in PBS buffer solution (10 mM, pH 7.4, containing 2.5 mM MgCl2) to complete the preparation of the modified electrode of the sensing interface.

[0047] 3. The construction method of triple-helix molecular switch probe (THMS) is as follows: ① Prepare the purchased DNA nucleic acid chains T1 (T1 has a base sequence of 5′-CTTCCTTCATTAGTTGGGGTTCAGTTGGACTTCCTTC-3′) and T2 (T2 has a base sequence of 5′-TTCATAGAAGGAAGTCTCAG-Fc-3′) to the required concentration of 10 μM using Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, and 5 mM KCl), anneal at 95°C for 5 min, and cool to room temperature.

[0048] ② Mix 50 μL T1 and 50 μL T2 in 400 μL Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, and 5 mM KCl), incubate at 30°C for 1 h to assemble THMS, and dilute to 1.0 μM. Store at 4°C for later use.

[0049] The displacement process of the Fc-labeled signal probe was verified using polyacrylamide gel electrophoresis (PAGE). Ultrapure water, 5×TBE, acrylamide (30%), TEMED, and APS (10%) were mixed in the specified proportions to prepare a 12% polyacrylamide gel. Then, 7 μL of each prepared sample was pipetted and mixed with 2 μL of loading buffer and 2 μL of LSYBR Gold fluorescent dye, respectively. After thorough mixing, the mixture was injected into the wells of the prepared gel plate using a microsyringe. 1×TBE buffer solution was added to the electrophoresis tank, ensuring the entire electrophoresis experiment was conducted in this buffer solution. The electrophoresis apparatus was set to 80 V and the electrophoresis time to 90 min. After the experiment, gel images were captured using a UVP gel imaging system. Figure 4 As shown, lane 1 is the DNA marker, and lanes 2, 3, and 4 correspond to T1, T2, and S1, respectively. Lane 5 corresponds to the reaction product of the mixture of T2 and S1. A new bright band appears in lane 6, proving the formation of THMS. Lane 7 shows the bands generated after the addition of AMOX. Compared with lane 6, a T2 band can be seen in lane 7, indicating that the target substance specifically recognizes the THMS loop sequence and successfully releases the T2 chain.

[0050] 4. The photoelectrochemical aptamer sensor includes a 1.0 μM triple-helix molecular switch probe solution, a modified electrode, and a Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, and 5 mM KCl).

[0051] To investigate the feasibility of the sensor, the photoelectric signal and EIS of the fabrication process were measured. For example... Figure 5As shown in (A, B), CulnS2-GR / ITO exhibits a strong cathodic photocurrent (curve a), which is attributed to the excellent photoelectric activity of CulnS2 and the addition of GR, which accelerates electron transfer and increases the photocurrent signal, thus resulting in a minimum Rct. Then, loading CS onto the electrode surface increases Rct and decreases the photocurrent (curve b). This is due to the weak conductivity of CS. The S1 chain is cross-linked onto the electrode via glutaraldehyde; the steric hindrance of the DNA chain leads to a decrease in the photocurrent signal and an increase in Rct (curve c). When the active sites on the electrode surface are blocked by MCH, the photocurrent continues to decrease because the introduction of organic molecules hinders electron transfer (curve d). Finally, the solution reacted with the target is dropped onto the electrode. The target binds to the triple helix molecule, releasing the ferrocene-modified signal probe T2, which further hybridizes with S1 and is immobilized on the electrode surface. Under illumination, electron transfer between CulnS2 and Fc increases the photocurrent. In the Nyquist plot, the formation of the DNA double helix structure and the steric hindrance effect of Fc hinder the transfer of electrons on the electrode surface, resulting in an increase in Rct (curve e).

[0052] Exploring the mechanism of PEC biosensing: To further investigate the charge transfer mechanism between CulnS2 and Fc, we used cyclic voltammetry (CV) to determine the reduction potential of Fc. Figure 6 As shown, curve a is the CV curve of the bare ITO electrode in 0.1 M PBS buffer solution, showing no obvious redox peaks. Curve b is the CV curve of the Fc-modified ITO electrode, clearly showing a pair of reversible ferrocene redox peaks, and the reduction potential of Fc is 0.11 V (using Ag / AgCl as a reference electrode). When using a standard hydrogen electrode (NHE) as a reference electrode, the reduction potential of Fc is 0.31 V.

[0053] Sensing mechanism such as Figure 7 As shown. Under visible light irradiation, the photoactive material CulnS2 can be excited, with electrons transitioning from VB to CB, generating photogenerated electron-hole pairs. Electrons on the ITO electrode are captured by holes on VB, producing a photocathode current. Figure 8 (A) It is known that the band gap (CB) of CulnS2 is -0.61 V, while the reduction potential of Fc is 0.31 V, which is higher than the band gap potential of CulnS2. This means that there is a potential gradient between the two, which causes electrons on the ITO electrode to transfer rapidly to Fc. Therefore, when the signal probe T2 modified with Fc is fixed on the electrode surface, the good energy level matching between CulnS2 and Fc will cause electrons to flow to the dissolved oxygen in the water more quickly, thus enhancing the photocurrent signal.

[0054] 5. The application of photoelectrochemical aptamer sensors in the detection of amoxicillin follows these steps: a. Add 10 μL of the triple helix molecular switch (THMS) and 10 μL of amoxicillin standard solutions of different concentrations sequentially to 80 μL of Tris-HCl buffer solution (10 mM, pH 6.0, containing 100 mM NaCl, 5 mM MgCl2, 5 mM KCl), and shake in a constant temperature shaker at 37°C for 2 h.

[0055] b. Add the mixed solution prepared in step a to the finally prepared electrode and incubate for 80 min. Clean the electrode three times and then measure the photoelectric signal.

[0056] Photoelectrochemical measurement conditions: Photoelectrochemical tests were performed on an electrochemical workstation using an LED lamp as the light source, with an output power greater than 20 Mw / cm². -2 A standard three-electrode system is used: the working electrode is ITO glass (with a modified area of ​​0.25 ± 0.01 cm²). 2 The reference electrode is saturated Ag / AgCl, and the counter electrode is platinum wire.

[0057] To better study the analytical performance of the constructed PEC aptamer sensor, the detection conditions were optimized. Specifically, the ratio of graphene dopant, the concentration of S1, and the reaction time between T2 and S1 were optimized. Figure 9 (A) shows the PEC response signals generated by different proportions of graphene-doped CulnS2. It can be seen that the photocurrent signal is largest when the added graphene proportion is 4%, while the photocurrent signal decreases when the added amount exceeds 4%. This is because excessive GO will produce multilayer graphene, which hinders electron transfer. Therefore, a 4% GO-CulnS2 composite material was used as the photoelectric substrate material in subsequent experiments. The concentration of S1 also plays a crucial role in the analysis and measurement of the sensor, and therefore it was optimized. Figure 9 (B) It can be seen that the photocurrent signal increases significantly when the concentration is between 0.25 and 1.00 μM, while the photocurrent change tends to stabilize after the concentration reaches 1.00 μM. This indicates that 1.00 μM is the optimal concentration for the S1 chain. Figure 9 (C) is the optimal hybridization time diagram for T2 and S1. It can be seen that the photocurrent increases significantly before 80 min, because more and more T2 and S1 bases pair up as time increases. After 80 min, the photocurrent signal becomes basically stable, indicating that the hybridization amount of T2 and S1 has approached saturation. Therefore, 80 min is selected as the optimal time for the formation of a double helix structure on the electrode.

[0058] Example of Implementation Results 1: Quantitative Analysis of Amoxicillin The performance of the constructed PEC aptamer sensor was investigated under optimized experimental conditions. Figure 10 As shown in (A), within the range of 100 fM to 50 nM, the PEC response continuously increases with increasing AMOX concentration. Figure 10 As shown in (B), the photoelectric signal and lg(C) AMOX The curves between ΔI and 0.6478 log(C) show a good linear relationship. The linear regression equation is ΔI = 0.6478 log(C) AMOX The correlation coefficient was 0.9903, and the limit of detection (LOD) was 22.41 pM (S / N=3). Here, ΔI = I−I0, where I0 is the photocurrent after incubation with MCH / S1 / CS / CulnS2-GR / ITO, and I is the photocurrent that appears after Fc is introduced onto the electrode surface. Compared with previously reported methods (Table 1), the constructed PEC sensor exhibits superior detection performance.

[0059] Table 1 compares the analytical performance of the constructed PEC aptamer sensor for detecting AMOX with other literature reports. Example 2 of Implementation Results: Investigation of Stability, Reproducibility, and Selectivity The prepared CulnS2-GR / ITO and T2 / MCH / S1 / CS / CulnS2-GR / ITO electrodes were continuously scanned under 10 switching illuminations, and the photocurrent changes were not significant. Figure 10 As shown in (C), this demonstrates the sensor's good stability. Simultaneously, three electrodes were prepared using the same method and used for measurements in 10 nM AMOX solution. The relative standard deviation (RSD) was 5.7%, indicating that the sensor has good reproducibility.

[0060] To investigate the specificity and selectivity of the constructed biosensor, 10 nM tobramycin (TOB), oxytetracycline (OTC), ampicillin (AMP), chloramphenicol (CPL), tetracycline (TET), and kanamycin (KNAN) were used as potential interfering substances and compared with AMOX.

[0061] Depend on Figure 10 (D) It can be seen that, apart from AMOX, other interfering substances have a small effect on the photocurrent change, which indicates that the constructed PEC biosensor has good selectivity for AMOX detection.

[0062] Application example: Analysis of actual samples Whether a sensor can be applied to practical measurements is also a standard for evaluating its quality. Therefore, this invention uses a PEC aptamer sensor to analyze milk and lake water samples to assess its feasibility and potential application prospects. In the experiment, different concentrations of AMOX were added to actual samples using the standard addition method, and the recovery rate was measured. Table 2 shows that the recovery rates of milk and lake water samples were between 95% and 105%, with small relative standard deviations (RSD). This demonstrates that the established method has high accuracy and is promising for the detection of AMOX content in actual samples.

[0063] Table 2 Results of spiked recovery experiments using PEC aptamer sensors to detect AMOX in milk and lake water. As shown in Table 2, the novel PEC aptamer sensor for amoxicillin determination of the present invention exhibits advantages such as high sensitivity and low detection limit when determining amoxicillin. Furthermore, the sensor also possesses good selectivity, high reproducibility, and stability, thus enabling its application in the detection of amoxicillin in actual samples such as milk and lake water.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 photoelectrochemical aptamer sensor, characterized in that: Includes modified electrodes and triple-helix molecular switch probes; The modified electrode is prepared as follows: 1) CulnS2-GR composite material was dispersed in ultrapure water to prepare CulnS2-GR dispersion; 2) The CulnS2-GR dispersion was drop-coated onto the pretreated electrode surface and dried, and then heated to a stable state to obtain the CulnS2-GR / ITO electrode; 3) Apply chitosan solution to the CulnS2-GR / ITO electrode, dry it, then add glutaraldehyde solution and let it stand; 4) After annealing and cooling, the S1 chain solution is dropped onto the electrode surface treated in step 3). After incubation at 37°C, the electrode is washed with PBS buffer solution to remove excess S1 chain and obtain the modified electrode. The nucleotide sequence of the S1 chain is shown in SEQ ID No.1, and its 5' end is connected to the -NH2-C6- group. 5) Drop 6-mercapto-1-hexanol solution onto the surface of the modified electrode from step 4), incubate at 25°C to eliminate non-specific active sites, and wash with PBS buffer solution to obtain the modified electrode; The preparation steps of the CulnS2-GR composite material are as follows: (1) Disperse GO in ethylene glycol and prepare GO dispersion by sonication; (2) Under stirring conditions, CuCl, InCl3·4H2O and H2NCSNH2 were added to the GO dispersion in sequence, and the mixture was stirred until it was completely dissolved to obtain a mixed solution; (3) After the mixed solution is reacted at high temperature, it is cooled to room temperature, and the resulting product is centrifuged, washed and dried to obtain CulnS2-GR composite material; The method for constructing the triple-helix molecular switch is as follows: ① Add T1 chain and T2 chain to Tris-HCl buffer solution respectively, and anneal and cool to obtain T1 chain solution and T2 chain solution; wherein the nucleotide sequence of T1 chain is shown as SEQ ID No.2 and the nucleotide sequence of T2 chain is shown as SEQ ID No.3, and the 3' end of T1 chain is modified with -Fc- group; ② After mixing the T1 chain solution and the T2 chain solution, Tris-HCl buffer solution was added, and the mixture was incubated and hybridized to obtain the triple helix molecular switch probe mother solution.

2. The photoelectrochemical aptamer sensor according to claim 1, characterized in that: In step (1), the mass concentration of the GO dispersion is 4%; in step (2), the molar ratio of CuCl, InCl3·4H2O and H2NCSNH2 in the GO dispersion is 1:1:4; in step (3), the high-temperature reaction is carried out at 200℃ for 24 h.

3. The photoelectrochemical aptamer sensor according to claim 2, characterized in that: In step 1), the concentration of the CulnS2-GR dispersion is 6 mg / mL. -1 In step 2), the pretreatment involves ultrasonically cleaning the ITO electrode with acetone, ethanol, and ultrapure water for 15 minutes, and then drying it at 60°C. The heating temperature is 170°C and the time is 2 hours.

4. The photoelectrochemical aptamer sensor according to claim 3, characterized in that: In step 3), the concentration of the chitosan solution is 0.1 mg / mL. -1 The mass fraction of glutaraldehyde solution is 2.5%; in step 4), the solvent in solution S1 is Tris-HCl buffer at pH 6.0, the concentration of S1 is 1.0 μM, and the annealing conditions are 95℃ for 5 min.

5. The photoelectrochemical aptamer sensor according to claim 4, characterized in that: In step 5), the concentration of the 6-mercapto-1-hexanol solution is 1 mM, and the solvent is ethanol; the PBS buffer solutions in steps 4) and 5) are both 10 mM PBS buffer solutions containing 2.5 mM MgCl2, pH 7.

4.

6. The photoelectrochemical aptamer sensor according to claim 5, characterized in that: In step ①, the concentrations of both the T1 chain solution and the T2 chain solution are 10 μM; in step ②, the volume ratio of the T1 chain solution, the T2 chain solution, and the Tris-HCl buffer solution is 1:1:

8.

7. The application of the photoelectrochemical aptamer sensor according to claim 1 in the detection of amoxicillin for non-disease diagnosis and / or treatment purposes, characterized in that, The steps are as follows: Add 1.0 μM triple helix molecular switch probe solution and the test solution sequentially to Tris-HCl buffer solution, shake at 37℃ for 2 h to obtain a mixed solution; wherein the volume ratio of triple helix molecular switch probe solution, test solution and Tris-HCl buffer solution is 1:1:

8. The mixed solution from step a was added dropwise to the modified electrode and incubated for 80 min. After washing the electrode with PBS buffer solution, the photoelectric signal was measured. Substituting the measured photoelectric signal into the linear regression equation, we get ΔI = 0.6478 log(C AMOX The value of amoxicillin in the test solution is calculated as 3.0253, with R being 0.9903.

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