Electrochemical sensor based on aptamer and bacteriophage dual recognition and application thereof

Abstract

The invention discloses an electrochemical sensor based on aptamer and bacteriophage dual recognition and application of the electrochemical sensor, and belongs to the technical field of food safety detection. The electrochemical sensor provided by the invention comprises a gold electrode modified by a DNA regular tetrahedron nanostructure and a calcined zeolite imidazate framework material anchored with phage and loaded with methylene blue, the DNA regular tetrahedron nanostructure comprises four DNA probes, and the four DNA probes are a chain A, a chain B, a chain C and a chain D; the bacteriophage is a salmonella typhimurium bacteriophage. The electrochemical sensor can perform dual recognition based on the aptamer and the bacteriophage, realizes high sensitivity, high specificity and rapid detection of salmonella typhimurium through the synergistic effect of all functional components, has biosafety prevention and control capability, and can meet the detection requirements of trace pathogenic bacteria in food.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to an electrochemical sensor based on dual recognition of aptamers and bacteriophages and its applications. Background Technology

[0002] Foodborne illnesses have always been a significant threat to human health, with those caused by Salmonella Typhimurium being particularly common. These illnesses primarily occur in poultry, eggs, and egg products. The pathogenic island-encoded T3SS complex protein induces an inflammatory response, disrupting the balance of the gut microbiota, thereby gaining a competitive advantage and colonizing the organism. Infection leads to symptoms such as acute diarrhea, fever, abdominal pain, and vomiting, and in severe cases, can be life-threatening. Therefore, highly sensitive detection of this pathogen is urgently needed.

[0003] Traditional plate culture remains the gold standard for Salmonella identification; however, this method is time-consuming and labor-intensive, and can no longer meet the current needs for rapid food safety screening. In recent years, immunological and molecular biological detection methods, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), have improved detection efficiency. However, ELISA typically relies on multiple incubation and washing steps, making the process cumbersome, and its sensitivity is limited, hindering rapid screening. While PCR technology offers higher sensitivity, its reliance on sophisticated thermal cycling equipment and complex sample pretreatment limits its application in on-site testing. Neither method can simultaneously meet the practical requirements of high sensitivity and on-site detection. In contrast, electrochemical biosensing technology, especially based on screen-printed electrodes (SPE), offers high sensitivity, ease of operation, and excellent portability, making it more suitable for low-concentration target analyte detection and rapid on-site screening scenarios.

[0004] Aptamers, as high-affinity and high-specificity oligonucleotide recognition elements, have gained increasing popularity in recent years as efficient recognition probes in SPE systems due to their structural stability, ease of modification, and low synthesis cost. However, the weak specific signals generated by trace pathogens are easily drowned out by background noise, posing a significant challenge to their reliable detection in food matrices. Introducing effective signal amplification mechanisms is crucial to improving the performance of electrochemical sensors in the detection of trace pathogens. While there are reports on signal amplification related to aptamer electrochemical systems, these methods are limited by detection time and sensitivity, failing to meet the current demand for rapid and highly sensitive detection.

[0005] Signal amplification via loading signal molecules is a relatively simple and time-efficient method to improve sensitivity. However, methods that directly couple electrochemically active labels to aptamers or antibodies have limited signal unit loading capacity and suffer from significant background interference, restricting further improvements in detection sensitivity. While zeolite imidazole ester frameworks (ZIFs) are ideal platforms for signal molecule loading and aptamer anchoring due to their high specific surface area, modifiability, and easy recyclability, most aptamer sensors typically require terminal modification with thiol or amino groups for covalent coupling with ZIFs. This process is not only cumbersome but can also affect the conformation and recognition activity of the aptamer, causing considerable inconvenience for the detection of trace amounts of Salmonella typhimurium. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an electrochemical sensor based on dual recognition of aptamers and bacteriophages, and its applications. This invention constructs an "aptamer..." target The "phage sandwich" structure enables dual recognition, and the electrochemical signal is amplified by carrying QZIF-67 loaded with methylene blue on the phage. At the same time, the lysis of the phage after recognition effectively inhibits the spread of pathogens in the waste being tested. This achieves the integration of highly sensitive detection and biosafety control of Salmonella typhimurium, providing a reliable means for on-site screening and post-event prevention of foodborne pathogens.

[0007] The technical solution of the present invention is as follows: The first aspect of this invention protects an electrochemical sensor based on dual recognition of aptamers and bacteriophages, the electrochemical sensor comprising a gold electrode modified with a DNA tetrahedral nanostructure and a calcined zeolite imidazole ester framework material anchored to bacteriophages and loaded with methylene blue. The DNA tetrahedral nanostructure includes four DNA probes, namely, strands A, B, C, and D; the nucleotide sequence of strand A is shown in SEQ ID No. 1; the nucleotide sequence of strand B is shown in SEQ ID No. 2; the nucleotide sequence of strand C is shown in SEQ ID No. 3; the nucleotide sequence of strand D is shown in SEQ ID No. 4; and the bacteriophage is a Salmonella typhimurium bacteriophage.

[0008] A second aspect of this invention protects a method for preparing the electrochemical sensor described in the first aspect, the method comprising the following steps: (1) Preparation of gold electrodes modified with DNA tetrahedral nanostructures: S1-1: Mix the A-chain solution, B-chain solution, C-chain solution, D-chain solution with TCEP solution, add TM buffer solution, and incubate at room temperature for 1-2 h; after high temperature treatment and rapid ice bath, obtain TDNs solution, which is the DNA tetrahedral nanostructure. S1-2: The TDNs solution was dropped onto the surface of the screen-printed electrode modified with gold nanoparticles and incubated at room temperature for 8-16 h to obtain the gold electrode TDNs / AuNPs / SPE modified with the DNA tetrahedral nanostructure. (2) Preparation of calcined zeolite imidazole ester framework material anchored to bacteriophage and loaded with methylene blue: S2-1: Mix a methanol solution of Co(NO3)2·6H2O with a methanol solution of 2-methylimidazole, stir at room temperature, let the reaction stand, collect the precipitate by centrifugation, wash with methanol and dry under vacuum to obtain ZIF-67 material. S2-2: ZIF-67 material was placed in a tube furnace and calcined in air atmosphere to obtain calcined zeolite imidazole ester framework material QZIF-67; then methylene blue and QZIF-67 were dispersed in deionized water and allowed to stand at room temperature for 24 h to load methylene blue onto QZIF-67; after centrifugation, precipitate collection, washing with deionized water, and vacuum drying, QZIF-67@MB composite material was obtained. S2-3: Incubate the QZIF-67@MB composite material with Salmonella Typhimurium phage stock solution at room temperature with shaking for 40-60 min, then centrifuge to collect the precipitate. After washing the precipitate with PBS buffer solution, redisperse the precipitate in PBS buffer solution to obtain Phage / QZIF-67@MB solution, which is the calcined zeolite imidazole ester framework material anchored to phage and loaded with methylene blue.

[0009] Preferably, in step S1-1, The concentrations of the A-chain solution, B-chain solution, C-chain solution, and D-chain solution are all 50–250 μmol / L; the concentration of the TCEP solution is 1–5 mmol / L. The volume ratio of the A-chain solution, B-chain solution, C-chain solution, D-chain solution, TCEP solution, and TM buffer solution is 1:1:1:1:10:86. The high-temperature treatment is performed at a temperature of 93-98°C for 5-10 minutes; the rapid ice bath is performed at a temperature of 3-5°C for 5-10 minutes.

[0010] Preferably, in steps S1-2, the concentration of the TDNs solution is 0.5~2.5 μmol / L and the volume is 25~35µL.

[0011] Preferably, in step S2-1, The concentration of the methanol solution of Co(NO3)2·6H2O is 0.017~0.020 g / mL; The concentration of the 2-methylimidazole methanol solution is 0.018~0.022 g / mL; The mass ratio of Co(NO3)2·6H2O to 2-methylimidazole is 1:(0.9~1.29). The stirring time is 55-65 seconds; The static reaction time is 22-26 h; The centrifugation speed is 10000~12000 rpm, and the time is 8~10 min; The vacuum drying temperature is 55~65 ℃, and the time is 12~14 h.

[0012] Preferably, in step S2-2, The calcination temperature is 250~300 ℃, and the time is 1~3 h; The mass ratio of methylene blue to QZIF-67 is 1:2; the concentration of methylene blue in deionized water is 0.5~1.5 mg / mL; The centrifugation speed is 10000~12000 rpm, and the time is 8~10 min; The vacuum drying temperature is 55~65 ℃, and the time is 12~14 h.

[0013] Preferably, in steps S2-3, The concentration of the Salmonella typhimurium bacteriophage stock solution was 10. 9 PFU / mL; The mass-to-volume ratio of the QZIF-67@MB composite material to the Salmonella typhimurium bacteriophage stock solution is 2 mg: (0.5~1.5) mL; The centrifugation speed is 5000~6000 rpm, and the time is 5~8 min; The concentration of the PBS buffer is 10 mmol / L, pH=7.4; The mass-to-volume ratio of the QZIF-67@MB composite material to PBS buffer is 2 mg: (0.5~1.5) mL.

[0014] The third aspect of this invention protects the application of an electrochemical sensor described in the first aspect or prepared by the preparation method described in the second aspect, wherein the electrochemical sensor is used to detect Salmonella typhimurium.

[0015] Preferably, the method for detecting Salmonella typhimurium using the electrochemical sensor includes the following steps: (1) Add 30 µL of the bacterial suspension to be tested to the surface of the TDNs / AuNPs / SPE electrode, incubate at 25 °C for 20~60 min, then rinse the electrode surface with deionized water and dry it; (2) Add 30 µL of the Phage / QZIF-67@MB solution (3 mg / mL) to the electrode surface, incubate at 25 °C for 3~20 min, rinse the electrode with deionized water again and dry it; (3) Immerse the dried electrode from step (2) in 10 mM PBS buffer solution with pH=7.4 and detect it by square wave voltammetry with a potential range of 0 V to -0.5 V and a pulse amplitude of 25 mV. (4) Calculate the concentration of Salmonella typhimurium in the sample based on the standard curve.

[0016] Preferably, in step (4), the standard curve is: ΔI = 43.57536 * log(C) - 24.4291, R² = 0.9938, where I represents the net peak current value after background subtraction, and C represents the concentration of Salmonella typhimurium.

[0017] The beneficial technical effects of this invention are as follows: This invention innovatively constructs a dual-recognition electrochemical biosensor based on aptamers and bacteriophages. Through the synergistic effect of various functional components, it achieves highly sensitive, specific, and rapid detection of Salmonella Typhimurium, while also possessing biosafety control capabilities. The method exhibits good linearity within a concentration range of 5–500 CFU / mL. I = 43.57536 * log(C) - 24.4291, R² = 0.9938), with a detection limit as low as 2.6 CFU / mL (calculated using 3σ / K, where σ represents the standard deviation of the blank group results and K represents the slope of the fitted curve), meeting the detection requirements for trace pathogenic bacteria in food.

[0018] This invention utilizes a DNA tetrahedral nanostructure as a scaffold to spatially fix a Salmonella typhimurium-specific aptamer. This three-dimensional rigid structure effectively avoids the disordered accumulation of aptamers on the electrode surface, significantly improving their binding efficiency and capture ability with the target bacteria, and laying the foundation for the subsequent formation of a stable "aptamer-bacteria-phage" sandwich detection structure.

[0019] This invention ingeniously employs calcined zeolite imidazole ester framework material (QZIF-67) as a highly efficient carrier for the signal molecule methylene blue (MB). The calcination process increases the specific surface area and loading capacity of the material, thereby achieving high-density loading of the signal molecule. More importantly, negatively charged Salmonella typhimurium-specific bacteriophages are directionally immobilized onto the positively charged QZIF-67@MB composite material through electrostatic adsorption, forming a signal probe. This method eliminates the need for complex chemical modifications to the recognition element, simplifying the probe preparation process. When the target bacterium is present, the aptamer on the electrode surface first captures it, and then the bacteriophage signal probe binds to the bacteria through its specific recognition, forming a "aptamer-bacteria-phage" sandwich complex. This process draws a large number of methylene blue molecules closer to the electrode surface, thereby generating a significantly enhanced electrochemical signal. This dual recognition mechanism not only improves the specificity of detection but also effectively amplifies the signal through the signal probe, giving the sensor ultra-high sensitivity.

[0020] The phage recognition element used in this invention, after identifying the target bacteria, can further exert its inherent lytic activity to effectively inactivate the captured bacteria. This characteristic significantly reduces the biosafety risks caused by live bacteria residue during the detection process, providing built-in safety assurance for rapid on-site detection. Through the synergistic function of each component, it overcomes the problems of insufficient sensitivity, cumbersome operation, or potential biosafety hazards in existing detection technologies, providing an efficient and integrated solution for the rapid and safe detection of trace pathogens in food. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the principle of the electrochemical sensor based on dual recognition of aptamers and bacteriophages used in this invention for detecting Salmonella typhimurium.

[0022] Figure 2 The electrophoresis results are shown for the electrochemical sensor in Example 1 of this invention.

[0023] Figure 3 The image shows the CV diagrams of electrodes with different modifications prepared according to the present invention.

[0024] Figure 4 EIS images of electrodes with different modifications prepared according to the present invention.

[0025] Figure 5 The image shows a transmission electron microscope (TEM) image of bacteriophage GB1-3 obtained in Example 2 of this invention.

[0026] Figure 6 Structural characterization diagrams of ZIF-67, QZIF-67, and QZIF-67@MB prepared in this invention; In the figure: (A) is a scanning electron microscope image of ZIF-67 prepared in Example 3; (B) is a scanning electron microscope image of QZIF-67 prepared in Example 3; (C) is a scanning electron microscope image of QZIF-67@MB prepared in Example 3.

[0027] Figure 7 The FT-TR spectra of ZIF-67 and QZIF-67 obtained in Example 3 of this invention are shown.

[0028] Figure 8 X-ray diffraction patterns of ZIF-67 and QZIF-67 obtained in Example 3.

[0029] Figure 9 The UV-Vis absorption spectra of MB, ZIF-67@MB and QZIF-67@MB prepared in Example 3 are shown.

[0030] Figure 10 The image shows the optimization results of TDNs concentration in the electrochemical sensor prepared according to this invention; the background-corrected data are normalized with the maximum value as a reference and expressed as a percentage.

[0031] Figure 11 The figure shows the optimized binding time of target bacteria and TDNs in the electrochemical sensor prepared in this invention; the background-corrected data are normalized with the maximum value as a reference and expressed as a percentage.

[0032] Figure 12 The image shows the optimized concentration results of the Phage / QZIF-67@MB signal probe; the background-corrected data are normalized with the maximum value as a reference and expressed as a percentage.

[0033] Figure 13 The image shows the optimized binding time between the signal probe (Phage / QZIF-67@MB) and the target bacteria; the background-corrected data were normalized with the maximum value as a reference and expressed as a percentage.

[0034] Figure 14 This is the SWV response diagram of the electrochemical sensor in Example 4 of the present invention to different concentrations of target.

[0035] Figure 15 This is a standard curve of the electrochemical sensor in Example 4 of the present invention for the relationship between the SWV peak current value and the concentration of Salmonella typhimurium.

[0036] Figure 16 The variation of SWV peak current of the electrochemical sensor at different times in Example 4 of this invention.

[0037] Figure 17 This is the electrochemical response of the electrochemical sensor to different types of bacteria in Example 4 of the present invention.

[0038] Figure 18 This refers to the bactericidal ability of the bacteriophage in the electrochemical sensor of Example 4 of the present invention against Salmonella typhimurium. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] An electrochemical sensor based on dual recognition of aptamers and bacteriophages, the electrochemical sensor comprising a gold electrode modified with a DNA tetrahedral nanostructure and a calcined zeolite imidazole ester framework material anchored to bacteriophages and loaded with methylene blue; The DNA tetrahedral nanostructure includes four DNA probes, namely, strands A, B, C, and D; the nucleotide sequence of strand A is shown in SEQ ID No. 1; the nucleotide sequence of strand B is shown in SEQ ID No. 2; the nucleotide sequence of strand C is shown in SEQ ID No. 3; the nucleotide sequence of strand D is shown in SEQ ID No. 4; and the bacteriophage is a Salmonella typhimurium bacteriophage.

[0041] The Salmonella typhimurium bacteriophage of this invention is named vB_Sal_YZCM1 and is classified as Salmonella typhimurium bacteriophage. Salmonella phage It was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 47002.

[0042] Table 1: Nucleotide sequences in the following examples

[0043] Note: Underlined sequences indicate characteristic sequences for identifying Salmonella typhimurium.

[0044] The underlined portion of strand D is the specific recognition region for Salmonella typhimurium. Strains A, B, C, and the ununderlined portions of strand D form part of the DNA tetrahedral (TDN) backbone. The SH-(CH2)6- modified at the 5' end of strands A, B, and C can form Au-S bonds with the gold electrode, thus immobilizing them on the electrode. The tetrahedral design of DNA prevents uneven distribution and entanglement caused by disordered single-strand immobilization, ensuring binding efficiency.

[0045] The fabrication method of the electrochemical sensor based on dual recognition of aptamers and bacteriophages includes the following steps: (1) Preparation of gold electrodes modified with DNA tetrahedral nanostructures: S1-1: Mix the A-chain solution, B-chain solution, C-chain solution, D-chain solution with TCEP solution, add TM buffer solution, and incubate at room temperature for 1-2 h; after high temperature treatment and rapid ice bath, obtain TDNs solution, which is the DNA tetrahedral nanostructure. S1-2: The TDNs solution was dropped onto the surface of the screen-printed electrode modified with gold nanoparticles and incubated at room temperature for 8-16 h to obtain the gold electrode TDNs / AuNPs / SPE modified with the DNA tetrahedral nanostructure. (2) Preparation of calcined zeolite imidazole ester framework material anchored to bacteriophage and loaded with methylene blue: S2-1: Mix a methanol solution of Co(NO3)2·6H2O with a methanol solution of 2-methylimidazole, stir at room temperature, let the reaction stand, collect the precipitate by centrifugation, wash with methanol and dry under vacuum to obtain ZIF-67 material. S2-2: ZIF-67 material was placed in a tube furnace and calcined in air atmosphere to obtain calcined zeolite imidazole ester framework material QZIF-67; then methylene blue and QZIF-67 were dispersed in deionized water and allowed to stand at room temperature for 24 h to load methylene blue onto QZIF-67; after centrifugation, precipitate collection, washing with deionized water, and vacuum drying, QZIF-67@MB composite material was obtained. S2-3: Incubate the QZIF-67@MB composite material with Salmonella Typhimurium phage stock solution at room temperature with shaking for 40-60 min, then centrifuge to collect the precipitate. After washing the precipitate with PBS buffer solution, redisperse the precipitate in PBS buffer solution to obtain Phage / QZIF-67@MB solution, which is the calcined zeolite imidazole ester framework material anchored to phage and loaded with methylene blue.

[0046] In some embodiments, in step S1-1, the TM buffer is prepared by weighing 6.057 g of Tris base and 2.033 g of magnesium chloride at room temperature, dissolving them in about 800 mL of ultrapure water, stirring thoroughly to dissolve them, slowly adding concentrated hydrochloric acid to adjust the pH to 7.4, and finally bringing the volume to 1 L with ultrapure water.

[0047] In steps S2-3, the mass-to-volume ratio of the QZIF-67@MB composite material to the PBS buffer is 2 mg:(0.5~1.5) mL. It should be understood that the PBS buffer here refers to the PBS buffer in which the precipitate is redispersed in the PBS buffer solution.

[0048] The Salmonella Typhimurium used in this invention was purchased from Shanghai Preservation Microbiology Co., Ltd., with accession number SMHCC D17213.

[0049] The screen-printed electrode (SPE) used in this invention is a gold nanoparticle-modified screen-printed electrode (AuNPs / SPE), which was purchased from Zhejiang Nazhihui Biotechnology Co., Ltd.

[0050] The LB liquid culture medium used in the following embodiments and comparative examples of the present invention was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; the LB semi-solid culture medium was prepared by adding 0.4% agarose to the LB liquid culture medium.

[0051] Figure 1 This diagram illustrates the fabrication process and detection principle of the electrochemical sensor based on dual aptamer and phage recognition, as described in this invention. The basic process involves the self-assembly of thiol-modified DNA tetrahedral nanostructures onto the surface of AuNPs / SPEs via Au-S bonds. Positively charged QZIF-67 is first loaded with methylene blue (MB) to form a QZIF-67@MB composite material. The negatively charged phage head binds to QZIF-67@MB through electrostatic interactions, forming a signal amplification probe. In the presence of Salmonella typhimurium, the target bacterium first binds to the aptamer immobilized on the electrode surface. Subsequently, the QZIF-67@MB-phage complex (Phage / QZIF-67@MB) is added, forming an "aptamer-target bacterium-phage" sandwich structure through specific phage binding, generating an electrochemical signal. Increased Salmonella typhimurium concentration leads to increased MB accumulation on the electrode surface, thereby enhancing the electrochemical signal intensity. By detecting changes in the characteristic oxidation peak current of the bacteriophage (MB), highly sensitive detection of Salmonella typhimurium was achieved. Furthermore, the inherent lytic ability of bacteriophages can inactivate Salmonella typhimurium, thereby achieving the goal of prevention and control.

[0052] Example 1: Preparation of gold electrodes modified with DNA tetrahedral nanostructures (TDNs / AuNPs / SPE) (1) Prepare solutions of four DNA strands with concentrations of 100 μmol / L in sequence (A strand: sequence as shown in SEQ ID No.1; B strand: sequence as shown in SEQ ID No.2; C strand: sequence as shown in SEQ ID No.3; D strand: sequence as shown in SEQ ID No.4). Then mix the prepared A strand solution, B strand solution, C strand solution, D strand solution, TCEP solution with a concentration of 3 mmol / L and TM buffer solution at a volume ratio of 1:1:1:1:10:86 and incubate at 25 °C for 1 h to obtain a mixed solution.

[0053] (2) Place the mixture obtained in step (1) in a PCR instrument and react at 95 °C for 10 min, then reduce the temperature to 4 °C and react for 10 min to complete the annealing assembly of the DNA tetrahedral nanostructure and obtain the TDNs solution.

[0054] The obtained TDNs solution was subjected to polyacrylamide gel electrophoresis (PAGE), and the electrophoresis pattern is shown below. Figure 2 As shown, lane M is the DNA Marker, lane 1 is the A chain, lane 2 is the AB chain, lane 3 is the ABC chain, and lane 4 is the ABCD chain. Lanes 1-4 correspond to the four assembly steps. The tetrahedral structure is in lane 4. Due to its larger biomolecule size and more complex three-dimensional structure, its migration rate is the slowest. The electrophoresis results indicate that the tetrahedral structure was successfully synthesized.

[0055] (3) 30 µL of the 1 μM TDNs solution prepared in step (2) was dropped onto the surface of the screen-printed electrode (AuNPs / SPE) modified with nano-gold and incubated at 25 °C for 12 h to fix the TDNs onto the electrode surface through the thiol groups at the apex (thiol groups on the A chain, B chain, and C chain) to obtain the TDNs / AuNPs / SPE electrode.

[0056] The AuNPs / SPE electrode and the TDNs / AuNPs / SPE electrode were placed in a 5 mmol / L [Fe(CN)6] solution. 3- / 4- The CV characteristic curves and electrochemical impedance spectroscopy were detected in 0.1 mol / L KCl, and the results are as follows: Figure 3 and Figure 4 As shown. From Figure 3 and Figure 4 As can be seen, compared with AuNPs / SPEs (black line), a significant decrease in peak CV current and charge transfer resistance (R) is observed on TDNs / AuNPs / SPEs (red line). ct The electron transfer between the redox probe and the electrode was significantly hindered, indicating successful fixation of TDNs.

[0057] Example 2: Preparation of Salmonella Typhimurium bacteriophage stock solution (1) Sample pretreatment: The mixed sample solution of chicken anal swabs collected from a market in Yangzhou and 10 mL of sterile PBS buffer (pH=7.4, 0.01 mol / L) was centrifuged at 12000 rpm and 4 ℃ for 10 min. The supernatant was collected and filtered with a 0.22 µm microporous membrane for sterilization.

[0058] (2) Phage enrichment: Mix 5 mL LB liquid medium, 1 mL of the filtrate obtained in step (1) with 50 µL of Salmonella Typhimurium suspension in the logarithmic growth phase, and incubate at 37 ℃ and 80 rpm for 18 h. Then centrifuge at 12000 rpm and 4 ℃ for 2 min. Filter the supernatant through a 0.22 µm microporous membrane to remove bacteria and obtain phage enrichment solution.

[0059] (3) Plaque identification: Mix 5 mL of warm LB semi-solid medium containing 0.4% agar with 350 µL of Salmonella Typhimurium suspension in logarithmic growth phase, spread it evenly on LB solid agar plate, and after solidification, spot 10 µL of the phage sample obtained in step (2), incubate at 37 ℃ upside down for 18 h, and observe the plaques.

[0060] (4) Phage purification and amplification: A single phage plaque was picked up with a sterile inoculation loop, immersed in 1 mL of SM buffer, and shaken at 4 ℃ for 12 h. Then, it was centrifuged at 4 ℃ and 12000 rpm for 2 min. After centrifugation, the supernatant was collected, and the filtrate was collected after passing through a 0.22 μm disposable needle filter to obtain purified phage. 100 µL of the purified phage solution was mixed with 5 mL of LB semi-solid medium containing 0.4% agar and 350 µL of Salmonella typhimurium bacterial solution, plated, and cultured at 37 ℃ for 18 h for amplification to obtain high-titer phage stock solution. To evaluate the morphology of the isolated Salmonella typhimurium phage, the phage was negatively stained with phosphotungstic acid solution and analyzed by transmission electron microscopy (TEM). The results are as follows: Figure 5 As shown, this bacteriophage exhibits a typical tadpole-shaped structure, with an icosahedral symmetrical head (approximately 60–80 nm in diameter) and a long, non-contractile tail (approximately 120–150 nm in length). A clear basal plate structure is visible at the end of the tail. These morphological features are consistent with the structural conservation of known Salmonella bacteriophages.

[0061] This bacteriophage is a Salmonella typhimurium bacteriophage, named vB_Sal_YZCM1, and classified as a Salmonella typhimurium bacteriophage. Salmonella phage It was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 47002.

[0062] Example 3: Preparation of Phage / QZIF-67@MB, a calcined zeolite imidazole ester framework material anchored to bacteriophages and loaded with methylene blue (MB). (1) Dissolve 0.546 g Co(NO3)2·6H2O and 0.616 g 2-methylimidazole in 30 mL of methanol respectively, and stir at 300 rpm for 30 min until completely dissolved; quickly pour the Co(NO3)2 solution into the 2-methylimidazole solution, stir manually for 60 s, let stand at room temperature for 24 h, centrifuge at 12000 rpm for 10 min, collect the precipitate, wash three times with methanol, and vacuum dry at 60 ℃ for 12 h to obtain ZIF-67 material. The scanning electron microscope image of ZIF-67 material is shown below. Figure 6 As shown in (A), the original ZIF-67 exhibits a smooth rhombic dodecahedral structure.

[0063] (2) The ZIF-67 material obtained by the above method was placed in a tube furnace and calcined at 250 °C for 3 hours in an air atmosphere to obtain the calcined zeolite imidazole ester framework material QZIF-67. The scanning electron microscope image of the QZIF-67 material is shown below. Figure 6 As shown in (B), QZIF-67 generally retains this dodecahedral morphology, although some surfaces collapse and gradually become rough due to ligand degradation during heat treatment.

[0064] (3) 5 mg methylene blue (MB) and 10 mg QZIF-67 were dispersed in 5 mL of deionized water, thoroughly mixed, and allowed to stand at room temperature for 24 h to achieve MB loading. The mixture was then transferred to a centrifuge tube, centrifuged at 12000 rpm for 10 min to collect the precipitate, washed three times with deionized water to remove free MB, and then vacuum dried at 60 ℃ for 12 h to obtain the QZIF-67@MB composite material. The scanning electron microscope image of the QZIF-67@MB composite material is shown below. Figure 6 As shown in (C).

[0065] (4) Take 2 mg of QZIF-67@MB composite material and 1 mL of 10 9 The PFU / mL Salmonella typhimurium phage stock solution was incubated with shaking at room temperature for 1 h, then centrifuged at 5000 rpm for 5 min to collect the precipitate. The precipitate was washed with PBS buffer (pH=7.4, 10 mM) and then redispersed in 1.0 mL of PBS buffer (pH=7.4, 10 mM) to obtain the Phage / QZIF-67@MB solution.

[0066] The synthesis of ZIF-67 and QZIF-67 was verified by Fourier transform infrared (FT-TR) spectroscopy, and the results are as follows: Figure 7 As shown. From Figure 7 It can be seen that at 1062 cm -1At the location, characteristic peaks of the C=N bond in the imidazoline ring (a typical structural unit of ZIFs) were observed in both ZIF-67 (black curve) and QZIF-67 (red curve); the FT-IR spectra of QZIF-67 and ZIF-67 were in high agreement. This spectral consistency indicates that the organic ligand framework was preserved and the metal-organic structure of ZIF-67 was maintained without significant ligand removal or lattice distortion.

[0067] The structures of QZIF-67 and ZIF-67 were analyzed by X-ray diffraction (XRD), and the results are as follows: Figure 8 As shown. From Figure 8 It can be seen that QZIF-67 retains the characteristic diffraction peaks of ZIF-67 in the 5-15° range, indicating that the material still retains some of the crystal structure characteristics of ZIF-67 after calcination. However, compared with ZIF-67, the intensity of the main diffraction peaks of QZIF-67 (especially at characteristic peak positions such as 2θ=7.3°, 10.4° and 12.7°) is significantly reduced. This phenomenon confirms the partial collapse of the ZIF-67 framework crystal structure during calcination.

[0068] The material properties of QZIF-67 and its precursor ZIF-67 were systematically characterized using ultraviolet-visible spectroscopy (UV-vis). Specifically, to verify the loading performance of the two materials on methylene blue (MB), 5 mg of MB and 10 mg of QZIF-67 were dispersed in 5 mL of deionized water. After standing at room temperature for 24 h, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was washed three times with deionized water to remove free MB and then redispersed in 5 mL of deionized water to obtain QZIF-67@MB and ZIF-67@MB solutions, respectively. UV-vis testing was performed on these solutions, with a 1 mg / mL MB solution used as a control. The results are as follows: Figure 9 As shown. From Figure 9 It can be seen that at the characteristic absorption wavelength of 680 nm, the absorbance values ​​of the supernatants of ZIF-67@MB and QZIF-67@MB are significantly lower than those of the control MB solution. This result indicates that MB molecules have been successfully loaded into ZIF-67 and QZIF-67 materials, establishing the ability of the two materials as effective signal carriers for electrochemical detection.

[0069] like Figure 3 and 4 As shown by the blue line, TDNs / AuNPs / SPE were incubated with Salmonella typhimurium (i.e., S. typhimruium / TDNs / AuNPs / SPE) leads to further attenuation of the CV signal and R ctThe increase in resistance at the interface, caused by the specific recognition between the aptamer (the D-chain of TDNs) and the target bacteria, signifies an additional increase in interfacial resistance. The aptamer-bacterial complex formed on the electrode surface spatially hinders the access of the redox probe, thereby reducing electron transfer efficiency. The continuous and consistent changes in CV and EIS data at each modification stage corroborate and characterize the stepwise assembly process of TDNs / AuNPs / SPE and its successful target capture, validating the feasibility of the proposed scheme.

[0070] like Figure 3 and 4 As shown by the green line, in Target / TDNs / AuNPs / SPE (i.e. S. typhimruium / The addition of Phage / QZIF-67@MB to the TDNs / AuNPs / SPEs composite introduced more non-conductive substrates, resulting in a maximum electrochemical impedance value, indicating the successful assembly of Phage / QZIF-67@MB on the electrode.

[0071] Example 4 An application method for an electrochemical sensor based on dual recognition of aptamers and bacteriophages, such as... Figure 10-13 As shown, optimal sensor performance is ensured through systematic optimization of key parameters. This optimization process, based on a pre-experimental design, includes the following steps: Optimization of TDNs Concentration: First, a series of DNA tetrahedral nanostructures (TDNs) solutions of different concentrations were prepared according to the method in Example 1, with concentration gradients of 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, and 2.5 μM. Each concentration of TDNs solution was dropped onto the surface of a gold nanoparticle-modified screen-printed electrode (AuNPs / SPE) and incubated at 25°C for 12 h to allow the TDNs to self-assemble and immobilize via Au-S bonds. Subsequently, the charge transfer resistance (R) at each concentration was measured using electrochemical impedance spectroscopy (EIS). ct The result is as follows: Figure 10 As shown, R ct The value increases with increasing TDNs concentration, and at a concentration of 1.0 μM, R... ct The near-saturation indicates that the electrode surface has been completely covered by TDNs. Therefore, the optimized TDNs concentration of 1.0 μM was used for subsequent electrode modification.

[0072] Optimization of the binding time between target bacteria and TDNs: AuNPs / SPEs immobilized with TDNs were incubated with a fixed concentration of Salmonella Typhimurium suspension at 25°C for different times (e.g., 20, 30, 40, 50, 60 min). After incubation, the electrodes were washed and the R value of EIS was measured. ct Value. Result as follows Figure 11 As shown, R ct The value increased with prolonged incubation time, reaching a stable value at 30 min, indicating that the binding of the aptamer and bacteria had reached equilibrium. Therefore, the optimized binding time between the target and TDNs was 30 min.

[0073] Optimization of Phage / QZIF-67@MB signal probe concentration: Phage / QZIF-67@MB signal probe solutions of different concentrations (0.5, 1.0, 2.0, 3.0, 4.0 mg / mL) were prepared. After bacterial capture by the electrode, each concentration of signal probe solution was added dropwise, incubated for 10 min, and then the characteristic peak current of methylene blue (MB) was measured using square wave voltammetry (SWV). The results are as follows: Figure 12 As shown, the SWV current increases with increasing probe concentration, reaching a maximum at 3.0 mg / mL; however, the current decreases at higher concentrations because excessive probe can cause blockage of the electrode surface. Therefore, the optimized signal probe concentration is 3.0 mg / mL.

[0074] Optimization of the interaction time between the signal probe (Phage / QZIF-67@MB) and target bacteria: At a fixed signal probe concentration (3.0 mg / mL), the probe was added to the electrode surface containing captured bacteria and incubated for different times (e.g., 3, 8, 5, 10, 15, 20 min). The SWV peak current was then measured. Results are shown below. Figure 13 As shown, the current value reaches its peak at 10 min, and extending the incubation time does not significantly enhance it. Therefore, the optimized interaction time is 10 min.

[0075] Based on the above optimization results, the application method of the electrochemical sensor based on dual recognition of aptamers and bacteriophages includes the following steps: (1) 30 µL of the bacterial suspension to be tested was dropped onto the surface of the TDNs / AuNPs / SPE electrode (TDNs solution concentration was 1.0 μM) prepared in Example 1 and incubated at 25 °C for 30 min; the electrode surface was rinsed with deionized water and dried. (2) Add 30 µL of Phage / QZIF-67@MB solution (3 mg / mL) prepared in Example 3 to the electrode surface and incubate at 25 °C for 10 min; rinse the electrode again with deionized water and dry it; (3) The square wave voltammetry was used to detect the potential in PBS buffer containing 10 mM pH=7.4, with a potential range of 0 V to -0.5 V and a pulse amplitude of 25 mV. (4) Calculate the concentration of Salmonella typhimurium in the sample based on the standard curve.

[0076] Before detecting the concentration of the target product, *Salmonella typhimurium*, a standard curve needs to be prepared using known concentrations of *Salmonella typhimurium*. The SWV response of different target concentrations is as follows: Figure 14 As shown. At the same time Figure 15 The linear relationship between the SWV peak current value and the concentration of Salmonella typhimurium was shown. The standard curve was: ΔI = 43.57536 * log(C) - 24.4291; the concentrations of Salmonella typhimurium were known to be 0 CFU / mL, 5 CFU / mL, 10 CFU / mL, 50 CFU / mL, 100 CFU / mL, and 500 CFU / mL.

[0077] Stability and specificity are among the most important characteristics of biosensors. To verify the stability of the sensor of this invention, the stability of the Salmonella Typhimurium electrochemical sensor constructed in this application was tested during a 216-hour storage period. Specifically, the TDNs / AuNPs / SPE electrode prepared in Example 1 and the Phage / QZIF-67@MB probe prepared in Example 3 were stored at 4°C. They were incubated with the target at different storage time periods and detected by square wave voltammetry. The stability of the sensor was evaluated by analyzing the changes in the current signal. The results are as follows: Figure 16 As shown, according to Figure 16 It can be seen that the sensor prepared by this invention can maintain good detection performance within 7 days, demonstrating good long-term stability and meeting the basic requirements for sensor storage stability in practical applications.

[0078] To verify the specificity of the sensor of the present invention, Staphylococcus aureus was detected according to the above application method. S. aureus ), Escherichia coli ( E. coli ), Bacillus cereus ( B. cereus ) and Listeria monocytogenes ( L. monocytogenes These are four common foodborne pathogens. The results are as follows: Figure 17 As shown, according to Figure 17 It is known that only Salmonella typhimurium (Salmonella typhimurium) S. typhimurium The system showed a significant increase in SWV peak current value, while the SWV peak current values ​​caused by the other four foodborne pathogens were smaller, indicating that the method has good specificity for the detection of Salmonella typhimurium.

[0079] In addition to specific recognition, the inherent lytic properties of bacteriophages also provide a role in biocontrol. The bactericidal efficiency of the sensor was quantified by assessing the survival rate of the target bacteria, as follows: TDNs / AuNPs / SPE were incubated with Salmonella Typhimurium for 30 min, followed by incubation with the signal probe (Phage / QZIF-67@MB), and then detection was performed. One hour after the signal probe (Phage / QZIF-67@MB) incubation was completed, plate counting of surviving Salmonella on the sensor was performed, and a control group without the signal probe was set up. Results are as follows... Figure 18 As shown. From Figure 18 As can be seen, compared with the control group, the viable bacterial count in the experimental group was significantly lower (p<0.001). Figure 18 This indicates that the sensor can reduce the risk of transmission by lysing the captured bacteria. This inherent safety feature of bacteriophages is particularly advantageous for field applications.

[0080] Test case Actual sample testing and spiked recovery rate test: To verify the applicability and reliability of the invented electrochemical sensor in actual sample detection, egg and chicken samples were prepared as detection matrices according to the requirements of the Chinese National Food Safety Standard "Salmonella Detection" (GB 4789.4-2024). Different concentrations (5.75~5.75×10⁻⁶) of the sensor were added to the egg and chicken samples. 2 The *Salmonella typhimurium* (CFU / mL) was used to simulate actual contamination conditions, and the detection method described in Example 4 was applied to systematically evaluate the performance of the developed detection method. Simultaneously, the traditional plate count method was used as a control method to verify the results and ensure data accuracy and comparability. Experimental results showed that the recoveries of egg and chicken samples were both between 90.10% and 108.70%, meeting the technical requirements for microbial detection method validation, and the detection results maintained high accuracy and reliability.

[0081] Table 2: Detection results of the sensor and the plate counting method of the present invention (n=3)

[0082] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. An electrochemical sensor based on dual recognition of aptamer and phage, characterized in that, The electrochemical sensor comprises a DNA tetrahedron nanostructure modified gold electrode and a calcined zeolitic imidazolate framework material anchoring phages and loaded with methylene blue; The DNA tetrahedron nanostructure comprises four DNA probes, which are A chain, B chain, C chain and D chain; the nucleotide sequence of the A chain is shown as SEQ ID No. 1; the nucleotide sequence of the B chain is shown as SEQ ID No. 2; the nucleotide sequence of the C chain is shown as SEQ ID No. 3; the nucleotide sequence of the D chain is shown as SEQ ID No. 4; and the phage is a Salmonella typhi phage.

2. A method of preparing an electrochemical sensor as claimed in claim 1, characterized in that, The preparation method comprises the following steps: (1) preparing a DNA tetrahedron nanostructure modified gold electrode: S1-1: mixing A chain solution, B chain solution, C chain solution, D chain solution and TCEP solution, adding TM buffer solution, and incubating at room temperature for 1-2 hours; then high-temperature treatment and rapid ice bath are performed to obtain TDNs solution, i.e. the DNA tetrahedron nanostructure; S1-2: dropping the TDNs solution on the surface of the nanogold modified screen-printed electrode and incubating at room temperature for 8-16 hours to obtain the DNA tetrahedron nanostructure modified gold electrode TDNs / AuNPs / SPE; (2) preparing a calcined zeolitic imidazolate framework material anchoring phages and loaded with methylene blue: S2-1: mixing Co(NO3)2·6H2O methanol solution and 2-methyl imidazole methanol solution, stirring at room temperature, standing for reaction, centrifuging to collect the precipitate, washing with methanol and vacuum drying to obtain ZIF-67 material; S2-2: placing the ZIF-67 material in a tube furnace and calcining under air atmosphere to obtain calcined zeolitic imidazolate framework material QZIF-67; then methylene blue is dispersed in deionized water with QZIF-67, and the mixture is left to stand at room temperature for 24 hours to load the methylene blue on the QZIF-67; then the mixture is centrifuged, the precipitate is collected, washed with deionized water, and vacuum dried to obtain QZIF-67@MB composite material; S2-3: incubating the QZIF-67@MB composite material with Salmonella typhi phage stock solution at room temperature for 40-60 minutes, then centrifuging to collect the precipitate, washing the precipitate with PBS buffer solution, and then dispersing the precipitate in PBS buffer solution again to obtain Phage / QZIF-67@MB solution, i.e. the calcined zeolitic imidazolate framework material anchoring phages and loaded with methylene blue.

3. The preparation method according to claim 2, characterized in that, In step S1-1, The concentrations of the A chain solution, B chain solution, C chain solution and D chain solution are all 50-250 μmol / L; and the concentration of the TCEP solution is 1-5 mmol / L; The volume ratio of the A chain solution, B chain solution, C chain solution, D chain solution, TCEP solution and TM buffer solution is 1:1:1:1:10:86; The high-temperature treatment is performed at a temperature of 93-98 ℃ for 5-10 minutes; and the rapid ice bath is performed at a temperature of 3-5 ℃ for 5-10 minutes.

4. The preparation method according to claim 2, characterized in that, The concentration of the TDNs solution in step S1-2 is 0.5-2.5 μmol / L, and the volume is 25-35 µL.

5. The preparation method according to claim 2, characterized in that, In step S2-1, The concentration of the Co(NO3)2·6H2O methanol solution is 0.017-0.020 g / mL; The concentration of the 2-methylimidazole methanol solution is 0.018-0.022 g / mL; The mass ratio of Co(NO3)2·6H2O to 2-methylimidazole is 1:(0.9-1.29); The stirring time is 55-65 s; The standing reaction time is 22-26 h; The centrifugation speed is 10000-12000 rpm, and the time is 8-10 min; The vacuum drying temperature is 55-65 ℃, and the time is 12-14 h.

6. The preparation method according to claim 2, characterized in that, In step S2-2, The calcination temperature is 250-300 ℃, and the time is 1-3 h; The mass ratio of methylene blue to QZIF-67 is 1:2, and the concentration of methylene blue in deionized water is 0.5-1.5 mg / mL; The centrifugation speed is 10000-12000 rpm, and the time is 8-10 min; The vacuum drying temperature is 55-65 ℃, and the time is 12-14 h.

7. The preparation method according to claim 2, characterized in that, In step S2-3, The concentration of the Salmonella typhimurium bacteriophage stock solution is 10 9 PFU / mL; The mass-volume ratio of QZIF-67@MB composite material to Salmonella typhimurium phage stock solution is 2 mg:(0.5-1.5) mL; The centrifugation speed is 5000-6000 rpm, and the time is 5-8 min; The concentration of PBS buffer is 10 mmol / L, and the pH is 7.4; The mass-volume ratio of QZIF-67@MB composite material to PBS buffer is 2 mg:(0.5-1.5) mL.

8. Use of an electrochemical sensor according to claim 1 or an electrochemical sensor produced by a method according to any one of claims 2 to 7, characterized in that, The electrochemical sensor is used for detecting Salmonella typhimurium.

9. Use according to claim 8, characterized in that, The method for detecting Salmonella typhimurium using the electrochemical sensor comprises the following steps: (1) Add 30 µL of the bacteria suspension to be detected on the surface of the TDNs / AuNPs / SPE electrode, incubate at 25 ℃ for 20-60 min, then rinse the electrode surface with deionized water and dry; (2) Add another 30 µL of the Phage / QZIF-67@MB solution (3 mg / mL) to the electrode surface, incubate at 25 ℃ for 3-20 min, rinse the electrode with deionized water again and dry; (3) Immerse the dried electrode of step (2) in 10 mM PBS buffer with pH=7.4, and detect by square wave voltammetry with a potential range of 0 V to -0.5 V and a pulse amplitude of 25 mV; (4) Calculate the concentration of Salmonella typhimurium in the sample according to the standard curve.

10. Use according to claim 9, characterized in that, In step (4), the standard curve is: ΔI = 43.57536 * log(C) - 24.4291, R²=0.9938, wherein I is the net peak current value after deducting the background, and C represents the concentration of Salmonella typhimurium.

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