Electrochemical and Photoelectrochemical Dual-Mode Aptasensor

Abstract

The present invention relates to an electrochemical and photoelectrochemical dual-mode aptasensor, belonging to the technical field of aptasensors. The aptasensor of the present invention includes an electrode, an electrode material coated on the surface of the electrode, and a nucleic acid aptamer anchored on the electrode material; the preparation method of the electrode material includes the following steps: dispersing a covalent triazine framework material and an iron salt in an organic good solvent of the iron salt, followed by solid-liquid separation, and then drying the obtained solid. The preparation method of the electrode material adopted by the aptasensor of the present invention can convert Fe ions into β-FeOOH nanocrystals and wrap them in the network of the triazine framework material. The combination of β-FeOOH nanocrystals and the triazine framework material can significantly promote electron transfer and enhance the separation of photogenerated electrons and holes. The aptasensor of the present invention can sensitively and selectively determine trace antibiotics and has broad application prospects in food safety and the environment.

Description

Technical Field

[0001] The present invention relates to an electrochemical and photoelectrochemical dual-mode aptamer sensor, belonging to the technical field of aptamer sensors. Background Art

[0002] Antibiotics such as penicillin G (PG), cephalosporins, carbapenems or tetracyclines have been synthesized and widely used to treat inflammation. However, in animal husbandry, poultry farming and aquaculture, in order to avoid bacterial infections, there is often overuse of antibiotics. Antibiotics can be excreted through feces into the environment and transferred to the human body through meat. Therefore, it causes serious environmental pollution and poses a serious threat to humans. PG is a broad-spectrum β-lactam antibiotic that exerts its bactericidal effect by destroying the cell wall of bacteria. During cooking processes such as frying and stewing, the PG residues in food will be partially decomposed to produce corresponding decomposition products, which are harmful to human life. The EU standard stipulates that the maximum residue limit of PG in milk is 4 μg·kg -1 , and the Chinese national standard stipulates it as 50 μg·kg -1 (GB 31650-2019). So far, various methods have been explored for the sensitive detection of PG, including high performance liquid chromatography-mass spectrometry, solid phase extraction, electrochemical biosensors, electrochemical surface plasmon resonance, fluorescence sensors, immunosensors, etc. However, traditional analytical methods for PG often have problems such as low determination sensitivity, cumbersome analytical steps, and expensive equipment. These disadvantages make it unable to meet the requirements for rapid and sensitive detection of trace PG in food and water environments. In addition, most of the determinations of PG rely only on a single detection method. The accurate analysis of PG in food is difficult to ensure accuracy in a complex environment. In this regard, combining the two to construct a dual-mode sensing detection can overcome this shortcoming. A variety of dual-mode detection methods have been established in the analysis of herbicides, pesticides, biomarkers, mycotoxins, etc., but there are few reports on the detection of trace PG.

[0003] Among various technologies, electrochemical methods (EC methods) have been widely used for the determination of many analytes (such as cancer biomarkers, heavy metal ions, antibiotics, biomolecules, DNA, or toxic and harmful small molecules) due to their advantages of fast response, small analyte dosage, feasible operation of EC devices, and simple data analysis. Compared with the method of directly detecting PG by host-guest interaction, the EC biosensor constructed by adsorbing the analyte-targeted antibody or aptamer on a sensitive platform often has a lower detection limit and higher selectivity due to the specific binding of PG to the corresponding probe. In particular, the label-free electrochemical biosensing strategy is usually used for the sensitive analysis of PG due to the feasibility of its construction. The optoelectrochemical biosensor, which uses light and electricity as two separable energy forms as the excitation source (input) and detection signal (output) respectively, has shown great potential in the sensitive analysis of different antibiotics such as tetracycline, oxytetracycline, sulfamethazine, nitrofurazone, chloramphenicol, tetracycline, doxorubicin hydrochloride, gentamicin sulfate, enrofloxacin, etc. It has the advantages of low cost, feasible simplified instrument, and low background noise. However, there has been no report on the preparation of a PEC biosensor for PG detection. Considering the advantages of EC and PEC technologies, if the two methods are combined to develop an EC-PEC dual-mode sensing technology, the accurate detection of PG in complex foods or environments can be achieved. In addition, the systematic evolution of ligands by exponential enrichment (SELEX) technology for screening single-stranded oligonucleotides (aptamers) has attracted extensive attention in the preparation of efficient aptasensors for detecting antibiotics. Compared with immunosensors developed by antigen-antibody specific interaction, aptasensors often have more excellent sensing performance, fast reaction speed, and low cost. In order to construct an excellent EC-PEC biosensor, a conductive material that can both transmit electron or photocurrent response as the determination signal and anchor a large number of biological probe molecules must be sought.

[0004] So far, various nanomaterials, such as porous organic polymers, poly(diallyldimethylammonium chloride)-functionalized graphene / Ag@Au nanosheets, Ti3C2-metal-organic frameworks (MOFs), TiO2-g-C3N4@AuNPs, or Co-MOF-on-TPN covalent-organic framework (COF) hybrid materials, have been used to construct EC aptasensors for detecting antibiotics. However, due to the lack of functional nanomaterials with enhanced EC activity and photoelectric conversion efficiency, a dual-mode EC-PEC aptasensor for antibiotic detection has not been developed yet. Summary of the Invention

[0005] The object of the present invention is to provide an electrochemical and optoelectrochemical dual-mode aptasensor, which can be used for the accurate detection of antibiotics such as PG in complex foods or environments.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] An electrochemical and photoelectrochemical dual-mode aptamer sensor, comprising an electrode, an electrode material coated on the surface of the electrode, and a nucleic acid aptamer anchored on the electrode material; the preparation method of the electrode material comprises the following steps: dispersing a covalent triazine framework material and an iron salt in an organic good solvent of the iron salt, performing solid-liquid separation, and then drying the obtained solid.

[0008] When preparing the electrode material of the electrochemical and photoelectrochemical dual-mode aptamer sensor of the present invention, doping of the triazine framework material with electrochemically active metal Fe is achieved by dispersing the iron salt and the covalent triazine framework material in a solvent, performing solid-liquid separation, and then drying the solid. During the preparation process, Fe ions are transformed into β-FeOOH nanocrystals and wrapped in the network of the triazine framework material. Covalent triazine-based frameworks (CTFs), as an important class of POPs, are composed of nitrile trimer building units, have low crystallinity, remarkable chemical and thermal stability, high electrochemistry activity, and excellent light absorption in the ultraviolet-visible light range. The formation of a heterojunction between β-FeOOH nanocrystals and the triazine framework material can significantly promote electron transfer and enhance the separation of photo-generated electrons and holes, which enables the electrode material to exhibit outstanding electrochemical (EC) and photoelectrochemical (PEC) capabilities, thus showing great potential as an electrode material for electrochemical and photoelectrochemical dual-mode aptamer sensors. This electrode material can expand the application of porous organic frameworks in biosensing and environmental monitoring. The electrochemical and photoelectrochemical dual-mode aptamer sensor of the present invention can sensitively and selectively detect trace antibiotics and has broad application prospects in food safety and the environment.

[0009] When the aptamer anchored on the electrode material of the aptamer sensor of the present invention is a penicillin-targeting aptamer, the detection limits of electrochemical and photoelectrochemical dual-mode label-free aptamer sensing are significantly lower than those of the PG biosensors reported by Wenjuan Guo et al. in "Microchemical Journal" and Rene Welden et al. in "Electrochemical Science Advances" (these PEC and EC technologies are as low as 0.7 and 0.08 fg·mL -1 ), and can be used for sensitive detection of trace PG.

[0010] Furthermore, the covalent triazine framework material is formed by reacting melamine with 2,5-dihydroxyterephthalaldehyde; the mass ratio of melamine to 2,5-dihydroxyterephthalaldehyde is 8:100 to 20:100, preferably 8:100 to 15:100, such as 10:100. The triazine framework material obtained using melamine (MA) and 2,5-dihydroxyterephthalaldehyde (Dha) as building units has a richer structure, higher porosity, and more excellent light absorption properties. Introducing Fe ions can improve the electrochemical activity of the electrode material, which all enable the finally obtained electrode material (Fe-MA-Dha-CTF) to have a high photoelectric conversion efficiency and good electrochemical activity. The aptamer sensor based on the triazine framework material (MA-Dha-CTF) formed by reacting melamine with 2,5-dihydroxyterephthalaldehyde shows an amplified sensing signal when detecting PG without the need for any EC or PEC indicators. In addition, the triazine framework material (MA-Dha-CTF) has a stable chemical structure, which can make the obtained electrode material (Fe-MA-Dha-CTF) have high stability, and further enable the aptamer sensor to have high sensitivity, high selectivity, good stability, excellent reproducibility, regenerability, and acceptable applicability.

[0011] Furthermore, the dispersion is carried out by mixing the suspension of the iron salt and the covalent triazine framework material and then performing a stirring treatment; the suspension is formed by dispersing the covalent triazine framework material in the good solvent; the time of the stirring treatment is 8 to 12 h, such as 10 h.

[0012] Furthermore, the iron salt is one or any combination of iron acetate, iron nitrate, and iron chloride; the good solvent for the iron salt is an alcohol solvent. Using an alcohol solvent has the advantage of good dispersibility. Further, the mass ratio of the covalent triazine framework material to the iron salt is 10:35 to 60:35, preferably 40:35 to 60:35, such as 50:35; controlling the mass ratio of the triazine framework material and the iron salt within this range can provide an appropriate doping amount. The volume of the organic good solvent corresponding to every 50 mg of the covalent triazine framework material is 10 to 30 mL, preferably 20 mL. The alcohol solvent is preferably methanol.

[0013] Furthermore, the drying treatment is a vacuum drying treatment. The temperature of the drying treatment is 40 to 80 °C, preferably 60 °C, and the time is 4 to 8 h, preferably 6 h.

[0014] Further, the covalent triazine framework material is prepared by a method including the following steps: reacting a mixed solution of melamine and 2,5-dihydroxyterephthalaldehyde at 150-200 °C for 48-96 h, for example, reacting at 180 °C for 72 h. After solid-liquid separation, the obtained solid is washed and dried. Further, for every 10 mg of melamine in the mixed solution, 2.0-4.0 mL of solvent is used, for example, 3.2 mL of solvent is used for 10 mg of melamine. The solvent of the mixed solution is preferably dimethyl sulfoxide.

[0015] Further, the mixed solution is obtained by mixing an organic solution of melamine and an organic solution of melamine and 2,5-dihydroxyterephthalaldehyde. The organic solution of melamine is obtained by dissolving melamine in dimethyl sulfoxide. The volume of dimethyl sulfoxide used for every 10 mg of melamine is 1.6 mL. The aqueous solution of 2,5-dihydroxyterephthalaldehyde is obtained by dissolving 2,5-dihydroxyterephthalaldehyde in dimethyl sulfoxide. The volume of dimethyl sulfoxide used for every 100 mg of 2,5-dihydroxyterephthalaldehyde is 1.6 mL.

[0016] Further, the electrode is a glassy carbon electrode. The glassy carbon electrode has good electrical conductivity, good inertness, and high chemical stability.

[0017] A large number of nucleic acid aptamers adsorbed by the electrode material can make the electrochemical and photoelectrochemical dual-mode aptasensor have high selectivity, and the nucleic acid aptamer targeted to the corresponding antibiotic can be selected according to the detection object. To achieve the detection of penicillin, the nucleic acid aptamer is a penicillin-targeted aptamer. The penicillin-targeted aptamer is preferably 5′-GGG TCTGAG GAG TGC GCG GTG CCA GTG AGT-3′.

[0018] Since the electrode material used in the aptasensor of the present invention has high photoelectric conversion efficiency, good electrochemical activity, high biological affinity for the targeted nucleic acid aptamer, and fast response speed, the aptasensor using the penicillin-targeted nucleic acid aptamer has a detection limit of 0.08 and 0.7 fg·mL -1 -10 ng·mL -1 in the PG concentration range, and the detection limits by electrochemical (EC) and photoelectrochemical (PEC) methods are 0.08 and 0.7 fg·mL -1 respectively. At the same time, the detection of PG has the advantages of high selectivity, good stability, and good repeatability, and has wide applicability in different real samples.

[0019] Further, the electrochemical and photoelectrochemical dual-mode aptamer sensor of the present invention is prepared by a method comprising the following steps: coating an electrode material on the surface of an electrode to obtain a modified electrode, then anchoring a nucleic acid aptamer on the modified electrode, and then performing a blocking treatment, thus obtaining the sensor.

[0020] Further, before coating the electrode material on the surface of the electrode, the surface of the electrode is pretreated; the pretreatment is to perform a polishing treatment on the surface, followed by washing, drying, and then an electroactivation treatment. The electroactivation treatment is carried out by cyclic voltammetry in 0.5 M H2SO4 at 100 mV·s -1 The surface of the electrode is scanned and activated in the range of -1.0 - 1.0 V until a stable cyclic voltammogram is obtained, then the surface of the electrode is washed and then dried.

[0021] Further, the method for coating the electrode material on the surface of the electrode comprises the following steps: coating a dispersion of the electrode material on the surface of the electrode, drying, cleaning the surface of the electrode, and then drying; the concentration of the electrode material in the dispersion of the electrode material is 0.1 - 2.0 mg·mL -1 For example, it is 1.0 mg·mL -1 .

[0022] Further, the method for anchoring the nucleic acid aptamer on the modified electrode comprises the following steps: incubating the modified electrode in a nucleic acid aptamer solution, and then washing to remove the loosely adsorbed nucleic acid aptamer; the nucleic acid aptamer is a penicillin-targeting nucleic acid aptamer. The concentration of the nucleic acid aptamer in the nucleic acid aptamer solution is 10 - 500 nmol·L -1 For example, it is 100 nmol·L -1 . The incubation time is 10 - 80 min, for example, 60 min.

[0023] Further, the penicillin-targeting nucleic acid aptamer is 5′-GGG TCT GAG GAG TGC GCG GTG CCAGTG AGT-3′.

[0024] The non-specific adsorption between the interfering substance and the modified electrode can be eliminated through the blocking treatment. Further, the blocking agent used in the blocking treatment is a 1% bovine serum albumin (BSA) solution by mass fraction. Further, during the blocking treatment, the modified electrode anchored with the nucleic acid aptamer is immersed in the blocking agent for incubation. The incubation time during the blocking treatment is 30 min. Description of the Drawings

[0025] Figure 1Figure showing the apparent morphology and elemental distribution characterization results of MA-Dha-CTF prepared in Example 1. Among them, 1(a) is a low-magnification SEM image, 1(b) is a high-magnification SEM image, 1(c) is a low-magnification TEM image, 1(d) is a high-magnification TEM image, 1(e) is a high-resolution TEM image, and 1(f) is the elemental (C, N, O) mapping images corresponding to the EDS mapping image of MA-Dha-CTF;

[0026] Figure 2 Figure showing the apparent morphology and elemental distribution characterization results of Fe-MA-Dha-CTF prepared in Example 1. Among them, 2(a) is a low-resolution TEM image, 2(b) is a high-resolution TEM image, and 2(c) is the EDS mapping image and the corresponding elemental (C, Fe, N, O) mapping images;

[0027] Figure 3 : (a) X-ray diffraction patterns of MA-Dha-CTF, Fe-MA-Dha-CTF, MA, and Dha prepared in Example 1, 3(b) nitrogen adsorption-desorption isotherm diagrams of (i) MA-Dha-CTF and (ii) Fe-MA-Dha-CTF prepared in Example 1, and 3(c) corresponding pore size distribution curves;

[0028] Figure 4 For the MA-Dha-CTF prepared in Example 1 13 13C NMR spectrum;

[0029] Figure 5 FT-IR spectra and EPR spectra of (i) MA-Dha-CTF and (ii) Fe-MA-Dha-CTF prepared in Example 1. Among them, 5(a) is the FT-IR spectrum and 5(b) is the EPR spectrum;

[0030] Figure 6 XPS diagrams of (i) MA-Dha-CTF and (ii) Fe-MA-Dha-CTF prepared in Example 1;

[0031] Figure 7 High-resolution XPS spectra of (a) Fe 2p, (b) C 1s, (c) O 1s, and (d) P 2p of Apt / Fe-MA-Dha-CTF prepared in Example 2;

[0032] Figure 8 For the (a) ultraviolet-visible diffuse reflectance absorption spectra and the relationship between (αhν) 2 and energy (hν) of (i) MA-Dha-CTF and (ii) Fe-MA-Dha-CTF prepared in Example 1. Among them, 8(a) is the ultraviolet-visible diffuse reflectance absorption spectrum and 8(b) is (αhν)2 Relationship diagram with energy (hν);

[0033] Figure 9 It is the EIS Nyquist diagram and equivalent circuit diagram simulated by Zview2 software in Experimental Example 2;

[0034] Figure 10 : (a) is the EIS Nyquist diagram of (i) bare GCE (Example 2), (ii) MA-Dha-CTF / GCE (comparative example), and (iii) Fe-MA-Dha-CTF / GCE (Example 2); (b) is the photocurrent response curve diagram of (i) bare GCE, (ii) MA-Dha-CTF / GCE, and (iii) Fe-MA-Dha-CTF / GCE in Example 2; (c) is the EIS Nyquist diagram of (i) bare GCE (Example 2), (ii) Fe-MA-Dha-CTF / GCE (Example 2), (iii) Apt / Fe-MA-Dha-CTF / GCE (Example 2), (iv) BSA / Apt / Fe-MA-Dha-CTF / GCE (Example 2), and (v) PG / BSA / Apt / Fe-MA-Dha-CTF / GCE; (d) is the photocurrent response curve diagram of (i) bare GCE (Example 2), (ii) Fe-MA-Dha-CTF / GCE (Example 2), (iii) Apt / Fe-MA-Dha-CTF / GCE (Example 2), (iv) BSA / Apt / Fe-MA-Dha-CTF / GCE (Example 2), and (v) PG / BSA / Apt / Fe-MA-Dha-CTF / GCE; (e) is the EIS Nyquist diagram of (i) bare GCE (Example 2), (ii) MA-Dha-CTF / GCE (comparative example), (iii) Apt / MA-Dha-CTF / GCE (comparative example), (iv) BSA / Apt / MA-Dha-CTF / GCE (comparative example), and (v) PG / BSA / Apt / MA-Dha-CTF / GCE; (f) is the photocurrent response curve diagram of (i) bare GCE (Example 2), (ii) MA-Dha-CTF / GCE (comparative example), (iii) Apt / MA-Dha-CTF / GCE (comparative example), (iv) BSA / Apt / MA-Dha-CTF / GCE (comparative example), and (v) PG / BSA / Apt / MA-Dha-CTF / GCE;

[0035] Figure 11 : (a) is ΔR at each stage when the aptasensor prepared in Examples 2 to 6 detects PG ctValue differences, (b) shows the ΔR of the aptasensors immobilized with different concentrations of aptamers prepared in Example 2 and Examples 7 - 11 after 1 h of detection of PG ct Value change diagram, (c) shows the EIS Nyquist diagrams of BSA / Apt / Fe - MA - Dha - CTF / GCE prepared in Example 2 incubated in PG solution for different times, (d) shows the ΔR ct Value change curve graph;

[0036] Figure 12 : (a) EIS Nyquist diagrams of BSA / Apt / Fe - MA - Dha - CTF / GCE prepared in Example 2 for detecting different concentrations of PG (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1 , 1 pg·mL -1 , 10 pg·mL -1 , 100 pg·mL -1 , 1 ng·mL -1 and 10 ng·mL -1 ), (b) shows the ΔR of BSA / Apt / Fe - MA - Dha - CTF / GCE prepared in Example 2 for detecting different concentrations of PG (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1 , 1 pg·mL -1 , 10 pg·mL -1 , 100 pg·mL -1 , 1 ng·mL -1 and 10 ng·mL -1 ), (c) shows the calibration curve between ΔR ct and the concentration of PG (Insert: Linear fitting graph of ΔR ct as a function of the logarithm of the PG concentration); (c) shows the detection of different concentrations of PG (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1 , 1 pg·mL -1 , 10 pg·mL -1 , 100 pg·mL -1 , 1 ng·mL -1 and 10 ng·mL -1) photocurrent response curve at [specific time]; (d) Calibration curve between ΔI and PG concentration when detecting different concentrations of PG (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1 , 1 pg·mL -1 , 10 pg·mL -1 , 100 pg·mL -1 , 1 ng·mL -1 and 10 ng·mL -1 ) by the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 (Insert: Linear fitting plot of ΔI as a function of the logarithm of PG concentration; Error bars are the standard deviations at n = 3);

[0037] Figure 13 : (a) ΔR ct value plots of detecting PG, different types of interferents, and the mixture of PG and all interferents by the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, (b) Photocurrent response change value plots of detecting PG, different types of interferents, and the mixture of PG and all interferents by the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, (c) EIS response value plots of detecting PG (1 fg·mL -1 ) by 5 independent aptasensors (BSA / Apt / Fe-MA-Dha-CTF / GCE) prepared in Example 2, (d) Photocurrent response change value plots of detecting PG (1 fg·mL -1 ) by 5 independent aptasensors (BSA / Apt / Fe-MA-Dha-CTF / GCE) prepared in Example 2, (e) EIS response change value plots of continuously detecting PG (1 fg·mL -1 ) every day for 20 days by the same BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, (f) Photocurrent response change value plots of continuously detecting PG (1 fg·mL -1 ) every day for 20 days by the same BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, (g) EIS response change value plots of the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 during the cycling process, (h) Photocurrent response change value plots of the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 during the cycling process (Error bars represent the standard deviations at n = 3). Detailed implementation manners

[0038] The technical solutions of the present invention will be further described below in conjunction with the detailed implementation manners.

[0039] Ferric acetate (C4H6FeO5) used in the following examples and comparative examples was purchased from Sinopharm Chemical Reagent Co., Ltd., phthalic anhydride, urea, melamine, and 2,5-dihydroxyterephthalaldehyde were all purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China), doxycycline (DOX), deoxynivalenol (DON), chloramphenicol (CAP), kanamycin (Kana), tobramycin (TOB), ampicillin (AMP), oxytetracycline (OTC), ofloxacin (OFLX), streptomycin (STRE), and penicillin G (PG) were all purchased from Solarbio Life Sciences Co., Ltd., and all solutions were prepared with ultrapure water (≥18.2 Ω·cm -1 ) as the solvent. The penicillin-targeting nucleic acid aptamer was: 5′-GGG TCT GAG GAG TGC GCG GTG CCA GTG AGT-3′.

[0040] Example 1

[0041] The preparation method of the electrochemical and photoelectrochemical dual-mode aptasensor electrode material in this example includes the following steps:

[0042] 1) Dissolve 10 mg of melamine in 1.6 mL of dimethyl sulfoxide (DMSO) to form solution A;

[0043] Dissolve 100 mg of 2,5-dihydroxyterephthalaldehyde in 1.6 mL of dimethyl sulfoxide to form solution B;

[0044] 2) After thoroughly mixing solutions A and B and sonicating for 5 min, transfer the mixed solution to a Pyrex tube (10 mL), degas 3 times, then react at 180 °C for 3 days. After centrifugation, wash the solid successively with acetone, tetrahydrofuran, and ethanol, and then dry the washed solid in a vacuum oven at 60 °C to obtain a solid material (denoted as MA-Dha-CTF);

[0045] 3) Disperse 50 mg of MA-Dha-CTF in 20 mL of methanol, sonicate for 10 min to form a homogeneous suspension, then add 35 mg of ferric acetate to the suspension and stir for 10 h. After filtration, wash with methanol 3 times and dry in vacuum at 60 °C for 6 h to obtain a powder (denoted as Fe-MA-Dha-CTF).

[0046] Example 2

[0047] The electrochemical and photoelectrochemical aptasensor in this example includes an electrode, an electrode material coated on the surface of the electrode, and a nucleic acid aptamer anchored on the electrode material; the preparation method of the electrochemical and photoelectrochemical dual-mode aptasensor in this example includes the following steps:

[0048] 1) Take a bare GCE with a diameter of 3 mm. After polishing the surface with alumina powder (particle size 0.05 μm), wash it with a mixed solution (HNO3 and ethanol, 1:1 (v / v)) and water for 10 min respectively. After drying in nitrogen at room temperature, activate it by cyclic voltammetry (CV) in 0.5 M H2SO4 by scanning in the range of -1.0 - 1.0 V until a stable cyclic voltammogram is obtained. Subsequently, rinse the pretreated bare GCE with water and dry it under N2 for standby. -1 In the range of -1.0 - 1.0 V until a stable cyclic voltammogram is obtained. Subsequently, rinse the pretreated bare GCE with water and dry it under N2 for standby.

[0049] 2) Disperse the Fe-MA-Dha-CTF powder prepared in Example 1 into pure water to obtain a Fe-MA-Dha-CTF suspension with a concentration of 1.0 mg·mL -1 of Fe-MA-Dha-CTF suspension;

[0050] Apply 10 μL of the Fe-MA-Dha-CTF suspension onto the surface of the pretreated bare GCE, then dry it overnight at room temperature, rinse it several times with pure water to remove the loosely attached Fe-MA-Dha-CTF, and dry it naturally in the air to obtain a modified electrode (denoted as Fe-MA-Dha-CTF / GCE).

[0051] 3) Incubate the obtained modified electrode in a penicillin-targeting nucleic acid aptamer solution (100 nmol·L -1 ) with the nucleic acid aptamer for 1 h, then wash it three times with pure water to remove the loosely adsorbed aptamer, to obtain an electrochemical and photoelectrochemical dual-mode aptamer sensor, denoted as Apt / Fe-MA-Dha-CTF / GCE.

[0052] 4) To eliminate the non-specific adsorption between PG and the modified electrode, perform a blocking treatment on Apt / Fe-MA-Dha-CTF / GCE in a bovine serum albumin (BSA) solution (mass concentration 1%), to obtain a blocked electrochemical and photoelectrochemical dual-mode aptamer sensor, denoted as BSA / Apt / Fe-MA-Dha-CTF / GCE.

[0053] Example 3

[0054] The electrochemical and photoelectrochemical dual-mode aptamer sensor of this example is only different from the electrochemical and photoelectrochemical dual-mode aptamer sensor of Example 2 in that: in step 2) of the preparation method of the aptamer sensor of this example, disperse the Fe-MA-Dha-CTF powder into pure water to obtain a Fe-MA-Dha-CTF suspension with a concentration of 0.1 mg·mL -1 of Fe-MA-Dha-CTF suspension, and then apply 10 μL of the Fe-MA-Dha-CTF suspension with a concentration of 0.1 mg·mL -1 onto the surface of the pretreated GCE.

[0055] Example 4

[0056] The electrochemistry and photoelectrochemistry dual-mode aptamer sensor of this example is only different from the electrochemistry and photoelectrochemistry dual-mode aptamer sensor of Example 2 in that: in step 2) of the preparation method of the aptamer sensor of this example, the Fe-MA-Dha-CTF powder is dispersed in pure water to obtain a Fe-MA-Dha-CTF suspension with a concentration of 0.2 mg·mL -1 Then, 10 μL of the Fe-MA-Dha-CTF suspension with a concentration of 0.2 mg·mL -1 is coated on the surface of the pretreated GCE.

[0057] Example 5

[0058] The electrochemistry and photoelectrochemistry dual-mode aptamer sensor of this example is only different from the electrochemistry and photoelectrochemistry dual-mode aptamer sensor of Example 2 in that: in step 2) of the preparation method of the aptamer sensor of this example, the Fe-MA-Dha-CTF powder is dispersed in pure water to obtain a Fe-MA-Dha-CTF suspension with a concentration of 0.5 mg·mL -1 Then, 10 μL of the Fe-MA-Dha-CTF suspension with a concentration of 0.5 mg·mL -1 is coated on the surface of the pretreated GCE.

[0059] Example 6

[0060] The electrochemistry and photoelectrochemistry dual-mode aptamer sensor of this example is only different from the electrochemistry and photoelectrochemistry dual-mode aptamer sensor of Example 2 in that: in step 2) of the preparation method of the aptamer sensor of this example, the Fe-MA-Dha-CTF powder is dispersed in pure water to obtain a Fe-MA-Dha-CTF suspension with a concentration of 2.0 mg·mL -1 Then, 10 μL of the Fe-MA-Dha-CTF suspension with a concentration of 2.0 mg·mL -1 is coated on the surface of the pretreated GCE.

[0061] Example 7

[0062] The electrochemistry and photoelectrochemistry dual-mode aptamer sensor of this example is only different from the electrochemistry and photoelectrochemistry dual-mode aptamer sensor of Example 2 in that: in step 3) of the preparation method of the aptamer sensor of this example, the concentration of the penicillin-targeting nucleic acid aptamer solution used is 10 nmol·L -1 .

[0063] Example 8

[0064] The electrochemical and photoelectrochemical dual-mode aptasensor of this example is only different from the electrochemical and photoelectrochemical dual-mode aptasensor of Example 2 in that: the concentration of the penicillin-targeting nucleic acid aptamer solution used in step 3) of the preparation method of the aptasensor in this example is 20 nmol·L -1 .

[0065] Example 9

[0066] The electrochemical and photoelectrochemical dual-mode aptasensor of this example is only different from the electrochemical and photoelectrochemical dual-mode aptasensor of Example 2 in that: the concentration of the penicillin-targeting nucleic acid aptamer solution used in step 3) of the preparation method of the aptasensor in this example is 50 nmol·L -1 .

[0067] Example 10

[0068] The electrochemical and photoelectrochemical dual-mode aptasensor of this example is only different from the electrochemical and photoelectrochemical dual-mode aptasensor of Example 2 in that: the concentration of the penicillin-targeting nucleic acid aptamer solution used in step 3) of the preparation method of the aptasensor in this example is 200 nmol·L -1 .

[0069] Example 11

[0070] The electrochemical and photoelectrochemical dual-mode aptasensor of this example is only different from the electrochemical and photoelectrochemical dual-mode aptasensor of Example 2 in that: the concentration of the penicillin-targeting nucleic acid aptamer solution used in step 3) of the preparation method of the aptasensor in this example is 500 nmol·L -1 .

[0071] Comparative Example

[0072] When preparing the aptasensor of this comparative example, only Fe-MA-Dha-CTF used in step 2) of Example 2 was replaced with MA-Dha-CTF prepared in step 2) of Example 1, and the content not mentioned is exactly the same as that of Example 2; among them, the modified electrode obtained in step 2) was denoted as MA-Dha-CTF / GCE.

[0073] Experimental Example 1 Basic Characterization of MA-Dha-CTF and Fe-MA-Dha-CTF Prepared in Example 1

[0074] The surface morphology of the synthesized samples was characterized using a JSM-6490LV field emission scanning electron microscope (FE-SEM, Japan) and a FEI TECNAI G2 F30 high-resolution transmission electron microscope (HR-TEM, USA) with a 200 kV field emission gun.

[0075] Elemental mapping was measured on the TEM using an energy dispersive spectrometer (EDS, XFlash-5030T, Beuke).

[0076] The specific surface area of the samples was determined by the Brunauer-Emmett-Teller (BET) method using a Micromeritics ASAP2022 instrument at liquid nitrogen temperature. Before measurement, all samples were degassed at 373 K for 8 hours.

[0077] The chemical composition was analyzed by Fourier transform infrared spectroscopy (FT-IR) using a Bruker TENSOR27 spectrometer (32 scans at 4 cm -1 resolution).

[0078] Powder X-ray diffraction (PXRD) was performed using a Rigaku D / Max-2500 x-ray diffractometer with a Cu K α target (λ = 0.15406 nm).

[0079] Raman spectra in the range of 50 - 4000 cm -1 were obtained at room temperature using a solid-state laser (excitation wavelength of 532 nm) on a Renishaw in Via Raman spectrometer.

[0080] X-ray photoelectron spectroscopy (XPS) data were collected using an AXIS HIS 165 spectrometer (Kratos Analytical, Manchester, UK) with a monochromatic Al K α X-ray source (1486.71 eV photons).

[0081] Scanning electron microscope (SEM) images of MA-Dha-CTF ( Figure 1 a) show a stacked blocky morphology and contain a large number of nanoparticles ( Figure 1 b). As Figure 1 shown in Figure 1 c, the transmission electron microscope (TEM) images confirm this result. The TEM images ( Figure 1 d) further show the ultrathin nanosheet shape of MA-Dha-CTF. In addition, no lattice fringes were found in the high-resolution TEM (HR-TEM) images (

[0082] For Fe-MA-Dha-CTF, the TEM image ( Figure 2 a) shows the combination of nanoparticles and nanorods. The porous accumulation of nanoparticles and nanorods confirms the high porosity of Fe-MA-Dha-CTF. In addition, clear lattice fringes of 0.25 nm can be observed in the HR-TEM image ( Figure 2 b) of Fe-MA-Dha-CTF, corresponding to the (110) plane of the cubic spinel structure β-FeOOH. This indicates that the adsorbed Fe ions are oxidized during the preparation of Fe-MA-Dha-CTF. These β-FeOOH nanocrystals not only contribute to improving the electrochemical activity but also greatly facilitate the immobilization of aptamers. In addition, scanning transmission electron microscopy combined with energy-dispersive X-ray spectroscopy (EDS) imaging shows that C, N, and O elements are uniformly distributed in MA-Dha-CTF and Fe-MA-Dha-CTF ( Figure 1 f), while Fe elements are present throughout the region of Fe-MA-Dha-CTF ( Figure 2 c).

[0083] The powder X-ray diffraction (PXRD) pattern ( Figure 3 a) of MA-Dha-CTF exhibits diffraction peaks significantly different from those of MA and Dha, indicating the formation of a new crystal phase after their reaction. The PXRD patterns of MA-Dha-CTF and Fe-MA-Dha-CTF show typical diffraction peaks at 2θ = 11.84° and 26.73°, which are attributed to the (110) and (310) crystal planes of β-FeOOH (JCPDS No. 34-1266), respectively. Through 13 13C solid nuclear magnetic resonance analysis, the conjugated structure connected by C=N bonds in MA-Dha-CTF is further confirmed ( Figure 4 ). The characteristic 13 13C resonance of the C=N imine group is observed at δ ≈ 165 ppm, and the characteristic peaks of the surrounding benzene rings are at δ ≈ 123 ppm and 146 ppm, respectively. However, due to the reaction with the amino group on MA, the typical aldehyde carbonyl 13 13C resonance at δ ≈ 195 ppm is missing. In addition, nitrogen (N2) adsorption-desorption measurements were carried out at 77 K to characterize the specific surface area and pore structure type of MA-Dha-CTF and Fe-MA-Dha-CTF. Their N2 adsorption-desorption isotherms ( Figure 3 b) show typical type-IV isotherms. The specific surface area of Fe-MA-Dha-CTF (145.76 m 2 2 / g -1 ) is smaller than that of MA-Dha-CTF (267.83 m 2 2 / g -1)。This indicates that β-FeOOH nanocrystals are embedded in the pores of MA-Dha-CTF, thereby reducing the surface area. From the pore size distribution curve ( Figure 3 c), it can be seen that the pore size distributions of MA-Dha-CTF and Fe-MA-Dha-CTF are in the range of 1 - 20 nm, indicating a mesoporous structure. The average pore size of MA-Dha-CTF is 12.74 nm, slightly larger than that of Fe-MA-Dha-CTF which is 9.89 nm.

[0084] The Fourier transform infrared spectroscopy (FT-IR) spectra of MA-Dha-CTF and Fe-MA-Dha-CTF ( Figure 5 a) show characteristic peaks at 1435 and 1529 cm -1 attributed to C=N on the triazine ring. Notably, a vibrational absorption at 592 cm -1 can be observed in the FT-IR spectrum of Fe-MA-Dha-CTF, corresponding to the stretching vibration of the Fe-O bond. In addition, considering that β-FeOOH nanocrystals can change the pore structure of CTF and introduce defects, it can be further verified by electron paramagnetic resonance (EPR). Generally, the formation of defects or vacancies in semiconductors generates unpaired electrons, thus producing characteristic peaks in the EPR spectrum. The EPR spectra of MA-Dha-CTF and Fe-MA-Dha-CTF ( Figure 5 b) both show an obvious peak with a g value of 2.004. This may be due to oxygen vacancies acting as electron traps, which is beneficial to improving the electrochemical activity and light absorption ability. In contrast, the peak intensity of Fe-MA-Dha-CTF is slightly higher than that of MA-Dha-CTF, indicating more defect vacancies. Therefore, Fe-MA-Dha-CTF will have excellent electrochemical activity, which is beneficial to improving the sensing performance.

[0085] In addition, X-ray photoelectron spectroscopy (XPS) characterization also shows that in the XPS measurement scanning spectra of MA-Dha-CTF and Fe-MA-Dha-CTF, signals of C 1s (284.6 eV), N 1s (400 eV) and O 1s (530 eV) coexist, while a weak Fe 2p signal can be obtained for Fe-MA-Dha-CTF ( Figure 6 ). To further understand the aptamer immobilization mechanism on Fe-MA-Dha-CTF, we also carried out XPS characterization on the Apt / Fe-MA-Dha-CTF complex. Figure 7A clear P 2p XPS signal is shown, which originates from the phosphate groups on the backbone of the aptamer chain oligonucleotide and can be regarded as an important indicator of aptamer adsorption. This result indicates that the prepared Fe-MA-Dha-CTF can serve as an effective platform for constructing aptamer sensors by immobilizing aptamers.

[0086] UV-vis diffuse reflectance spectroscopy (DRS) shows that the absorbance of MA-Dha-CTF even covers the entire UV-vis region and has a wide absorbance below 600 nm ( Figure 8 a), while Fe-MA-Dha-CTF well inherits the optical properties of MA-Dha-CTF and exhibits a similar optical absorbance. The calculated band gaps of MA-Dha-CTF and Fe-MA-Dha-CTF are 2.61 and 2.08 eV respectively ( Figure 8 b).

[0087] Experimental Example 2

[0088] The methods for electrochemical testing and photoelectrochemical testing in this experimental example are as follows:

[0089] Electrochemical testing: Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) analyses were carried out on a Solartron electrochemical analyzer. A traditional three-electrode system was used, where a GCE electrode with a diameter of 3 mm or a modified GCE electrode was used as the working electrode, an Ag / AgCl (saturated KCl) electrode was used as the reference electrode, and a platinum sheet was used as the counter electrode. In PBS (0.01 M, pH = 7.4) containing 5 mM [Fe(CN)6] 3- / 4- , 0.14 M NaCl and 0.1 M KCl, an EIS Nyquist plot was obtained at a potential of 0.21 V and a frequency range of 100 kHz to 0.1 Hz; the equivalent circuit during testing is shown in Figure 9 , including solution resistance (Rs), charge transfer resistance (Rct), constant phase element (CPE1) and Warburg impedance (W1). All electrochemical measurements were carried out at room temperature.

[0090] Photoelectrochemical (PEC) Test: PEC measurements were carried out on an electrochemical workstation using a three-electrode system, where a GCE electrode with a diameter of 3 mm or a modified GCE electrode was used as the working electrode, an Ag / AgCl (saturated KCl) electrode as the reference electrode, and a platinum sheet as the counter electrode. A 300 W xenon lamp (PLS-SXE300, Perfect Company, Beijing, China) was used as the light source, and the test was carried out in PBS (0.01 M, pH = 7.4). In the PEC test, the voltage was set to 0 V (vs. Ag / AgCl). After the current baseline was stable, the light source irradiated the working electrode at an interval of 30 s. The photocurrent response was recorded with a current-time (I-t) curve.

[0091] 1) Comparison of the EC and PEC performances of MA-Dha-CTF / GCE prepared in the comparative example and Fe-MA-Dha-CTF / GCE prepared in Example 2

[0092] The EIS Nyquist diagrams of the pre-treated bare GCE (Example 2), MA-Dha-CTF / GCE (comparative example), and Fe-MA-Dha-CTF / GCE (Example 2) are shown in Figure 10 a. As can be seen from Figure 10 a, the R ct value of the pre-treated bare GCE is very small, 30.9 Ω (curve i), indicating good conductivity. After modification, the R ct values of MA-Dha-CTF / GCE (curve ii) and Fe-MA-Dha-CTF / GCE (curve iii) are 254.5 and 167.5 Ω, respectively, both greater than that of the bare GCE. In comparison, due to the smaller R ct , the electrochemical conductivity of Fe-MA-Dha-CTF / GCE is better than that of MA-Dha-CTF / GCE. As mentioned above, the synthesized Fe-MA-Dha-CTF consists of abundant β-FeOOH nanocrystals and oxygen vacancies, has a porous network structure, and shows enhanced electron transfer ability.

[0093] In addition, the photocurrent response curves of the pre-treated bare GCE, MA-Dha-CTF / GCE, and Fe-MA-Dha-CTF / GCE in Example 2 are shown in Figure 10 b.

[0094] Figure 10b shows that the photocurrent response of Fe-MA-Dha-CTF / GCE (0.46 μA) is significantly higher than that of MA-Dha-CTF / GCE (0.17 μA) and bare GCE (0.03 μA). The results indicate that the prepared Fe-MA-Dha-CTF has a high photoelectric conversion efficiency due to its abundant defects and a large number of β-FeOOH nanocrystals. Therefore, we selected Fe-MA-Dha-CTF as the bifunctional EC and PEC electrode material for constructing a dual-mode aptasensor for PG detection.

[0095] 2) Performance of Fe-MA-Dha-CTF / GCE, Apt / Fe-MA-Dha-CTF / GCE, and BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2

[0096] EIS Nyquist diagrams of the bare GCE pretreated in Example 2, Fe-MA-Dha-CTF / GCE prepared in Example 2, Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, and BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, and the EIS Nyquist diagram of BSA / Apt / Fe-MA-Dha-CTF / GCE after detecting PG by incubating in PG solution (concentration of 1 fg·mL -1 ) for 30 min (PG / BSA / Apt / Fe-MA-Dha-CTF / GCE) are shown in Figure 10 e.

[0097] Figure 10 c shows the EIS Nyquist diagram of Apt / Fe-MA-Dha-CTF / GCE, and its R ct value is 381.9 Ω, which is greater than the R ct value (167.5 Ω) of Fe-MA-Dha-CTF / GCE, indicating that the aptamer is immobilized. Generally, there will be a strong repulsive interaction between the negatively charged phosphate groups on the aptamer chain and [Fe(CN)6] 3- / 4- , thus inhibiting the charge transfer at the electrode / electrolyte interface. Moreover, a large number of anchored aptamers increase the thickness of the conduction layer, further increasing the inhibitory effect on electron transfer. Similar aptamer immobilization behavior exists in MA-Dha-CTF / GCE ( Figure 10 e). The change in the R ct value (ΔR ct = R ct,1 - R ct,0 ) can represent the relative adsorption amount of the sensitive layer. The ΔR ct(214.4 Ω) is significantly greater than Apt / MA-Dha-CTF (148.1 Ω). This indicates that Fe-MA-Dha-CTF has a strong affinity and can immobilize a large number of aptamer chains on it, enhancing the detection ability for PG. In addition, the constructed Apt / Fe-MA-Dha-CTF electrode was incubated with BSA solution (1%) to block the non-specific adsorption of some interfering substances to the aptamer sensor (curve iii), and the R ct value increased slightly. The BSA / Apt / Fe-MA-Dha-CTF / GCE was incubated in PG solution (PG concentration was 1 fg·mL -1 ), and the Rct value of the obtained PG / Apt / Fe-MA-Dha-CTF / GCE gradually increased, resulting in a larger ΔR ct (737.5 Ω), which was significantly higher than the ΔR ct (535.3 Ω) of the MA-Dha-CTF-based aptamer sensor under the same conditions. Obviously, introducing Fe ions into MA-Dha-CTF can not only improve the conductivity but also provide more active sites to adsorb aptamers, enhancing the detection ability for PG. The results show that the modification of the CTF layer, the immobilization of aptamers, BSA adsorption, and the detection process of PG on the electrode, R ct gradually increase.

[0098] The construction and detection of an aptamer sensor for PG using Fe-MA-Dha-CTF as the electrode modification material were studied by PEC testing. The photocurrent curves of the bare GCE pretreated in Example 2, the Fe-MA-Dha-CTF / GCE prepared in Example 2, the Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2, and the photocurrent response curve of the BSA / Apt / Fe-MA-Dha-CTF / GCE after detecting PG by incubating in PG solution (concentration was 1 fg·mL -1 ) for 30 min (denoted as PG / BSA / Apt / Fe-MA-Dha-CTF / GCE) are shown in Figure 10 d.

[0099] As Figure 10As shown in d, the photocurrent response of Apt / Fe-MA-Dha-CTF / GCE (0.33 μA, curve iii) is lower than that of Fe-MA-Dha-CTF / GCE (0.46 μA, curve ii), which is related to the adsorption and fixation of the aptamer chain. The photocurrent response of PG / BSA / Apt / Fe-MA-Dha-CTF / GCE (curve iv) decreases to 0.21 μA, which can be attributed to the specific binding between the aptamer and PG. The change in photocurrent response during the detection of PG by the Fe-MA-Dha-CTF-based PEC aptasensor (ΔI = 0.02 μA) is significantly greater than that of the MA-Dha-CTF-based PEC aptasensor (ΔI = 0.01 μA) ( Figure 10 f). Considering that the Fe-MA-Dha-CTF type adapter has excellent dual-mode EC-PEC detection ability, it can be used for the sensitive analysis of PG.

[0100] In addition, in order to obtain the best sensing performance, the experimental and measurement conditions such as the usage of Fe-MA-Dha-CTF, the concentration of the aptamer, and the binding time between the aptamer and PG were optimized. Figure 11 Figure a shows different biosensors constructed by modifying the electrode with Fe-MA-Dha-CTF suspensions at different concentrations (0.1, 0.2, 0.5, 1, and 2 mg·mL -1 ) (corresponding to the blocked aptasensors in Examples 2 to 6 respectively) incubated in a PG solution (concentration of 1 fg·mL -1 ) for 60 min, and the change value (ΔR ct , that is, the change in the charge transfer resistance (R ct ) of the EIS response before and after detecting PG was obtained. The change value of the EIS response (ΔR ct ) increases with the increase in the dosage of Fe-MA-Dha-CTF. This indicates that a large number of aptamer chains can be anchored on Fe-MA-Dha-CTF, thus detecting PG more effectively. When the dosage of Fe-MA-Dha-CTF is greater than 1 mg·mL -1 , the observed EIS response reaches equilibrium.

[0101] The blocked aptasensors based on Fe-MA-Dha-CTF developed by anchoring aptamers at different concentrations in Examples 2, 7 to 11 were incubated in a PG solution (concentration of 1 fg·mL -1 ) for 1 h, and the change value (ΔRct value) of the EIS response before and after detecting PG was obtained. The results are as shown in Figure 11 Figure b. The ΔRct value increases with the increase in the concentration of the aptamer. When the concentration of the aptamer is greater than 100 nmol·L -1 , the ΔR ctThe value tends to be stable. When the modified electrode is incubated with the aptamer at a high concentration, more aptamer chains are immobilized due to the formation of the aptamer-PG complex. Therefore, the optimal aptamer concentration is 100 nmol·L -1 .

[0102] The binding time of PG on the aptasensor also affects the sensing performance. The BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 was incubated in a PG solution (concentration of 1 fg·mL -1 ); The EIS Nyquist diagrams after incubation for different times are shown in Figure 11 c and the corresponding change values (ΔR ct values) of the EIS response before and after detection are shown in Figure 11 d. As can be seen from Figure 11 c, as the time gradually extends from 0 min to 60 min, the diameter of the arc gradually increases to the maximum value. When the time is further extended, the radius of the arc slightly decreases. As can be seen from Figure 11 d, the ΔR ct value increases significantly at the beginning. When the binding time is greater than 1 h, the obtained ΔR ct value reaches equilibrium. Therefore, the optimal binding time for PG and the aptamer to be immobilized on the modified electrode is 1 h to ensure their sufficient binding.

[0103] The LOD of the biosensor of Fe-MA-Dha-CTF constructed under the optimal conditions (BSA / Apt / Fe-MA-Dha-CTF / GCE developed in Example 2) was evaluated by the EC (EIS) method. During the test, the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 was incubated in a PG solution (the concentration range of PG in the solution was 1 fg·mL -1 ~10 ng·mL -1 ) for 1 h to obtain a series of EIS Nyquist diagrams ( Figure 12 a) and the calibration curve between ΔR ct and the PG concentration (see Figure 12 b).

[0104] As can be seen from Figure 12 a, when the PG concentration increases from 1 fg·mL -1 to 1 pg·mL -1 , the R ct value derived from the EIS response gradually increases. Due to the formation of a complex between PG and the aptamer, the increased PG can be specifically recognized by the obtained aptasensor. When the PG concentration is further increased, a plateau is reached due to the saturated binding between PG and the aptamer.

[0105] From Figure 12It can be seen that the obtained ΔR ct value has a good linear relationship with the logarithm of the PG concentration (C PG ). The regression equation is ΔR ct = 0.38lgC PG + 1.53, and the correlation coefficient (R 2 ) value is 0.9967. Therefore, the calculated LOD of the EC aptasensor derived using EIS technology is as low as 0.08 fg·mL -1 .

[0106] The PEC method was used to evaluate the LOD of the electrochemical and photoelectrochemical dual-mode aptasensor developed in Example 2. During the test, the BSA / Apt / Fe-MA-Dha-CTF / GCE prepared in Example 2 was incubated in a PG solution (the concentration range of PG in the solution was 1 fg·mL -1 to 10 ng·mL -1 ) for 1 h, and the photocurrent response value after incubation was obtained, as shown in Figure 12 c. Figure 12 c shows that the photocurrent response decreases as the PG concentration increases in the range of 1 fg·mL -1 to 10 ng·mL -1 . There is a good linear relationship between the change in the photocurrent response value (ΔI) before and after detecting PG and the logarithm of the PG concentration ( Figure 12 d), and the obtained regression equation is ΔI (μA) = 0.0159lgC PG + 0.098, and the correlation coefficient (R 2 ) is 0.9905. Therefore, a low LOD of 0.7 fg·mL -1 can be obtained.

[0107] In summary, the electrochemical and photoelectrochemical dual-mode aptasensor based on Fe-MA-Dha-CTF has a wider linear range and a relatively low LOD, indicating that the sensor has good sensing ability for PG. As described above, Fe-MA-Dha-CTF has a large specific surface area, abundant functional groups, oxygen vacancies, and abundant active sites, which greatly promotes the anchoring of a large number of aptamer chains. In addition, the coupling of β-FeOOH nanocrystals, porous nanostructures, and triazine networks can endow Fe-MA-Dha-CTF with good electrochemical activity and high photoelectric conversion efficiency, thus increasing the response signal.

[0108] 3) Selectivity, stability, reproducibility, and regenerability of the electrochemical and photoelectrochemical dual-mode aptasensor (BSA / Apt / Fe-MA-Dha-CTF / GCE) constructed in Example 2

[0109] i) By analyzing various interferents that may coexist with PG, the selectivity of the electrochemical and photoelectrochemical dual-mode aptasensor was studied.

[0110] The selectivity of doxycycline (DOX), deoxynivalenol (DON), chloramphenicol (CAP), kanamycin (Kana), tobramycin (TOB), ampicillin (AMP), oxytetracycline (OTC), ofloxacin (OFLX), streptomycin (STRE) and the mixtures of all the above-listed interferents with PG that may coexist with PG in food were investigated. The concentration of each interferent and each interferent in the mixtures of all the listed interferents and PG was 100 fg·mL -1 , which is 100 times the concentration of PG (1 fg·mL -1 ).

[0111] The electrochemical and photoelectrochemical dual-mode aptasensor (BSA / Apt / Fe-MA-Dha-CTF / GCE) prepared in Example 2 was incubated in each interferent solution, PG solution and the mixed solution of 9 interferents and PG for 1 h by using the EC (EIS) method to obtain the change value (ΔR ct value) of the EIS response before and after detection. Figure 13 Figure a shows that the change in the EIS response (ΔR ct ) values observed for the determination of each interferent (including DOX, DON, CAP, Kana, TOB, AMP, OTC, OFLX and STRE) are not obvious. In contrast, the ΔR ct corresponding to the detection of PG is significant, which is comparable to the detection signal of the aptasensor for the mixture.

[0112] Under the same conditions, using the PEC method, the electrochemical and photoelectrochemical dual-mode aptasensor (BSA / Apt / Fe-MA-Dha-CTF / GCE) of Example 2 was incubated in each interferent solution, PG solution and the mixed solution of 9 interferents and PG for 1 h to obtain the change value (ΔI) of the photocurrent response before and after detection. Similar results can be obtained in ΔI ( Figure 13 Figure b).

[0113] Therefore, the electrochemical and photoelectrochemical dual-mode aptasensor of the present invention shows high selectivity for the detection of PG, even in a complex environment, which is attributed to the specific recognition between PG and the aptamer.

[0114] ii) By incubating 5 parallel BSA / Apt / Fe-MA-Dha-CTF / GCEs prepared in Example 2 in a PG solution (concentration of 1 fg·mL -1 ) for 1 h, the change (ΔR ct ) value of the EIS response before and after the detection of PG was obtained (seeFigure 13 c) and the values of the change in photocurrent response (ΔI) (see Figure 13 d) The relative standard deviation RSD values are 1.12% and 1.25% respectively. These results indicate that the dual-mode aptamer sensor has good repeatability.

[0115] iii) Using the electrochemistry and photoelectrochemistry dual-mode aptamer sensor (BSA / Apt / Fe-MA-Dha-CTF / GCE) prepared in the same Example 2, within 20 days, by continuously recording the changes in EIS response (ΔR -1 , incubation time is 1 h) and the change in photocurrent response (ΔI) value when detecting PG in the PG solution (PG solution concentration is 1 fg·mL ct ), to evaluate the stability of the sensor. The records of the changes in EIS response and photocurrent response for 20 consecutive days are shown in Figure 13 e and 13f. It can be seen that after 20 days, the attenuation is 2.3% and 3.4% compared with the original values.

[0116] iv) Immerse the electrochemistry and photoelectrochemistry dual-mode aptamer sensor (BSA / Apt / Fe-MA-Dha-CTF / GCE) prepared in Example 2 in the PG solution (concentration is 1 fg·mL -1 ) for 1 h (denoted as PG / BSA / Apt / Fe-MA-Dha-CTF / GCE after incubation), record the change in EIS response (ΔR ct ) value and the change in photocurrent response (ΔI) value before and after detecting PG, then soak PG / BSA / Apt / Fe-MA-Dha-CTF / GCE in 1 mM NaOH at room temperature for 2 min, and then rinse thoroughly with phosphate buffered saline (PBS). In this way, the aptamer-PG complex will dissociate, resulting in the removal of PG, and the dual-mode aptamer sensor can be regenerated. Immerse the regenerated electrochemistry and photoelectrochemistry dual-mode aptamer sensor into the PG solution (1 fg·mL -1 ) again, and the electrochemical signal and photocurrent response reach the original level. The whole regeneration process is repeated 10 times to evaluate the regeneration ability of BSA / Apt / Fe-MA-Dha-CTF / GCE. The results are shown in Figure 13 g and 13h.

[0117] Figure 13 g shows that there are slight fluctuations in the EIS response for detecting PG of the regenerated electrochemistry and photoelectrochemistry dual-mode aptamer sensor during 10 cycles, indicating good regeneration ability. Similar results can also be obtained by PEC measurement ( Figure 13 h).

[0118] In summary, the electrochemical and photoelectrochemical dual-mode aptasensor based on Fe-MA-Dha-CTF not only has an extremely low detection limit, high selectivity, good stability, and good reproducibility, but also can be regenerated for the detection of analytes, showing great potential for practical applications.

[0119] 4) Analysis of actual samples

[0120] The practicability of the dual-mode aptasensor was evaluated by detecting PG in different actual samples (raw milk and pork). First, the raw milk and pork were pretreated, and then the constructed dual-mode aptasensor was used to determine PG in different raw milk solutions and pork solutions by EC method (EIS) and PEC method, respectively. According to the calibration curve, the true concentration of PG in the solution was deduced from the obtained EIS and PEC responses. The simulated and true concentrations of PG determined by the EIS method are shown in Table 1-2.

[0121] Pretreatment of raw milk: Take raw milk (50 mL), mix it with 0.2 mL of NaOH (1 mM) and 10 mL of acetonitrile (containing 20% concentration of NH4OH), stir evenly, and filter through a 0.45 μm filter membrane to obtain the supernatant. Then, take a series of supernatant samples (each supernatant sample has a volume of 2 mL), and add different concentrations of PG (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1 , 1 pg·mL -1 , 10 pg·mL -1 , 0.1 ng·mL -1 , 1 ng·mL -1 , 10 ng·mL -1 ), and then use the constructed dual-mode aptasensor to detect PG in each solution by EIS and PEC methods, and then calculate the concentration of PG. The results are shown in Tables 1 and 3, respectively.

[0122] Pretreatment of pork samples: Take 1 g of chopped pork, then add 20 mL of ethyl acetate and 1.0 mL of K2CO3 (4 mol L -1 ) to the above pork samples, sonicate for 1 min, then shake for 2 h, and then centrifuge at 10000 rpm for 10 min. The obtained solid residue is dried at 40 °C, and then the dried solid residue is dispersed in a mixed solution of 1 mL of methanol and water (v:v = 1:1), and then redispersed in PBS (10 mM) to obtain a dispersion. Then, take a series of dispersion samples (each dispersion sample has a volume of 2 mL), and add different concentrations of PG standard solutions (1 fg·mL -1 , 10 fg·mL -1 , 100 fg·mL -1, 1 pg·mL -1 , 10 pg·mL -1 , 0.1 ng·mL -1 , 1 ng·mL -1 , 10 ng·mL -1 ), and then use the constructed dual-mode aptamer sensor to detect PG in each solution by EIS and PEC methods, and then calculate the concentration of PG. The results are shown in Tables 2 and 4 respectively.

[0123] Table 1 Detection of PG in milk by EIS method using a dual-mode aptamer sensor (n = 3)

[0124]

[0125]

[0126] Table 2 Detection of PG in pork by EIS method using a dual-mode aptamer sensor (n = 3).

[0127] <![CDATA[Add (pg·mL -1 )]]> <![CDATA[ΔR ct (Ω)]]> <![CDATA[Detected (pg·mL -1 )]]> Recovery rate (%) RSD (%) 0.001 390.13 0.001047 104.7 0.6 0.01 745.15 0.009333 93.3 0.3 0.1 1155.72 0.104713 104.7 0.4 1 1515.27 0.901571 90.2 0.3 10 1908.14 9.549926 99.5 0.6 100 2308.11 104.7129 104.7 0.9 1000 2672.65 993.2543 99.3 0.2 10000 3057.32 9332.543 93.3 0.4

[0128] It can be seen from the data in Tables 1 and 2 that the recovery rates of detecting PG content in milk and pork by using the electrochemical and photoelectrochemical dual-mode aptamer sensor of Example 2 by EIS method are relatively good, which are 92.0% - 109.6% and 90.2% - 104.7% respectively, and the RSDs are 0.2% - 1.2% and 0.2% - 0.9% respectively.

[0129] Table 3 Detection of PG in milk by PEC method using a dual-mode aptamer sensor (n = 3).

[0130]

[0131]

[0132] Table 4 Detection of PG in pork by PEC method using a dual-mode aptamer sensor (n = 3)

[0133] <![CDATA[Add (pg·mL -1 )]]> ΔI (μA) <![CDATA[Detected (pg·mL -1 )]]> Recovery rate (%) RSD (%) 0.001 0.0505 0.001047 104.7 0.2 0.01 0.0668 0.010965 109.7 0.6 0.1 0.0816 0.093325 93.3 0.7 1 0.0974 1.090687 109.1 0.5 10 0.1145 10.71519 107.2 1.5 100 0.1302 106.4143 106.4 0.8 1000 0.1462 1071.519 107.2 0.4 10000 0.1611 9306.792 93.1 1.6

[0134] It can be seen from Tables 3 and 4 that the recovery rates of detecting PG content in milk and pork by using the electrochemical and photoelectrochemical dual-mode aptamer sensor of Example 2 by PEC method are 93.3% - 109.6% for milk samples and 93.1% - 109.7% for pork samples respectively. The results show that the developed dual-mode aptamer sensor has high accuracy and can be used for the detection of PG in actual samples.

[0135] In summary, the electrochemical and photoelectrochemical dual-mode aptasensor electrode material of the present invention is used to construct an electrochemical and photoelectrochemical dual-mode aptasensor for PG detection in complex environments. The Fe-MA-Dha-CTF network structure composed of a large number of β-FeOOH nanocrystals and oxygen vacancies has a porous structure, a large specific surface area, multifunctionality, enhanced light absorption ability, and photogenerated electron and hole separation performance. Thereby improving the electrochemical conductivity, photoelectric conversion efficiency, and enhancing the biological affinity for the aptamer chain. When detecting PG, the electrochemical and photoelectrochemical dual-mode aptasensor based on Fe-MA-Dha-CTF exhibits better detection ability than the MA-Dha-CTF-based aptasensor, and the LODs obtained by EIS and PEC methods are 0.08 and 0.7 fg·mL -1 . Compared with the reported EC or PEC PG aptasensors, due to the use of label-free aptamers and without any electrochemical or PEC signal amplification reagents, this dual-mode aptasensor has significant advantages such as higher accuracy, lower detection limit, wider linear detection range, and more feasible construction. The present invention provides a new sensing strategy for the sensitive and selective detection of antibiotics based on the CTF network, which can expand the application of CTF in biosensing.

Claims

1. An electrochemical and optoelectrochemical dual-mode aptasensor, characterized in that: It includes an electrode, an electrode material coated on the surface of the electrode, and a nucleic acid aptamer anchored on the electrode material; the preparation method of the electrode material includes the following steps: dispersing a covalent triazine framework material and an iron salt in an organic good solvent of the iron salt, followed by solid-liquid separation, and then drying the obtained solid; the covalent triazine framework material is formed by reacting melamine and 2,5-dihydroxyterephthalaldehyde; the nucleic acid aptamer is a penicillin-targeting nucleic acid aptamer.

2. The electrochemical and optoelectrochemical dual-mode aptasensor according to claim 1, wherein: The mass ratio of the melamine to the 2,5-dihydroxyterephthalaldehyde is 8:100 to 20:

100.

3. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 1 or 2, characterized in that: The covalent triazine framework material is prepared by a method including the following steps: reacting a mixed solution of melamine and 2,5-dihydroxyterephthalaldehyde at 150 to 200 °C for 48 to 96 h, followed by solid-liquid separation, and then washing and drying the obtained solid.

4. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 1 or 2, characterized in that: The iron salt is one or any combination of iron acetate, iron nitrate, and iron chloride; the good solvent of the iron salt is an alcohol solvent; the mass ratio of the covalent triazine framework material to the iron salt is 10:35 to 60:

35.

5. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 1 or 2, characterized in that: The dispersion is to mix a suspension of the iron salt and the covalent triazine framework material and then perform a stirring treatment; the suspension is formed by dispersing the covalent triazine framework material in the good solvent; the time of the stirring treatment is 8 to 12 h.

6. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 1 or 2, characterized in that: The electrochemical and photoelectrochemical dual-mode aptamer sensor is prepared by a method including the following steps: coating the electrode material on the surface of the electrode to obtain a modified electrode, then anchoring the nucleic acid aptamer on the modified electrode, and then performing a blocking treatment to obtain it.

7. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 6, characterized in that: Method for coating an electrode surface with an electrode material, comprising the following steps: coating a dispersion of the electrode material on the electrode surface, cleaning the electrode surface after drying, and drying; the concentration of the electrode material in the dispersion of the electrode material is 0.1 to 2.0 mg·mL -1 .

8. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 6, characterized in that: Method for anchoring nucleic acid aptamer on modified electrode, comprising the following steps: incubating the modified electrode in a nucleic acid aptamer solution, and then washing to remove loosely adsorbed nucleic acid aptamer; the concentration of the nucleic acid aptamer in the nucleic acid aptamer solution is 10~500 nmol·L -1 .

9. The electrochemical and optoelectrochemical dual-mode aptamer sensor according to claim 8, characterized in that: The time of the incubation treatment is 10 to 80 min.

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