Gold-silver nanoclusters, preparation method and application thereof in electrochemiluminescence-electrochemical dual-mode detection of chloramphenicol

Abstract

This invention belongs to the field of electrochemiluminescence materials technology, and discloses a gold-silver nanocluster, its preparation method, and its application in the electrochemiluminescence-electrochemical dual-mode detection of chloramphenicol. The preparation method involves adding a silver source solution to a gold source solution, then adding an alkaline thiol β-cyclodextrin solution to obtain a mixed solution. Sodium bicarbonate is added to adjust the pH to 8.2-8.4, followed by incubation to obtain a crude product of gold-silver nanoclusters. The crude product is then purified by centrifugation and dialyzed to obtain the gold-silver nanoclusters. Furthermore, this invention uses these gold-silver nanoclusters as an ECL luminescent material to construct a sensor and applies it to the quantitative detection of chloramphenicol. This sensor innovatively achieves synergistic detection of ECL and DPV dual signals, with mutually verifiable detection results, exhibiting high sensitivity, excellent selectivity, good stability, and excellent repeatability.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence materials technology, and in particular relates to a gold and silver nanocluster, its preparation method, and its application in the electrochemiluminescence-electrochemical dual-mode detection of chloramphenicol. Background Technology

[0002] Chloramphenicol, a broad-spectrum antibiotic, was once widely used in livestock and aquaculture for the prevention and treatment of bacterial infections. However, its misuse or abuse can lead to residues in animal-derived foods (such as meat, eggs, and aquatic products) and environmental media (such as river water and soil). This substance has potential toxic side effects such as bone marrow suppression and aplastic anemia, posing a serious threat to human health. It can also disrupt the microbial balance in the ecological environment, leading to the proliferation of drug-resistant bacteria. Currently, many countries and regions have set strict limits on chloramphenicol residues in food, and some have even banned its use in animal products. Therefore, establishing efficient and accurate methods for detecting chloramphenicol residues is of great significance for ensuring food safety, maintaining ecological stability, and protecting public health. Currently, routine methods for detecting chloramphenicol residues mainly include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS / MS). While these methods offer advantages such as high sensitivity and accuracy, they also have limitations, including high equipment costs, complex sample pretreatment, long analysis cycles, and reliance on specialized operators. These limitations make it difficult to meet the practical needs of rapid on-site detection and large-scale sample screening, thus restricting their application in scenarios such as grassroots supervision and on-site quality control in production.

[0003] Electrochemical sensing technology, with its advantages of ease of operation, rapid response, low cost, and portable instruments, has shown broad application prospects in the field of antibiotic residue detection. Among them, electrochemiluminescence (ECL) and differential pulse voltammetry (DPV) are two highly representative electrochemical detection strategies. ECL detection relies on the luminescence behavior of electrogenerated excited-state substances, featuring high sensitivity and low background signal; DPV detection is based on the interaction between the target and the sensing interface, triggering changes in oxidation peak current, and boasts advantages of simple operation and rapid response. However, traditional electrochemical sensing technologies still face significant technical bottlenecks in chloramphenicol detection. Traditional ECL luminescent materials suffer from low luminescence efficiency, weak stability, and poor anti-interference ability; sensing materials lack specific binding sites, easily leading to non-specific adsorption in complex samples and poor detection selectivity; single-mode detection is susceptible to environmental interference, resulting in insufficient verification and accuracy; high-performance sensing materials require stringent preparation conditions, involve complex materials, are costly, and are prone to introducing impurities, affecting detection performance.

[0004] The properties of nanomaterials directly affect the detection performance of electrochemical sensors. Although single-metal nanoclusters have large specific surface areas and good conductivity, they suffer from drawbacks such as weak catalytic activity, poor stability, limited signal form, low luminescence efficiency, easy decay, and poor selectivity. There are no reports in the current technology of using mercapto-β-cyclodextrin-stabilized gold-silver bimetallic nanoclusters for chloramphenicol detection, and integrating simultaneous detection in both ECL and DPV modes. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides a gold and silver nanocluster, its preparation method, and its application in the electrochemiluminescence-electrochemical dual-mode detection of chloramphenicol. This material combines high sensitivity, strong selectivity, good stability, and repeatability, enabling synergistic detection and mutual verification of results in both ECL and DPV modes.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing gold and silver nanoclusters, wherein the preparation method comprises adding a silver source solution to a gold source solution, then adding an alkaline mercapto β-cyclodextrin solution to obtain a mixed solution, adding sodium bicarbonate to adjust the pH to 8.2-8.4, and then incubating to obtain a crude product of gold and silver nanoclusters. The crude product of gold and silver nanoclusters is purified by centrifugation and dialyzed to obtain gold and silver nanoclusters.

[0007] The silver source is silver nitrate, and the gold source is tetrachloroauric acid trihydrate; the pH of the alkaline mercapto-β-cyclodextrin solution is 8.0-8.4.

[0008] The molar ratio of the mercapto-β-cyclodextrin to the silver source and the gold source is 5.3-5.5:0.9-1.1:4.8-5.2.

[0009] The incubation temperature is 78-82℃, and the incubation time is 23-25h.

[0010] The alkaline thiol β-cyclodextrin solution is prepared by dissolving thiol β-cyclodextrin in sodium bicarbonate solution.

[0011] Sodium bicarbonate can regulate the pH of the system to be weakly alkaline. Under weakly alkaline conditions, some thiol groups dissociate into sulfide anions, which enhances the reactivity. If the pH is too low, the thiol groups are inert in the form of -SH; if the pH is too high, they are easily oxidized to form disulfide bonds and become inactive.

[0012] The present invention also protects a gold and silver nanoclusters prepared by the above method.

[0013] The present invention also protects an electrochemiluminescence-electrochemical dual-mode sensor comprising the gold and silver nanoclusters, the sensor comprising an MB / dsDNA probe and a gold and silver nanocluster / GCE electrode; The gold and silver nanoclusters / GCE electrode is prepared by drop-coating the gold and silver nanoclusters solution onto the surface of the GCE electrode. The MB / dsDNA probe includes: dsDNA and magnetic beads; The dsDNA is obtained by hybridization of the capture probe cDNA and the chloramphenicol aptamer Fc-CAP-aptamer modified with its complementary strand ferrocene; the biotin of the capture probe cDNA is linked to streptavidin-modified magnetic beads; the nucleotide sequence of the cDNA is shown in SEQ ID No. 1, and the nucleotide sequence of the chloramphenicol aptamer is shown in SEQ ID No. 2.

[0014] The hybridization temperature is 35-38℃, and the hybridization time is 25-35 min; In the preparation of the gold-silver nanocluster / GCE electrode, the concentration of the gold-silver nanocluster solution is 0.8-1.2 mg / mL; the drop volume of the gold-silver nanocluster solution is 8-12 μL.

[0015] The application of the electrochemiluminescence-electrochemical dual-mode sensor in the detection of chloramphenicol includes the following steps: the MB / dsDNA probe is incubated with the test solution containing chloramphenicol for the first time, and after magnetic separation, the supernatant is added to the gold-silver nanocluster / GCE electrode for the second incubation, and then ECL measurement and DPV measurement are performed respectively.

[0016] The first incubation is carried out at a temperature of 35-38℃ for 1.8-2.2 hours; the second incubation is carried out at a temperature of 35-38℃ for 45-55 minutes.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for preparing gold and silver nanoclusters of the present invention introduces silver elements to construct gold and silver bimetallic nanocluster cores, optimizes material properties by relying on the electronic synergy and lattice synergy effect of gold and silver bimetals, uses mercapto-β-cyclodextrin as ligand, and takes advantage of the supramolecular inclusion function of mercapto-β-cyclodextrin to prepare water-soluble bimetallic nanoclusters in one step. It does not require EDC / NHS crosslinking agents, multi-step acid-base adjustment and segmented incubation, and realizes high sensitivity, high selectivity, rapid and stable detection of chloramphenicol in electrochemiluminescence-electrochemical dual mode, which significantly improves the reliability of complex practical sample analysis.

[0018] (2) In this invention, thiol β-cyclodextrin has three core functions. First, it forms a stable metal-sulfur bond with gold and silver nanoclusters through thiol groups, acting as a structural stabilizer and improving the overall physicochemical stability of the material. Second, it achieves specific recognition and pre-enrichment of chloramphenicol molecules by relying on the internal hydrophobic cavity, thus constructing an endogenous recognition site. Third, it forms a spatial barrier on the outer layer, effectively inhibiting non-specific adsorption in complex samples and improving the anti-fouling ability of the sensing interface.

[0019] (3) The present invention uses gold and silver as two noble metals to achieve synergy of electronic energy levels, synergy of conductivity and catalysis, and synergy of structural stability.

[0020] Electronic energy level synergy: The energy levels of gold and silver are coupled to reduce the energy barrier for electronic excitation and transition, improve the quantum yield of electrochemiluminescence, and solve the problems of weak luminescence and easy signal attenuation of single metal gold clusters; Synergistic effect of conductivity and catalysis: Both gold and silver have excellent conductivity, and the bimetallic structure further enhances the interfacial electron transport rate, strengthens electrocatalytic ability, and realizes signal amplification; Synergistic structural stability: The high lattice matching degree of the two noble metals makes the overall structure of the nanoclusters more stable, and the photophysical and chemical stability is far superior to that of single metals and gold-cerium rare earth composite systems.

[0021] (4) The electrochemiluminescence-electrochemical dual-mode sensor constructed using gold and silver nanoclusters of the present invention exhibits good sensing potential when applied to the detection of chloramphenicol: the electrochemiluminescence mode can sensitively detect chloramphenicol in the range of 0.0005-100 ng / mL, with a detection limit of 1.5 × 10⁻⁶. −4 ng / mL; in electrochemical mode, chloramphenicol can be sensitively detected in the range of 0.0005-100 ng / mL, with a detection limit of 5.8 × 10⁻⁶ ng / mL. −4 ng / mL.

[0022] (5) The electrochemiluminescence-electrochemical dual-mode sensor constructed using gold and silver nanoclusters of the present invention is applied to the detection of chloramphenicol. This biosensor has excellent selectivity, stability and repeatability. Attached Figure Description

[0023] Figure 1 Here is a high-magnification transmission electron microscope image of the gold and silver nanoclusters prepared in this invention; Figure 2 The ultraviolet absorption and fluorescence spectra of the gold and silver nanoclusters prepared in this invention are shown. Figure 3 The electrochemiluminescence spectrum of the gold and silver nanoclusters prepared in this invention is shown. Figure 4 The FT-IR spectrum of the gold and silver nanoclusters prepared in this invention; Figure 5XPS spectrum of gold and silver nanoclusters prepared in this invention; Figure 6 The CV diagram shows the gold and silver nanoclusters prepared in this invention. Figure 7 ECL transient signal diagram of the gold and silver nanoclusters prepared in this invention; Where A is the potential step ECL transient curve of the reduction-oxidation annihilation process of gold and silver nanoclusters, and B is the potential step ECL transient curve of the oxidation-reduction annihilation process of gold and silver nanoclusters. Figure 8 The electrochemiluminescence response curves during the sensor assembly process of this invention are shown. Where a corresponds to naked GCE, b corresponds to gold and silver nanoclusters / GCE, and c corresponds to MB / dsDNA probe / gold and silver nanoclusters / GCE; Figure 9 This is the DPV curve of the sensor assembly process of the present invention; Where a corresponds to naked GCE, b corresponds to gold and silver nanoclusters / GCE, and c corresponds to MB / dsDNA probe / gold and silver nanoclusters / GCE; Figure 10 Electrochemiluminescence response curves of the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention to chloramphenicol at concentrations of 0.0005-100 ng / mL; Figure 11 Linear calibration curves of electrochemiluminescence-electrochemical dual-mode sensor prepared for this invention for chloramphenicol electrochemiluminescence at concentrations of 0.0005-100 ng / mL; Figure 12 The DPV response curves of the electrochemiluminescence-electrochemical dual-mode sensor prepared for this invention to chloramphenicol in the range of 0.0005-100 ng / mL; Figure 13 Linear calibration curves of the electrochemiluminescence-electrochemical dual-mode sensor prepared for this invention for chloramphenicol DPV in the range of 0.0005-100 ng / mL; Figure 14 ECL selectivity test results for the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention; Figure 15 The DPV selectivity test diagram of the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention; Figure 16 ECL stability test results for the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention; Figure 17 The DPV stability test diagram of the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention; Figure 18The repeatability test results are shown for the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention. Figure 19 This is a flowchart illustrating the fabrication process of the electrochemiluminescence-electrochemical dual-mode sensor of the present invention. Detailed Implementation

[0024] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] The electrochemiluminescence-electrochemical dual-mode sensor provided in this invention uses gold and silver nanoclusters as the ECL luminescent agent and triethylamine as the co-reactant. The gold and silver nanoclusters can generate a significant ECL emission signal. In the presence of chloramphenicol, the Fc-CAP-aptamer in the MB / dsDNA probe will be released; thiol β-cyclodextrin can specifically recognize the released Fc through guest-host interactions, thereby leading to quenching of the ECL signal and an increase in the DPV signal. The constructed sensing platform can sensitively and selectively detect chloramphenicol by monitoring changes in ECL and DPV signals (sensitive detection of chloramphenicol is crucial for food safety and environmental monitoring).

[0026] The streptavidin-modified magnetic beads used in subsequent embodiments were purchased from New England Biolabs.

[0027] Example 1: A method for preparing gold and silver nanoclusters Weigh 50 mg (43.44 μmol) of mercapto-β-cyclodextrin and dissolve it in 300 μL of 0.1 mol / L NaHCO3 solution. Mix thoroughly to obtain an alkaline mercapto-β-cyclodextrin solution with a pH of 8.2.

[0028] 8 μL of silver nitrate (1 mol / L) solution was slowly added dropwise to 40 μL of tetrachloroauric acid trihydrate (1 mol / L) solution, followed by the slow addition of the alkaline β-cyclodextrin solution prepared above, to obtain a mixed solution.

[0029] Add 3 mL of 0.1 mol / L NaHCO3 solution to the mixture to adjust the pH of the system to 8.3. After shaking well, place the mixture in an environment of 80℃ and heat it at a constant temperature for 24 h to obtain the crude product of gold and silver nanoclusters.

[0030] The obtained crude gold and silver nanoclusters were purified three times by centrifugation with ethanol at a volume ratio of 1:3, at a speed of 10,000 rpm for 10 min each time. The precipitate after three purifications was dispersed in ultrapure water to form a crude gold and silver nanoclusters solution with a concentration of 5 mg / mL.

[0031] The crude product solution of the gold and silver nanoclusters was then transferred to a dialysis bag (molecular weight cutoff 3500 Da), and dialyzed for 24 hours with deionized water as the dialysis medium to remove excess unreacted reactants and impurities.

[0032] After dialysis, the gold and silver nanoclusters were collected by centrifugation. A portion of the gold and silver nanoclusters were then re-dispersed using deionization to obtain a 1 mg / mL gold and silver nanocluster solution. This solution, along with the remaining gold and silver nanocluster solids, was placed in a sealed refrigerator at 4°C for later use in subsequent experiments.

[0033] The FT-IR and XPS spectra used solid gold and silver nanoclusters, while the other tests used solutions of gold and silver nanoclusters.

[0034] Example 2: Structural characterization of gold and silver nanoclusters 1. Transmission electron microscopy examination The 1 mg / mL gold and silver nanocluster solution prepared in Example 1 was examined using an HT7700 transmission electron microscope, and high-magnification transmission electron microscopy images were obtained, as shown below. Figure 1 As shown, by Figure 1 It can be seen that the obtained nanocluster particles are nearly spherical, with regular morphology and no obvious agglomeration, and have excellent dispersibility. Combined with the built-in scale of the electron microscope to measure and statistically analyze the particle size, the average particle size of the gold and silver nanocluster is 1.9±0.3nm.

[0035] 2. Ultraviolet absorption spectroscopy and fluorescence emission spectroscopy tests The 1 mg / mL gold and silver nanocluster solution prepared in Example 1 was subjected to UV absorption and fluorescence emission spectroscopy tests, and the results are shown in the figure. Figure 2 The ultraviolet absorption curve shows that the absorption intensity of the material decreases significantly with increasing incident wavelength in the ultraviolet region, and a shoulder peak appears near 300 nm; the fluorescence emission spectrum shows that it has a strong emission peak at 745 nm.

[0036] 3. Electrochemiluminescence spectroscopy test The 1 mg / mL gold and silver nanocluster solution prepared in Example 1 was subjected to electrochemiluminescence spectroscopy, and the results are shown in the figure. Figure 3 , Figure 3 As can be seen, the electrochemiluminescence peak is located at 870 nm; and Figure 2Compared to the fluorescence emission spectrum in the previous material, the electrochemiluminescence signal of this material exhibits a 125 nm redshift.

[0037] 4. FT-IR spectroscopy test To further verify the successful synthesis of gold and silver nanoclusters, FT-IR spectroscopy was performed on the gold and silver nanoclusters prepared in Example 1. The results are shown in [Figure 1]. Figure 4 Compared to free thiol β-cyclodextrin, the gold and silver nanoclusters prepared in this invention exhibit a higher growth rate at 645 cm⁻¹. -1 A new absorption peak characteristic of Au-S bond stretching vibration appeared at the point, which directly proves that Au-S covalent bonds were successfully constructed between thiol β-cyclodextrin and metal clusters, confirming the completion of the preparation of gold and silver nanoclusters.

[0038] 5. XPS spectral testing XPS spectroscopy was performed on the gold and silver nanoclusters prepared in Example 1. The spectra are shown in the figure. Figure 5 ,from Figure 5 It can be seen that the gold and silver nanoclusters are mainly composed of Au, Ag, S, C, N and O. High-resolution XPS of Au was observed at Au4f... 7 / 2 (84.4eV) and Au4f 5 / 2 Two strong peaks are observed at (88.1 eV), indicating the simultaneous presence of Au(I) and Au(0) metallic states within AuNCs. The fitted peaks at 374 and 368 eV in the Ag3d spectrum correspond to the Ag3d phases of Ag(0), respectively. 5 / 2 and Ag3d 3 / 2 Characteristic peaks.

[0039] The above test results show that the present invention successfully prepared gold and silver nanoclusters protected by thiol β-cyclodextrin.

[0040] Example 3: Electrochemical properties of surface-confined gold and silver nanoclusters First, gold-silver nanoclusters / GCE were prepared using the gold-silver nanoclusters and glassy carbon electrode (GCE) prepared in Example 1. The specific preparation method was as follows: 0.05 μm alumina powder was mixed with water to form a paste. A glassy carbon electrode with a diameter of 5 mm was polished until it was mirror-like and free of scratches. After verifying that the peak potential difference was qualified by the potassium ferricyanide system, ultrapure water and anhydrous ethanol were sonicated for 3 min each. Then, 10 μL of the 1 mg / mL gold-silver nanocluster solution prepared in Example 1 was dripped onto the electrode surface and allowed to dry naturally at room temperature to obtain a gold-silver nanocluster modified glassy carbon electrode (gold-silver nanoclusters / GCE).

[0041] Electrochemical performance was tested using a bare glassy carbon electrode (bare GCE) and gold-silver nanoclusters / GCE in a nitrogen-purged, oxygen-de-saturated blank phosphate buffer solution. The results are shown in [Figure number missing]. Figure 6 and Figure 7 .

[0042] Figure 6 As can be seen, compared with bare GCE, gold-silver nanoclusters / GCE exhibit a significant cathodic reaction process in blank phosphate buffer (pH 7.4, concentration 0.1M), with an onset potential of approximately -1.1V and a reduction peak potential of -1.29V. This result indicates that gold-silver nanoclusters can undergo electrochemical reduction to form a negatively charged state after electron injection into the lowest unoccupied molecular orbital (LUMO). During the reverse scan from 0 to +1.8V, an oxidation peak was detected in the system, with an onset potential of approximately +0.45V and a peak response potential of +0.91V, confirming that electrons released from the highest occupied molecular orbital (HOMO) can electrochemically oxidize gold-silver nanoclusters, transforming them into a positively charged state.

[0043] Figure 7 This is a graph of ECL transient signals. The blue line represents the step potential, and the black line represents the ECL transient signal.

[0044] Figure 7 A shows that no electrochemiluminescence signal was detected when the electrode potential was stable at -1.29V. However, a significant ECL response appeared immediately after the potential was switched to +0.91V, indicating that hole injection can effectively trigger annihilation-type electrochemiluminescence.

[0045] When the potential was adjusted back from +0.91V to -1.29V, the luminescent signal decayed rapidly. This indicates that electron injection annihilation of positively charged clusters is insufficient to maintain luminescence, while hole annihilation of negatively charged clusters is more conducive to ECL generation. The luminescence intensity gradually decreased with increasing hole injection duration, retaining 31% of the maximum signal intensity after 1 second. When the potential was switched from +0.91V to -1.29V in the second round, the residual luminescent signal dissipated rapidly within 0.35 seconds, indicating that the stability of the positively charged cluster state is weaker than that of the negatively charged state.

[0046] Repeated step tests between electron injection and hole injection potentials showed that the annihilation luminescence signal exhibited a decaying trend, but it still confirmed that the electrochemical redox reaction could repeatedly generate the luminescent excited state of gold and silver nanoclusters.

[0047] Combination Figure 7 At potential B, no luminescence was observed when the potential was constant at -1.29V. A significant ECL signal appeared after the potential was gradually increased to +0.91V, indicating radiative charge transfer during cluster oxidation. The luminescence intensity gradually decreased with repeated potential cycling. The gold and silver nanoclusters prepared in this invention have a longer negatively charged lifetime, effectively improving the electrochemiluminescence performance of the system. The electrode exhibits dual-pathway electrochemiluminescence characteristics, both cathodic and anodic.

[0048] Figure 6 , 7This invention demonstrates that the gold and silver nanoclusters prepared by this invention can reversibly generate two charged intermediates, positive and negative, through electrochemical oxidation-reduction. The stability of the two intermediates is significantly different. Hole-dominated annihilation-type ECL is achieved by relying on the annihilation of positive and negative charges. The oxidation process is accompanied by radiative charge transfer. The material has both the dual-pathway ECL luminescence characteristics of anode and cathode and the repeatable photoelectric conversion performance.

[0049] Example 4: Fabrication of an electrochemiluminescence-electrochemical dual-mode sensor: 1. Probe preparation: The cDNA of 5′-SH-(CH2)6-TTTTCTACC GACTCGC-Biotin-3′, which is modified with a thiol group at the 5′ end and biotin at the 3′ end, was added to streptavidin-modified magnetic beads and incubated. The biotin in the cDNA interacted with streptavidin at 37°C for 1 h to form cDNA-modified magnetic beads (MB / cDNA). 10 mg / mL MB / cDNA was prepared using 0.01 mol / L PBS buffer (pH 7.4).

[0050] Then, 20 μL of MB / cDNA at a concentration of 10 mg / mL was dispersed in 200 μL of ferrocene-modified chloramphenicol aptamer solution with the sequence 5′-Fc-dT10-TTAGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCAC GGTCGGCGAGTCGGTG-3′ at a concentration of 1 μmol / L. DNA hybridization was performed at 37 °C for 30 min to form MB / dsDNA probe. A 1 μM MB / dsDNA probe solution was prepared using 0.01 mol / L PBS buffer (pH 7.4).

[0051] The nucleotide sequences of the above cDNA and chloramphenicol aptamer are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0052] 2. The electrode drop-coating process is as follows: First, 0.05 μm alumina powder was mixed with water to form a paste, which was then used to polish a 5 mm diameter glassy carbon electrode until it was smooth and free of scratches. After verifying the peak potential difference using a potassium ferricyanide system, the electrode was sonicated for 3 min each with ultrapure water and anhydrous ethanol. 10 μL of a 1 mg / mL aqueous solution of mercapto-β-cyclodextrin-stabilized gold and silver nanoclusters was then dropped onto the electrode surface and allowed to air dry at room temperature to obtain a gold and silver nanocluster-modified glassy carbon electrode (gold and silver nanocluster / GCE electrode).

[0053] Example 5: Application of the above-mentioned electrochemiluminescence-electrochemical dual-mode sensor in the detection of chloramphenicol. 1. ECL and DPV Measurement After incubating 10 μL of the above-mentioned MB / dsDNA probe solution (1 μM) with 90 μL of the test solution containing chloramphenicol (chloramphenicol concentrations of 0.0005, 0.001, 0.005, 0.01, 0.1, 1, 10, and 100 ng / mL, respectively) at 37°C for 2 h, and then magnetically separating the solution, 10 μL of the supernatant was added to the above-mentioned gold-silver nanoclusters / GCE electrode for a second incubation at 37°C for 50 min. ECL and DPV measurements were then performed.

[0054] The above-mentioned chloramphenicol-containing test solution was prepared using 0.01 mol / L PBS buffer (pH 7.4).

[0055] The ECL measurement was performed in a phosphate buffer solution containing 0.1 mol / L triethylamine (concentration 0.1 mol / L, pH 7.4); the DPV test was performed in a phosphate buffer solution containing 0.1 mol / L potassium chloride (concentration 0.1 mol / L, pH 7.4).

[0056] Test conditions for ECL: ECL scan range is 0-1.4V, scan speed is 0.05V / s, photomultiplier tube voltage is 500V; Test conditions for DPV: scan range is 0-0.6V, scan speed is 0.05V / s.

[0057] The following tests involving ECL and DPV shall all use the test methods, test environment and test conditions described above.

[0058] cDNA assembles onto streptavidin-modified magnetic beads (MBs) via a specific streptavidin-biotin interaction, and then hybridizes with Fc-CAP-aptamer to form a dsDNA probe containing a specific recognition sequence for chloramphenicol. In the presence of the target chloramphenicol, the MB / dsDNA probe releases Fc-CAP-aptamer. After magnetic separation, thiol β-cyclodextrin can recognize ferrocene (Fc) released from the MB via guest-host interaction, causing Fc to quench ECL emission and increase DPV signal.

[0059] Figure 8 The figures show the electrochemiluminescence response curves corresponding to the ECL sensor assembly process. Data shows that the bare GCE did not exhibit a significant electrochemiluminescence signal (curve a), while the ECL intensity was significantly enhanced after GCE was modified with gold and silver nanoclusters (curve b). When the supernatant from the reaction with chloramphenicol was incubated with gold and silver nanoclusters / GCE, the ECL signal of the resulting Fc / gold and silver nanoclusters / GCE showed a significant decrease compared to that of the gold and silver nanoclusters / GCE (curve c). These dynamic changes in the ECL signal perfectly match the assembly and reaction process of the biosensor, confirming the successful fabrication of the target biosensor.

[0060] Figure 9 The figures show the DPV curves corresponding to the assembly process of the electrochemical sensor. Data shows that no DPV response was observed on the bare GCE (curve a) because there was no electrochemically active material on this electrode. Similarly, the DPV signal of the gold-silver nanocluster modified GCE was almost zero in the 0-0.6V range (curve b). When the supernatant containing Fc-CAP-aptamer was incubated with gold-silver nanoclusters / GCE / GCE, a higher DPV response was generated due to the presence of electroactive Fc (curve c). Fc inserts into the lumen of the thiol β-cyclopaste through host-guest interactions. As an electroactive molecule, Fc is an effective medium for electron transfer in electrochemical measurements and an ideal guest for forming the complex. These results demonstrate the successful fabrication of the biosensor.

[0061] 2. Electrochemiluminescence response test of the electrochemiluminescence-electrochemical dual-mode sensor prepared in this invention to different concentrations of chloramphenicol. The electrochemiluminescence response curves of different concentrations of chloramphenicol (0.0005, 0.001, 0.005, 0.01, 0.1, 1, 10, and 100 ng / mL) were detected using the above method, as shown in the figure. Figure 10-13 .

[0062] Figure 10 The image shows the electrochemiluminescence response curves of the ECL sensor for chloramphenicol in the concentration range of 0.0005-100 ng / mL. Figure 10 It can be seen that within the concentration range of 0.0005 ng / mL to 100 ng / mL, the response signal of the ECL sensor gradually weakens as the concentration of chloramphenicol gradually increases.

[0063] Figure 11 Linear calibration curves of the ECL sensor for chloramphenicol in the range of 0.0005-100 ng / mL are shown below. Figure 11 As shown, the ECL signal exhibits a very strong linear correlation with the logarithm of chloramphenicol concentration, and the corresponding linear regression equation is I. ECL =0.4871–0.1433lgC(R) 2 =0.9933), the calculated detection limit (LOD) is 1.5 × 10⁻⁶. -4 The obtained biosensor, at ng / mL, enables sensitive detection of chloramphenicol.

[0064] Figure 12 The DPV response curves of the electrochemical sensor to chloramphenicol in the range of 0.0005-100 ng / mL are shown below. Figure 12As shown, from 0.0005 ng / mL to 100 ng / mL, with the increase of chloramphenicol concentration, the electroactive substance Fc on the electrode surface gradually increases, and therefore the DPV signal gradually increases.

[0065] Figure 13 Linear calibration curves of the electrochemical sensor for chloramphenicol in the concentration range of 0.0005-100 ng / mL are shown. Data indicates a good linear correlation between the sensor's DPV signal and the logarithm of the chloramphenicol concentration, with the corresponding linear regression equation being I. DPV =24.0610+5.7530lgC(R 2 =0.9965). The calculated LOD of this sensor is 5.8 × 10⁻⁶. -4 The result of ng / mL indicates that the constructed biosensor can achieve highly sensitive detection of chloramphenicol.

[0066] 3. Selective testing Interference group: Tetracycline, streptomycin, oxytetracycline, gentamicin, kanamycin, and nemoxicillin were each prepared into a 10 ng / mL solution using 0.01 mol / L PBS buffer (pH 7.4); Chloramphenicol group: Prepared into a 1 ng / mL solution using 0.01 mol / L PBS buffer (pH 7.4).

[0067] Control group: 0.01 mol / L PBS buffer (pH 7.4) Mixed group: The above 6 interfering substances and chloramphenicol were mixed in 0.01 mol / L PBS buffer (pH 7.4). The final concentration of chloramphenicol in the mixture was 1 ng / mL, and the final concentration of each interfering substance was 10 ng / mL.

[0068] The test method is as follows: 10 μL of the above MB / dsDNA probe solution (1 μM) and 10 μL of the test solution are incubated at 37°C for 2 h for the first time. After magnetic separation, 10 μL of the supernatant is added to the above gold and silver nanoclusters / GCE electrode for a second incubation of 50 min. ECL and DPV are then measured respectively.

[0069] Figure 14 The results presented show the selectivity test results of the ECL sensor. The test data indicate that the ECL intensity decrease is minimal for interfering substances and blank samples, while the target analyte chloramphenicol causes a significant decrease in ECL intensity. Furthermore, the response signal of the ECL sensor to chloramphenicol alone exhibits good consistency with the response signal in the mixture of chloramphenicol and potential interfering substances, confirming the excellent selectivity of this biosensor.

[0070] Figure 15The results presented show the selectivity test results of the DPV sensor. The test data indicate that the DPV intensity increases minimally with the interfering substances and the blank sample, while the target analyte chloramphenicol causes a significant increase in DPV intensity. Furthermore, the response signal of the DPV sensor to chloramphenicol alone exhibits good consistency with the response signal in the mixture of chloramphenicol and potential interfering substances, further confirming the excellent selectivity of this biosensor.

[0071] 4. Stability Test Figure 16 The results presented show the stability test results of the constructed ECL sensor. The test data shows that when the sensor performs 10 consecutive potential step operations within the 0-1V potential range, the relative standard deviation (RSD) for detecting 0.0005 ng / mL chloramphenicol is 3.86%, which indicates that the ECL sensor has excellent operational stability.

[0072] Figure 17 The results presented show the stability test results of the constructed DPV sensor. The test data shows that when the sensor continuously performs 12 cycles of potential testing within the potential range of 0-0.6V, the relative standard deviation (RSD) for the detection of 10ng / mL chloramphenicol is 3.16%, which further demonstrates that the DPV sensor has excellent operational stability.

[0073] 5. Repeatability testing Figure 18 The repeatability test results of the constructed ECL sensor were presented, and the reproducibility performance of the sensor was evaluated by detecting a 0.1 ng / mL chloramphenicol standard solution. Data showed that the response signals of the seven parallel prepared electrodes were not significantly different under the same detection conditions (consistent with the detection conditions described above), with an RSD of only 3.02% at a concentration of 0.1 ng / mL. This result indicates that the biosensor designed in this invention possesses excellent reproducibility.

[0074] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing gold and silver nanoclusters, characterized in that: The preparation method involves adding a silver source solution to a gold source solution, then adding an alkaline thiol β-cyclodextrin solution to obtain a mixed solution. Sodium bicarbonate is added to adjust the pH to 8.2-8.4, and then incubation is performed to obtain a crude product of gold and silver nanoclusters. The crude product of gold and silver nanoclusters is purified by centrifugation and dialyzed to obtain gold and silver nanoclusters.

2. The method for preparing gold and silver nanoclusters according to claim 1, characterized in that: The silver source is silver nitrate, and the gold source is tetrachloroauric acid trihydrate; the pH of the alkaline mercapto-β-cyclodextrin solution is 8.0-8.

4.

3. The method for preparing gold and silver nanoclusters according to claim 1, characterized in that: The molar ratio of the mercapto-β-cyclodextrin to the silver source and the gold source is 5.3-5.5:0.9-1.1:4.8-5.

2.

4. The method for preparing gold and silver nanoclusters according to claim 1, characterized in that: The incubation temperature is 78-82℃, and the incubation time is 23-25h.

5. The method for preparing gold and silver nanoclusters according to claim 1, characterized in that: The alkaline thiol β-cyclodextrin solution is prepared by dissolving thiol β-cyclodextrin in sodium bicarbonate solution.

6. A gold-silver nanocluster prepared by the method according to any one of claims 1-5.

7. An electrochemiluminescence-electrochemical dual-mode sensor comprising the gold and silver nanoclusters of claim 6, characterized in that: The sensor includes an MB / dsDNA probe and a gold-silver nanocluster / GCE electrode. The gold and silver nanocluster / GCE electrode is prepared by drop-coating the gold and silver nanocluster solution of claim 6 onto the surface of the GCE electrode. The MB / dsDNA probe includes: dsDNA and magnetic beads; The dsDNA is obtained by hybridization of the capture probe cDNA and the chloramphenicol aptamer Fc-CAP-aptamer modified with its complementary strand ferrocene; the biotin of the capture probe cDNA is linked to streptavidin-modified magnetic beads; the nucleotide sequence of the cDNA is shown in SEQ ID No. 1, and the nucleotide sequence of the chloramphenicol aptamer is shown in SEQ ID No.

2.

8. The electrochemiluminescence-electrochemical dual-mode sensor according to claim 7, characterized in that: The hybridization temperature is 35-38℃, and the hybridization time is 25-35 min; In the preparation of the gold-silver nanocluster / GCE electrode, the concentration of the gold-silver nanocluster solution is 0.8-1.2 mg / mL; the drop volume of the gold-silver nanocluster solution is 8-12 μL.

9. The application of the electrochemiluminescence-electrochemical dual-mode sensor according to claim 7 in the detection of chloramphenicol, characterized in that: The process includes the following steps: the MB / dsDNA probe is incubated with the test solution containing chloramphenicol for the first time. After magnetic separation, the supernatant is added to the gold-silver nanocluster / GCE electrode for the second incubation. Then, ECL and DPV measurements are performed respectively.

10. The application of the electrochemiluminescence-electrochemical dual-mode sensor according to claim 9 in the detection of chloramphenicol, characterized in that: The first incubation is carried out at a temperature of 35-38℃ for 1.8-2.2 hours; the second incubation is carried out at a temperature of 35-38℃ for 45-55 minutes.

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