A dual-signal aptamer sensor and its preparation method and application

Abstract

The present invention discloses a dual-signal aptamer sensor, a preparation method thereof, and its application in detecting foodborne pathogens. The dual-signal aptamer sensor comprises a cathode electrode, an anode electrode 1, an anode electrode 2, an electrode interface, and a working electrode lead. The cathode electrode, anode electrode 1, and anode electrode 2 form two sets of bipolar electrodes; a reservoir 1 is provided on the cathode electrode, a reservoir 2 is provided on the anode electrode 1, and a reservoir 3 is provided on the anode electrode 2; the anode electrode 1 and the anode electrode 2 are screen-printed electrodes modified with a polydopamine film, and the polydopamine film also includes amino-modified capture DNA. The dual-signal aptamer sensor effectively utilizes the quantitative relationship of the bipolar system, avoids interference from the instrument or environment, improves the signal-to-noise ratio, and enhances the sensitivity and detection range of the device; the detection sensitivity is high and the specificity is good; the detection cost is low, and the detection is rapid, simple, sensitive, and specific.

Description

Technical Field

[0001] The present invention relates to a dual-signal aptamer sensor and a preparation method and application thereof, and in particular to a bipolar electrode colorimetric dual-mode sensing platform and a detection method thereof, belonging to the field of biosensor technology. Background Art

[0002] In the past two years, food hygiene has become a major concern. According to the World Health Organization, foodborne diseases cause over 400,000 deaths annually, with children under five accounting for approximately one-third of these deaths. Therefore, food safety has become a top public safety and health concern worldwide. Human foodborne illnesses are caused by a variety of factors, including viruses, bacteria, parasites, toxins, and chemicals in food, with foodborne pathogens being a major factor. Currently, the main foodborne pathogens include Salmonella typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Shigella. These foodborne pathogens have been linked to outbreaks of foodborne infections in humans and animals. Therefore, the detection of foodborne pathogens is crucial for ensuring food safety and hygiene, and the development of efficient methods for detecting these pathogens is of great significance.

[0003] Electrochemiluminescence (ECL) is a technology that combines electrochemistry and chemiluminescence. When voltage is applied, highly active free radicals near the electrode surface generate excited states through electron transfer, and the excited states jump back to the ground state to produce luminescence. In ECL research, organic compound molecules (Luminol) and inorganic metal complexes (Ru(bpy)3 2+) were the most commonly used luminescent reagents in the early days. Both categories of luminescent reagents have been extensively studied and reported in both basic research and commercial applications. Compared to photoluminescence (PL), electrochemiluminescence (ECL) does not require an additional excitation light source, so the measured signal is not affected by stray light interference, which significantly improves the sensitivity and selectivity of ECL analysis. Furthermore, there is now increasing attention on determining the ratio of dual-signal ECL. Compared with traditional single-signal ECL sensing strategies, ECL dual-signal ratiometric sensing strategies offer the advantages of high sensitivity and low background signal, making them suitable for the quantitative analysis of trace targets in complex biological systems. A bipolar electrode (BPE) is a conductive material that promotes electrochemical reactions at its two electrodes even when not in direct contact with the driving electrode. A closed BPE is a type of BPE that physically separates the two electrodes of the BPE. This configuration is ideal for use in combination with ECL for the fabrication of scale and visual ECL biosensors. However, these types of biosensors currently cannot achieve high-sensitivity detection of foodborne pathogens, primarily due to limited ECL reagent options, insufficient visible ECL intensity, and insufficient current through the BPE. Therefore, finding a reagent and method that can significantly improve the sensitivity and accuracy of ratiometric and visual BPE-ECL biosensors has become a major issue in solving the problem of high-sensitivity detection of foodborne pathogens.

[0004] It has been reported in the literature that ferrocene (Fc), a substance with excellent electroactivity commonly used in electrochemical detection, has a strong electrochemical effect on Luminol and Ru(bpy)3 2+ The luminescence intensity of Fc and its derivatives has attracted widespread attention from chemists and materials scientists due to their unique properties when combined with other molecules. A multicolor luminescence sensor has been constructed by adding ferrocene to the cathode and a dual-color luminescent material to the anode. This sensor can detect three prostate biomarkers by detecting changes in the anode ECL caused by increases and decreases in the Fc current at the BPE cathode.

[0005] Based on this, if a simple and sensitive biosensor technology combining visual ECL evaluation and ratiometric detection principles could be developed for the quantitative detection of foodborne pathogens in food, it would greatly improve the reliability of biosensors and enable wider practical applications of biosensor technology. Currently, existing technologies directly add the target substance or a substance related to the target substance directly to the sensor surface. The redox reaction on the electrode surface may affect the amount of the target substance. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dual-signal aptamer sensor.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the dual-signal aptamer sensor.

[0008] Another technical problem to be solved by the present invention is to provide an application of the dual-signal aptamer sensor in detecting foodborne pathogens.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is: a dual-signal aptamer sensor, including a cathode electrode, an anode electrode 1 and an anode electrode 2, an electrode interface and a working electrode lead, the cathode electrode, the anode electrode 1 and the anode electrode 2 constitute two groups of bipolar electrodes; a reservoir 1 is provided on the cathode electrode, a reservoir 2 is provided on the anode electrode 1, and a reservoir 3 is provided on the anode electrode 2; the anode electrode 1 and the anode electrode 2 are screen-printed electrodes modified with a polydopamine film, and the polydopamine film also includes amino-modified capture DNA.

[0010] The sequence of the captured DNA is shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6, and is combined with the corresponding Fc-tagged bacterial aptamer for detecting S. typhimurium, S. aureus and E. coli O157:H7.

[0011] The present invention also includes a method for preparing a dual-signal aptamer sensor, comprising the following steps:

[0012] 1) Preparation of bipolar electrodes: A negative electrode, anode electrode 1, and anode electrode 2, working electrode leads respectively connected to the negative electrode, anode electrode 1, and anode electrode 2, as well as an electrode standard layer and an electrode insulating layer are printed on a substrate using a light-curing insulating paste, and then cured by ultraviolet light;

[0013] 2) Preparation of dual-signal aptasensor:

[0014] 2.1. Reservoir 1 is set on the cathode electrode of the bipolar electrode, reservoir 2 is set on the anode electrode 1, and reservoir 3 is set on the anode electrode 2;

[0015] 2.2. Dopamine solution was added dropwise to anode electrode 1 and anode electrode 2, respectively, and the scanning potential was set to -0.2~0.9V. Cyclic voltammetry was performed, and the electrodes were rinsed with PBS buffer and dried with N2 to obtain screen-printed electrodes modified with polydopamine film.

[0016] 2.3. The captured DNA was added dropwise onto the surface of the polydopamine-modified screen-printed electrode, placed in a humid environment, rinsed with PBS buffer, dried with N2, and the unbound active sites were blocked with BSA and rinsed with PBS buffer to obtain a dual-signal aptamer sensor.

[0017] 3) Electrode interface 1 is connected to the cathode electrode, and electrode interface 2 is connected to the anode electrode 2 to obtain a dual-signal aptamer sensor.

[0018] Wherein, the substrate in step 1) is an inert material.

[0019] The concentration of the captured DNA in step 2.3 is 1 μM, and the amount used is 10 μL.

[0020] The present invention also includes the application of a dual-signal aptamer sensor in detecting foodborne pathogens.

[0021] The present invention also includes a method for detecting foodborne pathogens using a dual-signal aptamer sensor, comprising the following steps:

[0022] 1) Mixing and incubating the sample solution containing the Fc-tagged bacterial aptamer and bacteria, and centrifuging to obtain the Fc-aptamer supernatant;

[0023] 2) Add the supernatant of the Fc-aptamer dropwise to the anode electrode 1 and anode electrode 2 of the dual-signal aptamer sensor, incubate, and rinse the electrodes with PBS buffer;

[0024] 3) Ru(bpy)3 was added dropwise to anode electrode 1 and anode electrode 2 respectively. 2+ -TprA solution and Luminol-H2O2 solution, add PBS buffer on the cathode, and perform ECL signal detection under the action of constant potential;

[0025] 4) When anode electrode 1 does not generate light and anode electrode 2 generates blue light, it proves that the sample solution does not contain foodborne pathogens; when anode electrode 1 generates red light, it proves that the sample solution contains foodborne pathogens.

[0026] Wherein, the concentration of the Luminol-H2O2 solution is 0.5-3mM, the Ru(bpy)3 2+ The concentration of the -TprA solution was 3.5 to 6 mM.

[0027] Preferably, Ru(bpy)3 2+ The concentration of -TPrA is 3.5mM. At this time, the Ru(bpy)3 2+ The red ECL signal was almost completely suppressed by Fc; the concentration of Luminol-H2O2 was 2.5 mM, at which time the blue ECL emission intensity of Luminol on the anode electrode 2 was large, and no self-quenching effect occurred.

[0028] Wherein, the Fc-labeled bacterial aptamer is a DNA fragment such as SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5; the concentration of the Fc-labeled bacterial aptamer is 0-1 μM.

[0029] Preferably, the concentration of Fc-labeled bacterial aptamer is 1 μM, at which point Ru(bpy)3 2+ The red ECL signal of Luminol was almost completely suppressed, while the blue ECL signal of Luminol was the strongest.

[0030] The incubation time of the bacterial sample solution and the Fc-labeled bacterial aptamer in step 2) is 15 to 90 minutes, and the constant potential voltage in step 3) is 3.5 to 6V.

[0031] Preferably, in step 2), the incubation time of the bacterial sample solution and the Fc-labeled bacterial aptamer is 60 min, at which time Ru(bpy)3 2+ The red ECL signal intensity of Luminol reaches the maximum, and the blue ECL signal intensity of Luminol reaches the minimum; the constant potential voltage in step 3) is 5.5V, the blue luminescence signal of Luminol is stronger, and the blue luminescence signal of Ru(bpy)3 2+ The red luminescence signal is weak, which is convenient for subsequent observation.

[0032] Working principle: Add ferrocene (Fc) modified aptamer to the anode, and introduce ferrocene (Fc) modified aptamer to the BPE anode surface through hybridization; add Ru (bpy) 3 to anode electrode 1 and anode electrode 2 respectively. 2+ -TPA and Luminol-H2O2 solution, PBS buffer was added to the cathode. Ferrocene can not only inhibit Ru(bpy)3 2+ -TPA oxidation, and its oxidation product Fc + It can also be achieved through the excited state Ru(bpy)3 2+* To Fc + efficient energy transfer to quench Ru(bpy)3 2+ -TPA ECL, Ru(bpy)3 2+ The ECL intensity of Luminol is quenched due to the oxidation product of ferrocene Fc + The catalysis of the co-reactant H2O2 is greatly enhanced. Therefore, by evaluating Ru(bpy)3 2+ The RGB intensity ratio of Luminol can detect foodborne pathogens with ultra-sensitive sensitivity.

[0033] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. It effectively utilizes the quantitative relationship of the bipolar system, avoiding interference from the instrument or environment, improving the signal-to-noise ratio, and enhancing the sensitivity and detection range of the device; 2. It has high detection sensitivity and good specificity; 3. It is low-cost, rapid, simple, sensitive, and has good specificity; 4. It utilizes the electrochemically active substance ferrocene as an intermediate medium, effectively avoiding the influence of redox reactions on the analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the proportional dimensions of the bipolar electrodes of the dual-signal aptamer sensor;

[0035] Figure 2 A is the ECL image of the dual-signal aptasensor without modified ferrocene; Figure 2 B is the ECL image when free ferrocene was added to the dual-signal aptamer sensor; Figure 2 C is the ECL image of the dual-signal aptamer sensor modified with ferrocene far away from the electrode surface; Figure 2 D is the ECL image of the dual-signal aptasensor modified with ferrocene close to the electrode surface;

[0036] Figure 3 A is the CV curve of electropolymerized dopamine; Figure 3 B is Luminol and Ru(bpy)3 2+ ECL spectrum of

[0037] Figure 4 A1 is Ru(bpy)3 under different concentration conditions 2+ ECL spectrum of Figure 4 A2 is Ru(bpy)3 under different concentration conditions 2+ RGB intensity change map; Figure 4 B1 is the ECL spectra of Luminol at different concentrations; Figure 4 B2 is the RGB intensity change diagram of Luminol under different concentration conditions;

[0038] Figure 5 A is the ECL image under different concentrations of ferrocene aptamer; Figure 5 B is different concentrations of Luminol and Ru(bpy)3 2+ RGB intensity changes under conditions;

[0039] Figure 6 A is the ECL image of foodborne pathogens and ferrocene aptamer under different incubation time conditions; Figure 6 B is the corresponding Luminol and Ru(bpy)3 under different incubation time conditions of foodborne pathogens and ferrocene aptamer 2+ RGB intensity changes;

[0040] Figure 7 Luminol and Ru(bpy)3 at different voltages 2+ ECL image;

[0041] Figure 8 Flowchart of the dual-signal aptasensor method for detecting foodborne pathogens;

[0042] Figure 9 A is the concentration of Luminol and Ru(bpy)3 under different conditions of Salmonella typhimurium 2+ ECL image; Figure 9 B shows the Luminol and Ru(bpy)3 concentrations under different Salmonella typhimurium concentrations 2+ RGB intensity changes; Figure 9 C is the Ru(bpy)3 under different concentrations of Salmonella typhimurium 2+ Changes in the RGB intensity ratio with Luminol;

[0043] Figure 10 A is the expression of Luminol and Ru(bpy)3 under different concentrations of Staphylococcus aureus 2+ ECL image; Figure 10 B is the ECL image of E. coli O157:H7 at different concentrations; Figure 10 C is Ru(bpy)3 under different concentrations of Staphylococcus aureus 2+ Changes in the RGB intensity ratio with Luminol; Figure 10 D is the Ru(bpy)3 under different concentrations of E. coli O157:H7 2+ Changes in the RGB intensity ratio with Luminol;

[0044] Figure 11 A is the ECL image of the dual-signal aptasensor in the specific detection of foodborne pathogens at different concentrations; Figure 11 B is the specific detection of Ru(bpy)3 by the dual-signal aptasensor for foodborne pathogens at different concentrations. 2+ Ratio of RGB intensity to Luminol;

[0045] Figure 12 A is the ECL image of different concentrations of Salmonella typhimurium in actual samples; Figure 12 B is the Ru(bpy)3 under different concentrations of Salmonella typhimurium in actual samples 2+ Ratio of RGB intensity to Luminol;

[0046] Figure 13 A is the ECL image of Staphylococcus aureus at different concentrations in actual samples; Figure 13 B is the Ru(bpy)3 in different concentrations of Staphylococcus aureus in actual samples 2+ Ratio of RGB intensity to Luminol;

[0047] Figure 14 A is the ECL image of different concentrations of E. coli O157:H7 in actual samples; Figure 14 B Ru(bpy)3 in actual samples under different concentrations of E. coli O157:H7 2+ Ratio of RGB intensity to Luminol. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0049] Reagents required for the present invention:

[0050] 1. Salmonella typhimurium (ATCC14028), Staphylococcus aureus (ATCC 29213), and Escherichia coli O157:H7 strain (CICC 10907) were preserved in our laboratory. Ferrocene-labeled aptamers and capture DNA were purchased from Shanghai Sangon Biotechnology Co., Ltd., Luminol was purchased from Shanghai Sigma High-Tech Co., Ltd., and Ru(bpy)3 2+ Tripropylamine (TPrA) and tripropylamine (TPrA) were purchased from Shanghai Aladdin Reagent Co., Ltd., and dopamine (DA) was purchased from Nanjing Wanqing Chemical Glass Instrument Co., Ltd. Hydrogen peroxide (H2O2), sodium chloride (NaCl), peptone, bovine serum albumin (BSA), yeast extract powder, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Luria-Bertani liquid medium was purchased from Nanjing Wanqing Chemical Glass Instrument Co., Ltd.

[0051]

[0052] 2. Beef, bread, and pure milk were purchased from farmers' markets and supermarkets in Nanjing;

[0053] The instrument selected for use in the present invention:

[0054] 1. Electrochemical workstation (CHI750E);

[0055] 2. Fluorescence spectrophotometer (F-4700);

[0056] 3. Vortex mixer (IKA German);

[0057] 4. Centrifuge (Eppendorf German);

[0058] 5. Combined shaking table (HYL-C2).

[0059] Example 1 Preparation of Bipolar Electrode Aptamer Dual-Signal Aptamer Sensor

[0060] Preparation of bipolar electrodes: A 47.2mm×20mm polyethylene terephthalate (PET) inert material is used as the substrate, and then working electrode leads 1-8 with a width of 0.6mm are printed on the substrate. Next, a circular carbon electrode and an arc-shaped carbon layer are made with a photocurable insulating paste and cured by ultraviolet light to serve as a bipolar electrode. After drying, the electrode specification layer is printed with a photocurable insulating paste and cured with ultraviolet light. Finally, the electrode insulation layer is printed with a photocurable insulating paste and cured with ultraviolet light. The proportional dimensions of the bipolar electrode are as follows: Figure 1 As shown in the figure, 40 electrode plates are printed each time. The printed electrode plates are dried under vacuum at 60°C for 12 hours to completely evaporate the solvent and solidify the electrode strips. When ready for use, the screen-printed sheets can be cut and used as individual electrodes.

[0061] Preparation of the dual-signal aptasensor: Reservoirs 1-3 were placed on the cathode electrode, anode electrode 1, and anode electrode 2, respectively. The cathode electrode and anode electrode 1, and the cathode electrode and anode electrode 2, respectively, formed the sensor's two sets of bisphenol A polyoxyethylene ethers (BPEs). A 20 μL solution of 3 mM dopamine (DA) (prepared in 10 mM PBS, pH 6.5, with N2 flow for 15 minutes to remove O2) was added dropwise to anode electrodes 1-2. The scanning potential was set from -0.2 to 0.9 V, and cyclic voltammetry (CV) was performed at a scan rate of 20 mV s for 20 revolutions. -1 . The electrode was then rinsed with the PBS and blown dry under N2 to obtain a polydopamine (PDA) film-modified screen-printed electrode. Then, the two anodes were functionalized with capture DNA of the sequence shown in SEQ ID NO.2. 10 μL of 1 μM capture DNA of the sequence shown in SEQID NO.2 was dropped onto the surface of the PDA-modified screen-printed electrode and placed in a humid environment at room temperature overnight. Under weakly alkaline conditions, the amino-modified capture DNA was covalently bound to the PDA film-modified electrode through a Schiff base reaction in one step, and then the electrode was rinsed with PBS buffer and blown dry with nitrogen. Finally, the unbound active sites were blocked with 2% BSA for 2 hours and rinsed with PBS to obtain a dual-signal aptamer sensor for detecting S. typhimurium.

[0062] The same method was used to prepare a dual-signal aptamer sensor for detecting S. aureus with two anodes functionalized by the capture DNA of the sequence shown in SEQ ID NO.4 and a dual-signal aptamer sensor for detecting E. coli O157:H7 with two anodes functionalized by the capture DNA of the sequence shown in SEQ ID NO.6.

[0063] Example 2 Ferrocene inhibits Ru(bpy)3 2+ Verification of Luminol Luminescence and Catalytic Luminol Luminescence

[0064] First, the intensity of the luminescent agent was observed when the aptamer was not modified with ferrocene: the reservoirs of the cathode electrode and the anode electrode 1 and the anode electrode 2 were filled with pH 6.5, 10mM PBS buffer, 3.5mM Ru(bpy)3 2+ -TPrA (by Ru(bpy)3 2+ TPrA was mixed and vortexed) and 10 μL of Luminol-H2O2 solution (mixed and vortexed with Luminol and H2O2), and a constant potential of 5.5 V was applied to the dual-signal aptamer sensor using an electrochemical workstation. The blue ECL intensity of Luminol in anode electrode 2 was very dim, while the blue ECL intensity of Ru(bpy)3 in anode electrode 1 was very dim. 2+ The red ECL intensity is very strong ( Figure 2 A). When 10 μL of free 1 μM capture DNA of SEQ ID NO.2 was directly added to the two anode electrodes of the sensor, the blue ECL intensity of Luminol was slightly enhanced, while Ru(bpy)3 2+ The red ECL intensity is slightly reduced ( Figure 2 B) When 10 μL of ferrocene labeled aptamer (1 μM) was added to the two anode electrodes of the sensor, which were away from the electrode surface. Figure 2 C) and the ferrocene-labeled aptamer near the electrode surface ( Figure 2 D), compared with the ferrocene far from the electrode surface, the ferrocene near the electrode surface has a significant promoting effect on the ECL of Luminol, while the ferrocene far from the electrode surface has a significant promoting effect on the ECL of Ru(bpy)3 2+ This control experiment confirmed that the ferrocene near the electrode surface has the most significant effect on the luminescent agent.

[0065] Example 3 Study on dopamine electropolymerization and ECL characterization

[0066] Dopamine (3,4-dihydroxyphenylethylamine) is an important neurotransmitter, and its electrochemical reaction is irreversible or quasi-reversible. Electrochemical polymerization of dopamine monomers can be achieved by potential sweep electrolysis in a 10 mM PBS buffer at pH 6.5. This electrochemical deposition in PBS buffer at pH 6.5 was chosen because dopamine can undergo self-polymerization under weakly alkaline conditions (pH 8.0), making this type of chemical deposition non-site-selective. Figure 3A is the CV curve of electrochemical polymerization of dopamine monomer in PBS buffer. As can be seen from the figure, there are oxidation peaks at 0.3V and 0.6V, while there is no redox peak in the blank control group where PBS buffer was added to the electrode. This indicates that the oxidation products of the electrode reaction have accumulated on the screen-printed BPE surface. Luminol and Ru(bpy)3 2+ The ECL spectrum of Figure 3 As shown in B, Luminol has a characteristic emission peak at 425nm, while Ru(bpy)3 2+ There is a characteristic emission peak at 625 nm, which is consistent with literature reports.

[0067] Example 4 Study on factors affecting ECL intensity

[0068] To meet the detection performance of the aptamer sensor, the concentration of luminescent reagent, Fc-aptamer concentration, bacteria / aptamer incubation time and driving voltage were studied.

[0069] Effect of luminescent reagent concentration on the intensity of its own ECL signal: 10 μL of 1 μM Fc-aptamer of SEQ ID NO. 1 was added to each of the two anode electrodes of the dual-signal aptamer sensor for detecting S. typhimurium prepared in Example 1 and incubated for 1 hour. The electrodes were rinsed with PBS buffer. Then, 10 μL of 2.5 mM Luminol-H2O2 solution was added to anode electrode 2, and 10 μL of 6.0 mM, 5.5 mM, 5.0 mM, 4.5 mM, 4.0 mM, and 3.5 mM Ru(bpy)3 were added to anode electrode 1, respectively. 2+ -TPrA solution, 10 μL PBS buffer was added to the cathode electrode, and a constant potential of 5.5 V was applied to the dual-signal aptamer sensor using an electrochemical workstation to perform ECL signal detection. The ECL image based on the dual-signal bipolar electrode was obtained. The results are shown in FIG. Figure 4 A1 and Figure 4 As shown in A2. With Ru(bpy)3 2+ With the decrease of -TPrA concentration, the red ECL intensity gradually decreased. 2+ When the concentration of -TPrA was 3.5 mM, Ru(bpy)3 2+ The ECL signal is almost completely inhibited by Fc, so Ru(bpy)3 is selected 2+ The optimal concentration of -TPrA solution was 3.5 mM.

[0070] Effect of Luminol-H2O2 concentration on the intensity of its own ECL signal: 10 μL of 1 μM Fc-aptamer of SEQ ID NO.1 was added to each of the two anode electrode ends of the bipolar electrode aptamer dual-signal aptamer sensor for detecting S. typhimurium prepared in Example 1 and incubated for 1 hour. The electrodes were rinsed with PBS buffer; 3.5 mM Ru(bpy)3 was then added to the anode electrode 1. 2+ 10 μL of Luminol-H2O2 solution was added to the anode electrode 2, and 10 μL of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, and 3.0 mM Luminol-H2O2 solution was added to the cathode electrode. 10 μL of PBS buffer was added to the cathode electrode. An electrochemical workstation was used to apply a constant potential of 5.5 V to the dual-signal aptamer sensor for ECL signal detection. The ECL image based on the dual-signal bipolar electrode was obtained. The results are shown in FIG. Figure 4 B1 and Figure 4 B2. As the Luminol-H2O2 concentration increased from 0.5 mM to 2.5 mM, the blue ECL emission intensity increased accordingly. With further increases in Luminol-H2O2 concentration, the ECL intensity decreased slightly due to self-quenching. Therefore, the optimal concentration of the Luminol-H2O2 solution was determined to be 2.5 mM.

[0071] Effect of Fc-aptamer concentration on Ru(bpy)3 2+ Effect of Luminol on the ECL signal intensity: 10 μL of 0 μM, 0.1 μM, 0.3 μM, 0.5 μM, 0.75 μM and 1.0 μM Fc-aptamer of SEQ ID NO. 1 was added to each of the two anode ends of the bipolar electrode aptamer dual-signal aptamer sensor for detecting S. typhimurium prepared in Example 1 and incubated for 1 hour. The electrodes were rinsed with PBS buffer; 3.5 mM Ru(bpy)3 was then added to the anode electrode 1. 2+ 10 μL of TPrA solution was added, 10 μL of 2.5 mM Luminol-H2O2 solution was added to the anode electrode 2, and 10 μL of PBS buffer was added to the cathode electrode. A constant potential of 5.5 V was applied to the dual-signal aptamer sensor using an electrochemical workstation to perform ECL signal detection. The ECL image based on the dual-signal bipolar electrode was obtained. The results are shown in Figure 5 As the concentration of Fc-aptamer increased from 0μM to 1.0μM, Ru(bpy)3 2+ The red ECL emission intensity of Luminol decreased, while the blue ECL emission intensity of Luminol increased. When the Fc-aptamer concentration was 1 μM, Ru(bpy)3 2+The red ECL signal of Luminol was almost completely suppressed, and the blue ECL signal of Luminol was the strongest. Therefore, the final concentration of Fc-aptamer was determined to be 1 μM.

[0072] Effect of incubation time on Ru(bpy)3 2+ Effect of Luminol on the ECL signal intensity: 10 μL of 1 μM Fc-aptamer of SEQ ID NO. 1 was added to each of the two anode electrode ends of the bipolar electrode aptamer dual-signal aptamer sensor for detecting S. typhimurium prepared in Example 1 for incubation for 15 min, 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. The electrodes were rinsed with PBS buffer; 3.5 mM Ru(bpy)3 was then added to the anode electrode 1. 2+ 10 μL of TPrA solution was added, 10 μL of 2.5 mM Luminol-H2O2 solution was added to the anode electrode 2, and 10 μL of PBS buffer was added to the cathode electrode. A constant potential of 5.5 V was applied to the dual-signal aptamer sensor using an electrochemical workstation to perform ECL signal detection. The ECL image based on the dual-signal bipolar electrode was obtained. The results are shown in Figure 6 When the incubation time is 15 to 60 minutes, Ru(bpy)3 2+ The red ECL intensity of Luminol increased with the extension of incubation time, while the blue ECL intensity of Luminol decreased with the extension of incubation time; when the incubation time reached 60 minutes, the red ECL intensity of Ru(bpy)3 2+ The red ECL signal intensity of Luminol reached its maximum, and the blue ECL signal intensity of Luminol reached its minimum; after more than 60 minutes, the ECL intensities of both changed slowly, so 60 minutes was selected as the optimal incubation time.

[0073] Optimization of the applied detection voltage: 10 μL of 1 μM Fc-aptamer of SEQ ID NO. 1 was added to each of the two anode electrode ends of the bipolar electrode aptamer dual-signal aptamer sensor for detecting S. typhimurium prepared in Example 1 and incubated for 60 min. The electrodes were rinsed with PBS buffer; 3.5 mM Ru(bpy)3 was then added to the anode electrode 1. 2+ -TPrA solution 10 μL, 2.5 mM Luminol-H2O2 solution 10 μL was added to the anode electrode 2, 10 μL PBS buffer was added to the cathode electrode, and an electrochemical workstation was used to apply constant potentials of 3.5 V, 4.0 V, 4.5 V, 5.0 V, 5.5 V, and 6.0 V to the dual-signal aptamer sensor, respectively, to perform ECL signal detection and obtain ECL images based on the dual-signal bipolar electrode. Figure 7As shown, with the increase of applied voltage, Luminol and Ru(bpy)3 2+ Considering that the modification of the dual-signal aptamer sensor by Fc-aptamer will lead to the 2+ The red ECL signal of Luminol was weakened, while the blue ECL signal of Luminol was enhanced, and the detection voltage of 5.5V was finally selected. At 5.5V, the blue luminescence signal of Luminol was stronger, while that of Ru(bpy)3 2+ The red luminescence signal is weak, which is convenient for subsequent observation. At a constant potential of 6V, Ru(bpy)3 2+ The red ECL signal was also relatively strong and could not be clearly distinguished from the case of unmodified Fc-aptamer. Finally, a detection voltage of 5.5 V was selected for subsequent experiments to obtain the optimal performance of the dual-signal aptamer sensor.

[0074] Optimal experimental conditions: Ru(bpy)3 2+ The concentration of -TPrA was 3.5 mM, the concentration of Luminol-H2O2 was 2.5 mM, the concentration of Fc-aptamer was 1 mM, the incubation time of bacteria and Fc-aptamer was 60 minutes, and the detection voltage was 5.5 V.

[0075] Example 5 Verification of the Ability of Dual-Signal Aptamer Sensor to Detect Foodborne Pathogens

[0076] The steps for culturing Salmonella typhimurium are as follows:

[0077] ① Remove Salmonella typhimurium strains stored in 50% (v / v) glycerol from a -80°C freezer, thaw, and inoculate 1% of the strain into 10 mL of LB (Luria-Bertani) liquid medium. Incubate at 37°C, 160 rpm, in a shaker for 18-24 hours.

[0078] ② The obtained bacterial solution was transferred into 10 mL of LB liquid medium at a 1% inoculum volume, and cultured in a shaker at 37°C and 160 rpm for 2.5 hours to obtain a Salmonella typhimurium bacterial solution;

[0079] ③ Transfer 6 mL of bacterial solution to a 10 mL centrifuge tube and centrifuge at 10,000 rpm for 10 min at 4°C using a refrigerated centrifuge. Discard the supernatant to obtain the bacterial pellet.

[0080] ④ Add 6 mL of sterile saline (0.85% NaCl) to the bacterial pellet, resuspend the cells, centrifuge at 10,000 rpm for 10 min at 4°C to wash away the residual culture medium, and then add 6 mL of sterile saline to resuspend the cells. Repeat this operation twice to obtain a bacterial suspension.

[0081] ⑤ The obtained bacterial suspension was diluted with sterile physiological saline to obtain a concentration of 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU mL -1 of Salmonella typhimurium suspension.

[0082] The culture steps for Staphylococcus aureus and Escherichia coli O157:H7 were the same as those described above.

[0083] Take 300 μL of 1 mM Fc-tagged bacterial aptamer solution of SEQ ID NO.1 and mix with 300 μL of 0, 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU mL -1 The Salmonella typhimurium suspension was mixed, incubated in a centrifuge tube at 37°C for 1 hour, and then centrifuged at 8000 rpm for 10 minutes to obtain nine groups of supernatants containing Fc-tagged aptamers, which were negatively correlated with the original bacterial suspension concentration. Figure 8 As shown in the steps, the supernatant containing Fc-tagged aptamers was dripped onto the two anode electrodes of the dual-signal aptamer sensor, incubated for 1 h, and the electrodes were rinsed with PBS buffer; then 10 μL of 3.5 mM Ru(bpy)3 was dripped onto anode electrode 1 and anode electrode 2, respectively. 2+ -TPrA and 2.5mM Luminol-H2O2, add 10μL PBS buffer to the cathode electrode, use an electrochemical workstation to apply a 5.5V constant potential on the dual-signal aptamer sensor, and perform ECL signal detection. The ECL image based on the dual-signal bipolar electrode can be identified by the naked eye or analyzed using a mobile phone camera and software. Figure 9 As shown in A, with the increase of Salmonella typhimurium concentration, Ru(bpy)3 2+ The red ECL signal intensity of Luminol gradually increases, while the blue ECL signal intensity of Luminol gradually decreases; below 10 0 CFU mL -1 When Ru(bpy)3 2+ No red ECL signal is generated, and the blue ECL signal of Luminol is the strongest; when the concentration of Salmonella typhimurium is 10 0CFU mL -1 When Ru(bpy)3 2+ The red ECL signal is weak, while the blue ECL signal of Luminol is strong. Figure 9 As shown in B, Ru(bpy)3 2+ The RGB intensity of the Luminol gradually increases, while the RGB intensity of the Luminol gradually decreases. The naked eye can make a preliminary judgment on the concentration of Salmonella Typhimurium. A more accurate quantitative assessment is Figure 9 As shown in C, with the increase of Salmonella typhimurium concentration, Ru(bpy)3 2+ The ratio of the RGB intensity of Luminol to the RGB intensity of Luminol gradually increases. 0 ~10 7 CFU mL -1 The experiment was conducted on Salmonella typhimurium. 0 ~10 6 CFU mL -1 Ru(bpy)3 2+ The ratio of the ECL intensity of Luminol is linear, and the linear equation is y = 0.3001logx + 0.08567, R 2 =0.9963; at a concentration of 10 7 CFU mL -1 When the Ru(bpy)3 2+ The ratio of the ECL intensity of Luminol to the linear equation is far from that of the linear equation and is not within the detection range. 0 CFU mL -1 That is, the minimum concentration of the substance to be tested can be determined in this experiment, which is the detection limit, and the dynamic detection range is 10 0 ~10 6 CFU mL -1 .

[0084] The same method was used to detect the concentration of 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU mL -1 S. aureus bacterial suspension and 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 CFU mL -1 E.coli O157:H7 bacterial suspension, the test results are as follows Figure 10 Similarly, the linear equation obtained by S. aureus detection is y=0.2997logx+0.08443, R 2 =0.9955, the linear equation obtained by E.coli O157:H7 detection was y=0.3027logx+0.07353, R 2 =0.9954. 7 CFU mL -1 When Ru(bpy)3 was measured in S.aureus and E.coli O157:H7 2+ The ratio of the ECL intensity to Luminol is also far from the linear equation and is out of the detection range. 0 CFU mL -1 When Ru(bpy)3 2+ No red ECL signal is generated, and the blue ECL signal of Luminol is the strongest; when the concentration of S. aureus and E. coli O157:H7 is 10 0 CFUmL -1 When Ru(bpy)3 2+ The red ECL signal is weak, and the blue ECL signal of Luminol is strong. 0 CFU mL -1 It is also the detection limit of S. aureus and E. coli O157:H7, with a detection range of 10 0 ~10 6 CFU mL -1 Therefore, the detection limit of the dual-signal aptasensor is 10 0 CFU mL -1 , the detection range is 10 0 ~10 6 CFU mL -1 In summary, the dual-signal aptasensor dual-signal sensing detection method for foodborne pathogens is suitable for the detection of S. typhimurium, S. aureus and E. coli O157:H7.

[0085] Compared with the traditional method of observing ECL intensity, the dual-signal ECL intensity ratio detection adjusts to environmental changes by self-calibrating the ratio of the two signals, effectively avoiding the interference of external factors on the traditional molecular probe signal, and enhancing the accuracy and sensitivity of the sensor in detecting foodborne pathogens.

[0086] Example 6 Specific Detection of Dual-Signal Aptamer Sensor

[0087] According to the method of Example 5, 12 test samples were prepared: (1) 10 CFU mL -1 Fc-aptamer-S.typhimurium and S.typhimurium; (2)10CFU mL -1 Fc-aptamer-S. aureus and S. aureus; (3) 10 CFU mL -1 Fc-aptamer-E.coli O157:H7 and E.coli O157:H7; (4) 10 3 CFU mL -1 Fc-aptamer-S.typhimurium and S.typhimurium; (5)10 3 CFU mL -1 Fc-aptamer-S. aureus and S. aureus; (6) 10 3 CFU mL -1 Fc-aptamer-E.coli O157:H7 and E.coli O157:H7; (7) 10 6 CFUmL -1 Fc-aptamer-S.typhimurium and S.typhimurium; (8)10 6 CFU mL -1 Fc-aptamer-S. aureus and S. aureus; (9) 10 6 CFU mL -1 Fc-aptamer-E.coli O157:H7 and E.coli O157:H7; (10) 10 CFU mL -1 A mixture of Fc-aptamer-S.typhimurium, S.typhimurium, Fc-aptamer-S.aureus, S.aureus, Fc-aptamer-E.coli O157:H7 and E.coli O157:H7; (11) 10 3 CFU mL -1 A mixture of Fc-aptamer-S.typhimurium, S.typhimurium, Fc-aptamer-S.aureus, S.aureus, Fc-aptamer-E.coli O157:H7 and E.coli O157:H7; (12) 10 6 CFU mL -1A mixture of Fc-aptamer-S.typhimurium, S.typhimurium, Fc-aptamer-S.aureus, S.aureus, Fc-aptamer-E.coliO157:H7 and E.coli O157:H7.

[0088] Detection of blank group: 10μL Luminol-H2O2 and Ru(bpy)3 were added to the two anode electrodes of the dual-signal aptamer sensor respectively. 2+ -TPrA luminescent agent, the cathode is 10μL PBS buffer, each group of test samples are added to the two anode electrodes respectively, and a constant voltage of 5.5V is applied to both ends of the dual-signal aptamer sensor using an electrochemical workstation to obtain Ru(bpy)3 at different concentrations. 2+ Ratio of RGB intensity to Luminol ( Figure 11 B).

[0089] According to the steps of Example 5, solutions (1) to (12) were added dropwise to the two anode electrodes of the dual-signal aptamer sensor containing the captured DNA sequence such as SEQ ID NO. 2, incubated for 1 h, and the electrodes were rinsed with PBS buffer; then 10 μL of Luminol and Ru(bpy)3 were added dropwise. 2+ To the two anode electrodes, 10 μL PBS buffer was added to the cathode, and ECL signal detection was performed under a constant potential of 5.5 V. The negative sample without S. typhimurium added in Example 5 was used as a control to compare the Ru(bpy)3 2+ The RGB intensity ratio of Luminol is as follows: Figure 11 As shown: Ru(bpy)3 of group (1)(2)(3) and group (10)(11)(12) 2+ The RGB intensity ratios of Luminol are basically the same, while the Ru(bpy)3 2+ The RGB intensity ratios with Luminol were very small, consistent with the negative sample area. Therefore, the dual-signal aptamer sensor containing the captured DNA sequence such as SEQ ID NO. 2 is suitable for the specific detection of S. typhimurium.

[0090] Using the same method, it was determined that the dual-signal aptamer sensor containing the captured DNA sequence of SEQ ID NO. 4 was suitable for the specific detection of S. aureus, and the dual-signal aptamer sensor containing the captured DNA sequence of SEQ ID NO. 6 was suitable for the specific detection of E. coli O157:H7.

[0091] Example 7 Actual sample detection

[0092] The actual samples were processed according to the China National Food Safety Standard (GB 4789.4-2016) Food Microbiology Test for Salmonella Typhimurium. The actual samples were beef, bread, and pure milk, all of which were purchased from local supermarkets.

[0093] For liquid samples (pure milk), homogenization is not required and oscillation is used to mix directly. For solid samples (beef, bread), 2.5 g of each sample was weighed and placed in a sterile homogenization bag containing 22.5 mL of 1× PBS buffer. Beat for 1 to 2 minutes to make a 1:10 sample solution. Two groups of each sample were prepared. Aseptically transfer the sample to a 100 mL conical flask and add 2.5 mL of 0, 10, and 10% PBS solution to the sample. 0 and 10 1 CFU mL -1 typhimurium; 300 μL of the actual sample containing Salmonella typhimurium was incubated with 300 μL of 1 μM Fc-tagged aptamer with the sequence shown in SEQ ID NO.1 at 37°C for 1 hour, followed by centrifugation at 8000 rpm for 10 minutes to obtain a supernatant containing the Fc-tagged aptamer. The supernatant containing the Fc-tagged aptamer was dripped onto the two anode electrodes of the dual-signal aptamer sensor, incubated for 1 hour, and the electrodes were rinsed with PBS buffer; then 10 μL of 3.5 mM Ru(bpy)3 was dripped onto anode electrode 1 and anode electrode 2, respectively. 2+ -TPrA and 2.5mM Luminol-H2O2, 10μL PBS buffer was added to the cathode, and a constant potential of 5.5V was applied to the dual-signal aptamer sensor using an electrochemical workstation to obtain Ru(bpy)3 2+ ECL images with Luminol ( Figure 12 A) and Ru(bpy)3 2+ Ratio of RGB intensity to Luminol ( Figure 12 B). Figure 12 As shown, the ECL images of the three actual samples at different Salmonella typhimurium concentrations are consistent with the ECL signals of Salmonella typhimurium detected in Example 5. 2+ The RGB intensity ratio trend mirrored that of Luminol. These results demonstrate that the influence of the food matrix on ECL signal intensity is negligible in the dual-signal aptasensor device. At the same S. Typhimurium concentration, the ECL signal intensities of different food samples were very similar. These experimental results demonstrate that the dual-signal aptasensor exhibits excellent accuracy and sensitivity and can be used as a quantitative detection method for S. Typhimurium in food samples.

[0094] The dual-signal aptamer sensor containing the corresponding captured DNA was used to detect the concentrations of 0 and 10 0 and 10 1 CFU mL -1 Staphylococcus aureus and Escherichia coli O157:H7, the obtained Ru(bpy)3 2+ ECL images with Luminol and Ru(bpy)3 2+ The RGB intensity ratios of Luminol are as follows: Figure 13 and Figure 14 The ECL images of the three actual samples at different concentrations of Staphylococcus aureus and Escherichia coli O157:H7 are consistent with the ECL signals of Staphylococcus aureus and Escherichia coli O157:H7 detected in Example 5. 2+ The RGB intensity ratio trend mirrored that of Luminol. These results demonstrate that the influence of the food matrix on ECL signal intensity is negligible in the dual-signal aptasensor device. At the same concentration of Staphylococcus aureus and E. coli O157:H7, the ECL signal intensities of different food samples were very similar. In summary, the dual-signal aptasensor exhibits excellent accuracy and sensitivity and can be used as a quantitative detection method for Salmonella Typhimurium, Staphylococcus aureus, and E. coli O157:H7 in food samples.

Claims

1. A method for detecting foodborne pathogens based on a dual-signal aptamer sensor, characterized in that: The dual-signal aptamer sensor includes a cathode electrode, an anode electrode 1 and an anode electrode 2, an electrode interface, and a working electrode lead. The cathode electrode, anode electrode 1, and anode electrode 2 form two sets of bipolar electrodes; a reservoir 1 is provided on the cathode electrode, a reservoir 2 is provided on the anode electrode 1, and a reservoir 3 is provided on the anode electrode 2; the anode electrode 1 and the anode electrode 2 are screen-printed electrodes modified with a polydopamine film, and the polydopamine film also includes amino-modified capture DNA; The method comprises the following steps: 1) The Fc-labeled bacterial aptamer and the bacterial sample solution are mixed, incubated, and centrifuged to obtain the Fc-aptamer supernatant; 2) The supernatant of the Fc-aptamer was added dropwise to the anode electrode 1 and the anode electrode 2 of the dual-signal aptamer sensor, respectively, incubated, and the electrodes were rinsed with PBS buffer; 3) Add Ru(bpy)3 to anode electrode 1 and anode electrode 2 respectively 2+ -TprA solution and Luminol-H2O2 solution, add PBS buffer on the cathode, and perform ECL signal detection under the action of constant potential; 4) When anode electrode 1 does not emit light and anode electrode 2 produces blue light, it proves that the sample solution does not contain foodborne pathogens; when anode electrode 1 produces red light, it proves that the sample solution contains foodborne pathogens; 5) According to Ru(bpy)3 2+ The content of foodborne pathogens was detected by the RGB intensity ratio of Luminol.

2. The method according to claim 1, characterized in that The concentration of the Luminol-H2O2 solution is 0.5~3 mM, and the Ru(bpy)3 2+ The concentration of the -TprA solution is 3.5~6 mM.

3. The method according to claim 1, characterized in that The Fc-labeled bacterial aptamer is a DNA fragment as shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5; the concentration of the Fc-labeled bacterial aptamer is 0-1 μM.

4. The method according to claim 1, wherein The incubation time of the bacteria-containing sample solution and the Fc-labeled bacterial aptamer in step 1) is 15-90 minutes; the constant potential voltage in step 3) is 3.5-6V.

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