Preparation method and application of electrochemical dynamic detection platform with multi-stage signal amplification

Abstract

The application provides a preparation method and application of an electrochemical dynamic detection platform with multi-stage signal amplification, gold / polydopamine nanoparticles are prepared, the gold / polydopamine nanoparticles are modified on the surface of a gold electrode, and the gold electrode with the surface modified with the gold / polydopamine nanoparticles is used as a working electrode, is assembled with a counter electrode and a reference electrode in an electrolytic cell device, is connected with an electrochemical workstation, and thus the electrochemical dynamic detection platform with multi-stage signal amplification is obtained. The gold / polydopamine nanoparticles are used for modifying the gold electrode to realize first-stage signal amplification, the polydopamine surface phenolic hydroxyl group mediated redox double electron cycle system is used to realize second-stage signal amplification, and in application, the specific recognition and third-stage signal amplification are realized by using the beta-galactosidase secreted by Escherichia coli to catalyze a reaction. The electrochemical dynamic detection platform has the advantages of high sensitivity, strong anti-interference, and the ability of specific detection and continuous dynamic monitoring of Escherichia coli in actual samples by multi-stage signal amplification.

Description

Technical Field

[0001] This invention relates to the field of Escherichia coli detection technology, and in particular to a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. Background Technology

[0002] Escherichia coli (E. coli) is a common foodborne microorganism in daily life. Infection with it can easily cause diarrhea and other diseases. Therefore, early monitoring of E. coli to prevent related diseases is of great significance and importance. The national standard for E. coli detection is the agar plate method, which involves incubating the bacteria on a culture medium for 24 hours and counting the number of bacterial colonies to determine whether infection has occurred. However, the agar plate method has significant drawbacks: the counting method results in a high detection limit, and the long incubation and bacterial proliferation time makes it insufficient in terms of sensitivity and timeliness.

[0003] Currently, several new technologies have been proposed to address the problems of the agar plate method, such as paper-based fluorescence and colloidal gold methods. These methods primarily utilize the surface charge of bacteria to induce changes in fluorescence or visible light, but they are all qualitative or semi-quantitative, with limited detection sensitivity. Furthermore, electrochemical impedance spectroscopy or redox methods are used to detect bacteria, utilizing changes in the system's potential, resistance, and current caused by bacterial metabolites. While these methods offer improved sensitivity, they still have many limitations. They cannot meet the requirements for detecting low-concentration bacteria and require pretreatment of the bacteria, such as specific labeling, to address specificity issues. This process is relatively time-consuming and complex, and they cannot achieve specific detection of *E. coli*.

[0004] Electrochemical detection technology converts the chemical signal generated between the sensitizing material on the working electrode surface and the target analyte into an electrical signal. Due to its advantages such as high sensitivity, good stability, and low pollution, it has been widely used in environmental monitoring, food safety, and other fields. The working components of an electrochemical detection device mainly include: a sensing element, a conversion element, and a signal output device. The sensing element converts the changes in various parameters during the reaction between the sensitizing material on the working electrode and the analyte into an electrical signal and transmits the obtained electrical signal to the conversion element. The conversion element converts the electrical signal into a digital signal. The signal output device amplifies and displays the signal, enabling qualitative and quantitative analysis of the target analyte. Currently, there are reports of using electrochemical detection technology to detect *Escherichia coli*.

[0005] An invention patent (application number CN202111005201.7) discloses a method for preparing an electrochemical biosensor and its application in the detection of *E. coli*. The method involves dropping CDs-Fe3O4 nanocomposite material onto the surface of a glassy carbon electrode to prepare a CDs-Fe3O4 / GCE working electrode. After activation, the electrode is immersed in probe DNA to obtain a DNA / CDs-Fe3O4 / GCE probe electrode. The probe electrode is then immersed in *E. coli* O157:H7 solutions of different concentrations for *E. coli* detection. This electrochemical biosensor is specific only to *E. coli* O157:H7 strain, and the reaction time between the probe electrode and the strain is relatively long, resulting in poor detection timeliness and efficiency. Furthermore, this biosensor cannot perform long-term continuous dynamic monitoring of *E. coli* concentration and proliferation activity.

[0006] In view of this, it is necessary to design an improved method for preparing and applying an electrochemical dynamic detection platform with multi-stage signal amplification and multiple signal amplification functions to solve the above problems. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. This electrochemical dynamic detection platform uses gold / polydopamine nanoparticles to modify the surface of a gold electrode to achieve the first stage of signal amplification, utilizes the redox two-electron cycle system mediated by the phenolic hydroxyl groups on the polydopamine surface to achieve the second stage of signal amplification, and utilizes the β-galactosidase catalytic reaction specifically secreted by E. coli to achieve specific recognition and the third stage of signal amplification. Through the multi-stage signal amplification effect, highly sensitive and specific detection of E. coli is achieved, enabling long-term continuous dynamic monitoring of the concentration and proliferation activity of E. coli in food and beverages.

[0008] To achieve the above-mentioned objectives, this invention provides a method for preparing a multi-stage signal amplification electrochemical dynamic detection platform, comprising the following steps:

[0009] S1. Polydopamine nanoparticles are uniformly dispersed in deionized water to obtain a polydopamine solution. Chloroauric acid solution is added to the polydopamine solution under shaking condition, and then shaken at constant temperature for 20-28 hours. After multiple centrifugation treatments and washing, gold / polydopamine nanoparticles are obtained.

[0010] S2. The gold / polydopamine nanoparticles obtained in step S1 are modified onto the surface of the gold electrode to obtain a gold electrode with surface-modified gold / polydopamine nanoparticles.

[0011] S3. Using the gold electrode with surface-modified nano-gold / polydopamine obtained in step S2 as the working electrode, the platinum electrode as the counter electrode, and the silver or silver chloride electrode as the reference electrode, the three are assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain an electrochemical dynamic detection platform with multi-stage signal amplification.

[0012] As a further improvement of the present invention, in step S1, the particle size of the polydopamine nanoparticles is 50-200 nm; the concentration of the polydopamine solution is 0.5%-1.5%; and the concentration of the chloroauric acid solution is 0.05%-0.2%.

[0013] As a further improvement of the present invention, the volume ratio of the chloroauric acid solution to the polydopamine solution is (10-20):1.

[0014] As a further improvement of the present invention, in step S1, the method for preparing the polydopamine nanoparticles includes the following steps:

[0015] S11. Dissolve dopamine hydrochloride in deionized water to obtain a dopamine hydrochloride solution with a concentration of 1-2 mg / mL; place the magnetic ball into the dopamine hydrochloride solution and heat and stir to 45-60°C;

[0016] S12. Add a 0.8-1.5M NaOH solution to the hydrochloric acid dopamine solution being stirred in step S11, continue stirring for 1-3 minutes, let it stand for 4-6 hours after it becomes uniform, and then perform multiple centrifugations and washing to obtain the polydopamine nanoparticles.

[0017] As a further improvement of the present invention, in step S2, the method of modifying the gold / polydopamine nanoparticles on the surface of the gold electrode is as follows: the gold / polydopamine nanoparticles are uniformly mixed with a solvent to prepare a nano-gold / polydopamine solution, and the nano-gold / polydopamine solution is uniformly dispersed on the surface of the gold electrode by electrodeposition or dropwise addition. After drying, the gold electrode with surface-modified nano-gold / polydopamine is obtained.

[0018] As a further improvement of the present invention, in step S12, the volume ratio of the dopamine hydrochloride solution to the NaOH solution is (200-250):1.

[0019] As a further improvement of the present invention, in step S1, the constant temperature is 25°C; the centrifugation speed of the centrifugation process is 6000-10000 rpm, and the time is 8-15 min.

[0020] As a further improvement of the present invention, in step S12, the centrifugation speed is 8000-12000 rpm and the time is 8-15 min.

[0021] The present invention also provides an application of a multi-stage signal amplification electrochemical dynamic detection platform, which is used to perform electrochemical detection on an Escherichia coli detection solution; the Escherichia coli detection solution includes Escherichia coli and solution A; solution A is a solution that can undergo an enzymatic reaction with β-galactosidase.

[0022] As a further improvement of the present invention, the Escherichia coli detection solution further includes a ruthenium tripyridine solution, wherein solution A is a 4-aminophenyl-β-D-galactopyranoside solution.

[0023] The beneficial effects of this invention are:

[0024] 1. The present invention provides a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. First, gold / polydopamine nanoparticles are prepared; then, the gold / polydopamine nanoparticles are modified on the surface of a gold electrode to obtain a gold electrode with surface-modified gold / polydopamine nanoparticles; finally, the gold electrode with surface-modified gold / polydopamine nanoparticles is used as the working electrode, a platinum electrode as the counter electrode, and a silver or silver chloride electrode as the reference electrode, and assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain a multi-stage signal amplification electrochemical dynamic detection platform. The electrochemical dynamic detection platform of this invention employs gold / polydopamine nanoparticles to modify the surface of a gold electrode to achieve the first level of signal amplification, utilizes the redox two-electron cycle system mediated by the phenolic hydroxyl groups on the polydopamine surface to achieve the second level of signal amplification, and utilizes the β-galactosidase catalytic reaction specifically secreted by E. coli to achieve specific recognition and the third level of signal amplification. This electrochemical dynamic detection platform achieves highly sensitive and specific detection of E. coli through multiple signal amplification effects, and simultaneously enables long-term continuous dynamic monitoring of the concentration and proliferation activity of E. coli in food and beverages.

[0025] 2. This invention achieves first-level signal amplification by modifying electrode materials with gold / polydopamine nanoparticles. Modification with gold / polydopamine nanoparticles increases the electrode's specific surface area and active sites, forming a conductive nanonetwork structure that significantly enhances electron transfer efficiency. Furthermore, by controlling the size of the gold and / or polydopamine nanoparticles, the electrode's specific surface area and electron transfer efficiency can be further improved. Simultaneously, this invention utilizes a redox multi-electron cycle system mediated by the phenolic hydroxyl groups on the polydopamine surface, introducing two electron carriers with different redox potentials to achieve a second-level signal amplification effect. When applied to a multi-stage signal amplification electrochemical dynamic detection platform, the catalytic reaction of E. coli's specific galactosidase not only achieves a third-level signal amplification and efficient electron transfer but also enables specific detection of the substrate. The triple signal amplification effect of this invention achieves highly sensitive monitoring of E. coli, reducing detection time to less than 30 minutes, eliminating the need for pretreatment, and demonstrating good practical value and application prospects.

[0026] 3. The preparation steps of this invention are simple and highly feasible; when the prepared electrochemical dynamic detection platform is used, no pretreatment of bacteria is required, and specific enzymes and their substrates can be directly detected, eliminating the detection steps and improving the overall detection efficiency; at the same time, it realizes dynamic and highly sensitive detection of Escherichia coli proliferation activity, and can perform long-term continuous dynamic monitoring of Escherichia coli in actual samples such as food and beverages. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the preparation process and characterization of the gold / polydopamine nanoparticles of the present invention.

[0028] Figure 2 This diagram illustrates the highly sensitive and rapid detection of Escherichia coli using the electrochemical dynamic detection platform with multiple signal amplification functions of this invention.

[0029] Figure 3 This is a schematic diagram illustrating the operation of the electrochemical dynamic detection platform of the present invention for bacterial detection.

[0030] Figure 4 This is a comparative analysis of the amplification effect of the electrochemical detection signal of Escherichia coli at different time points in Example 1.

[0031] Figure 5 This is a comparative analysis of the amplification effect of the electrochemical detection signal of Escherichia coli at different time points in Comparative Example 1.

[0032] Figure 6 for Figure 4 and Figure 5 The changing trend of the peak value of the intermediate oxidation peak.

[0033] Figure 7This is a comparative analysis of the amplification effect of the electrochemical detection signal of Escherichia coli at different time points during the detection of Comparative Examples 2 and 3.

[0034] Figure 8 The graph shows the electrochemical detection results of different concentrations of bacterial markers in the three liquid samples of Examples 3-5.

[0035] Figure 9 The graph shows the results of continuous electrochemical monitoring of the concentration of Escherichia coli added to the three liquid samples of Examples 3-5. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] It should also be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0038] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0039] A method for preparing a multi-stage signal amplification electrochemical dynamic detection platform includes the following steps:

[0040] S1. Polydopamine nanoparticles (PDA) are uniformly dispersed in deionized water to obtain a polydopamine solution. Chloroauric acid solution is added to the polydopamine solution under shaking condition, and then shaken at constant temperature for 20-28 hours. After multiple centrifugation treatments and washing, gold / polydopamine nanoparticles (Au / PDAjanus nanoparticles) are obtained.

[0041] The polydopamine nanoparticles have a particle size of 50–200 nm; the polydopamine solution has a concentration of 0.5%–1.5%; the chloroauric acid solution has a concentration of 0.05%–0.2%; the volume ratio of chloroauric acid solution to polydopamine solution is (10–20):1; the constant temperature is 25℃; the centrifugation speed is 6000–10000 rpm, and the time is 8–15 min. By adjusting the above parameters, the size of the prepared polydopamine nanoparticles can be controlled, further improving the specific surface area and electron transfer efficiency of the electrode.

[0042] S2. Modify the gold / polydopamine nanoparticles obtained in step S1 onto the surface of the gold electrode to obtain a gold electrode with surface-modified nano-gold / polydopamine.

[0043] S3. Using the gold electrode with surface-modified nano-gold / polydopamine obtained in step S2 as the working electrode, the platinum electrode as the counter electrode, and the silver or silver chloride electrode as the reference electrode, the three are assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain an electrochemical dynamic detection platform with multi-stage signal amplification.

[0044] Specifically, in step S2, the method for modifying the gold / polydopamine nanoparticles on the surface of the gold electrode is as follows: the gold / polydopamine nanoparticles are uniformly mixed with a solvent to prepare a nano-gold / polydopamine solution, and the nano-gold / polydopamine solution is uniformly dispersed on the surface of the gold electrode by electrodeposition or dropwise addition. After drying, the gold electrode with surface-modified nano-gold / polydopamine is obtained.

[0045] Please see Figure 1 As shown, in step S1, the preparation method of polydopamine nanoparticles includes the following steps:

[0046] S11. Dissolve dopamine hydrochloride in deionized water to obtain a dopamine hydrochloride solution with a concentration of 1-2 mg / mL; place the magnetic ball into the dopamine hydrochloride solution and heat and stir to 45-60℃;

[0047] S12. Add a 0.8-1.5M NaOH solution dropwise to the dopamine hydrochloride solution being stirred in step S11, and continue stirring for 1-3 minutes. After it becomes homogeneous, let it stand for 4-6 hours, and then perform multiple centrifugations and washing to obtain polydopamine nanoparticles. The volume ratio of dopamine hydrochloride solution to NaOH solution is (200-250):1, the centrifugation speed is 8000-12000 rpm, and the time is 8-15 minutes.

[0048] The gold / polydopamine nanoparticle (Au / PDAjanus) modified electrode material proposed in this invention improves the specific surface area and active sites of the electrode, forming a conductive nanonetwork structure, which greatly increases the electron transfer efficiency. This nanoelectrode realizes the first-level signal amplification function. Moreover, the preparation steps of gold / polydopamine nanoparticles are simple and the size is controllable. The polydopamine nanoparticles are about 50-200 nm in size, and the gold particles are about 20-60 nm in size.

[0049] Please see Figure 3As shown, this invention also provides an application of a multi-stage signal amplification electrochemical dynamic detection platform, which is used to perform electrochemical detection of an Escherichia coli detection solution. The E. coli detection solution includes E. coli and solution A; solution A is a solution that can undergo an enzymatic reaction with β-galactosidase. When applying the multi-stage signal amplification electrochemical dynamic detection platform, this invention takes advantage of the characteristic that E. coli can secrete a special β-galactosidase through its own proliferation and metabolism, and uses solution A, which can undergo an enzymatic reaction with β-galactosidase, as the substrate. The β-galactosidase produced by E. coli can catalyze the production of p-aminophenol (PAP) from solution A, and PAP participates in the electrochemical cycle system, which not only achieves a third level of signal amplification and efficient electron transfer, but also achieves specific detection of E. coli.

[0050] Specifically, the E. coli detection solution also includes ruthenium tripyridine (Ru) solution; solution A is 4-aminophenyl-β-D-galactopyranoside (PAPG) solution. Please refer to [link / reference]. Figure 2 As shown, this invention utilizes a redox multi-electron cycling system mediated by the phenolic hydroxyl groups on the surface of polydopamine, introducing an electron transporter (ruthenium tripyridine) with different redox potentials as an independent cycling system; simultaneously, a 4-aminophenyl-β-D-galactopyranoside solution undergoes an enzymatic reaction with β-galactosidase released by Escherichia coli, producing p-aminophenol (PAP), which participates in the redox cycling system as another independent cycling system; when the two substances are present simultaneously, they can synergistically amplify the redox reaction signal, achieving a second signal amplification effect. Applying the dual-cycle electron cycling system to electrochemical bacterial detection improves detection sensitivity.

[0051] In some specific implementations, during E. coli culture, isopropyl-β-D-thiogalactoside (IPTG) inducer is added to E. coli, followed by isothermal incubation. The addition of the inducer can promote the specific release of β-galactosidase from E. coli.

[0052] In some specific implementations, the method for culturing *E. coli* is as follows: Prepare 500 mL of LB medium (5 g tryptone, 2.5 g yeast extract, 5 g sodium chloride, and 500 mL deionized water), prepare two Erlenmeyer flasks, and cover the flask mouths with aluminum foil. Prepare pipette tips and centrifuge tubes. Place the above materials in an autoclave for 2 hours for sterilization. After sterilization, place the materials in a UV transfer window and irradiate with UV light for 30 minutes. Then, transfer the materials to the bacteriological chamber, remove them, and place them on the operating table. Take 170 mL of LB medium from a beaker and place it in an Erlenmeyer flask. Allow it to cool, then remove the frozen *E. coli* inoculum from the refrigerator and allow it to thaw. Use a pipette to add 200 μL of *E. coli* to the Erlenmeyer flask containing the LB medium. Place the flask in a constant temperature incubator at 37°C and a rotation speed of 180 rpm for 12 hours to cultivate the first generation of *E. coli*. Repeat the culture process to culture second-generation *E. coli* (note that when culturing second-generation *E. coli*, 200 μL should be taken from the first-generation *E. coli* and added to a new container containing 170 mL of LB medium). After culturing for 5 hours, it is ready for use (5 hours is optimal). After removing the *E. coli*, perform OD600 analysis (note that the *E. coli* in the medium is analyzed directly without centrifugation; the blank group used is LB medium), and add LB medium until the OD600 value of the *E. coli* is 1. At this point, the *E. coli* solution is ready.

[0053] The electrochemical dynamic detection platform of this invention achieves first-level signal amplification by modifying the gold electrode surface with gold / polydopamine nanoparticles, and second-level signal amplification by utilizing the redox two-electron cycle system mediated by the phenolic hydroxyl groups on the polydopamine surface. In the detection of Escherichia coli, it achieves specific recognition and third-level signal amplification by utilizing the β-galactosidase catalytic reaction specifically secreted by E. coli. Thus, through multiple signal amplification effects, this electrochemical dynamic detection platform not only achieves highly sensitive and specific detection of E. coli, shortening the detection time to less than 30 minutes, but also enables long-term continuous dynamic monitoring of the concentration and proliferation activity of E. coli in food and beverages.

[0054] It should be noted that the multi-stage signal amplification electrochemical dynamic detection platform of the present invention has the characteristics of strong anti-interference, high sensitivity and high efficiency when detecting Escherichia coli, which can shorten the detection time to less than 30 minutes, and when the detection time is not required, the lowest detection limit concentration can be as low as 10. 1 CFU / mL.

[0055] Example 1

[0056] This embodiment provides a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform, including the following steps:

[0057] S1. Polydopamine nanoparticles were uniformly dispersed in 20 mL of deionized water to obtain a 1% (w / w) polydopamine solution. 3 mL of the polydopamine solution was taken out using a pipette, and 0.2 mL of chloroauric acid solution (w = 0.1%) was added while shaking to ensure thorough mixing. The mixture was then placed in a constant temperature shaking incubator and shaken continuously for 24 h. The product was then centrifuged multiple times (8000 rpm, 10 min) and washed to obtain gold / polydopamine nanoparticles. The preparation method of the polydopamine nanoparticles is as follows:

[0058] S11. Take 180 mg of dopamine hydrochloride, dissolve it in 90 mL of deionized water, put the magnetic ball into the solution and use a heating stirrer to stir and heat the dopamine hydrochloride solution to 50°C.

[0059] S12. Add 0.4 mL of 1 M NaOH solution dropwise to the dopamine hydrochloride solution being stirred in step SS. When the NaOH solution is added, the color of the solution immediately turns pale yellow, and gradually turns dark brown over time. Continue stirring for 3 min, and after it becomes homogeneous, let it stand for 5 h. Then, put the polydopamine solution into 50 mL centrifuge tubes and centrifuge them (10000 rpm, 10 min). During the centrifugation, rinse with deionized water several times to finally obtain polydopamine nanoparticles.

[0060] S2. The gold / polydopamine nanoparticles obtained in step S1 are modified on the surface of the gold electrode by the dropwise addition method. Specifically, the gold / polydopamine nanoparticles are uniformly mixed with a solvent to prepare a nano gold / polydopamine solution. 8 μL of the solution is taken with a pipette and uniformly dispersed on the surface of the gold electrode. After the water is completely evaporated, the gold electrode with nano gold / polydopamine surface modified is obtained.

[0061] S3. Using the gold electrode with surface-modified nano-gold / polydopamine obtained in step S2 as the working electrode, the platinum electrode as the counter electrode, and the silver or silver chloride electrode as the reference electrode, the three are assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain a multi-stage signal amplification electrochemical dynamic detection platform.

[0062] S4. Electrochemical detection of the *E. coli* test solution: Take 20 mL of *E. coli* with an OD600 of 1, add 3 mL of a pre-prepared 1 mM 4-aminophenyl-β-D-galactopyranoside (PAPG) solution, and incubate at 37°C for 30 min. Remove and place in the electrolytic cell of the electrochemical dynamic detection platform, simultaneously adding 1 mL of ruthenium tripyridine solution. Then, place one end of the nitrogen-purging tube into the electrolytic cell solution (i.e., the test solution), and purge with nitrogen for three minutes to remove hydrolyzed oxygen from the solution. Turn on the electrochemical workstation, open the computer, use the chi66e software, select the method, cyclic voltammetry (CV), and set the parameters, such as initial voltage 0V, maximum voltage 0.55V, minimum voltage -0.45V, cycle rate 0.1V / s, and 7 cycles. Click the test button; the electrochemical workstation will begin working and perform a cyclic scan of the solution. After the scan is complete, click "Finish" and save the data.

[0063] Please see Figure 4 As shown, this embodiment tested E. coli at different time points (0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 24h, 36h, and 48h) after adding 3mL of PAPG, starting from 0h. A comparative analysis of the electrochemical detection signal amplification effect was obtained. Figure 4 It can be seen that after the addition of the substrate PAPG, the bacterial proliferation and metabolism produce β-galactosidase, which can effectively decompose the substrate PAPG to produce PAP molecules. The PAP molecules then diffuse to the electrode surface and participate in the two-electron oxidation cycle system, working synergistically with ruthenium terpyridine to achieve a cascade amplification effect of the signal. With the increase of time, a detection signal appears at the post-oxidation peak at 0.5 h, and the signal continuously strengthens. This result indicates that the electrochemical system can be used to dynamically detect, record, and analyze the proliferation and metabolism process of E. coli.

[0064] Comparative Example 1

[0065] Comparative Example 1 provides a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. The difference from Example 1 is that PAPG solution was not added to E. coli in step S4. The rest is roughly the same as Example 1 and will not be repeated here.

[0066] Please see Figure 5 As shown, Comparative Example 1 tested E. coli at different time points (0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 24h, 36h, and 48h) after adding 3mL of PAPG, starting from 0h. A comparative analysis of the electrochemical detection signal amplification effect was obtained. Figure 5It can be seen that without the addition of the substrate PAPG, the electrochemical response signal of its oxidation peak position does not change significantly with time. Combined with the results of Example 1, the addition of PAPG will generate PAP that can participate in the redox cycle reaction, thus effectively amplifying the detection signal. It also shows that β-galactosidase produced by bacterial proliferation metabolism does not directly participate in the electrode reaction. At this time, the signal is weak and cannot effectively detect Escherichia coli.

[0067] Please see Figure 6 As shown in the figure, the red curve represents Figure 4 The signal intensity of the oxidation peak position changes over time when the substrate contains PAPG. The blue curve represents the change in signal intensity over time. Figure 5 The signal intensity at the mid-oxidation peak position changes over time. The results show that after the addition of the substrate PAPG, the β-galactosidase produced by bacterial proliferation metabolism can decompose the substrate PAPG into PAP through an enzymatic reaction, thereby realizing a two-electron cycling system. Compared with the case without substrate addition, this signal is significantly amplified, which can greatly improve the sensitivity of Escherichia coli detection and obtain a lower detection limit.

[0068] Comparative Example 2

[0069] Comparative Example 2 provides a method for preparing an electrochemical dynamic detection platform and its application. The difference from Example 1 is that, in step S4, when performing electrochemical detection on the Escherichia coli detection solution, ruthenium terpyridine solution was not added. The rest is roughly the same as in Example 1, and will not be repeated here.

[0070] Comparative Example 3

[0071] Comparative Example 3 provides a method for preparing an electrochemical dynamic detection platform and its application. Compared with Example 1, the difference is that steps S1 to S2 are not performed. Instead of the surface-modified nano-gold / polydopamine gold electrode, a gold electrode is directly used as the working electrode. The rest is roughly the same as in Example 1, and will not be described again here.

[0072] Please see Figure 7 As shown, Comparative Examples 2-3 characterized the detection results of *E. coli* at different time points (0.5h, 1h, 1.5h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 24h, 36h, and 48h). The experimental results showed that without the addition of ruthenium terpyridine (Ru) solution and using a standard blank unmodified gold electrode, no effective detection signal could be obtained in the early stage (0h–6h). Although the concentration of β-galactosidase secreted by bacteria increased with time, gradually resulting in a detection signal, the signal was weak (compared to...). Figure 6(Results comparison) and the process is time-consuming, making it difficult to achieve rapid and accurate detection of bacteria. Therefore, comprehensive results show that the addition of ruthenium terpyridine solution can realize a two-electron circulation system, and the gold electrode modified with gold / polydopamine nanomaterials can provide a larger specific surface area. Both of these methods can enhance the detection signal and effectively combine, thereby achieving a multi-amplification effect of the signal, ultimately improving the bacterial detection limit and shortening the detection time.

[0073] Example 2

[0074] Example 2 provides a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. The difference from Example 1 is that 3 mL of 0.5 mM isopropyl-β-D-thiogalactoside solution is added to the cultured Escherichia coli. The rest is roughly the same as in Example 1 and will not be repeated here.

[0075] Examples 3-5

[0076] Examples 3-5 provide a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. The difference from Example 2 is that in Examples 3-5, the cultured Escherichia coli is added to three samples—lake water, beer, and fresh milk—in equal amounts, with 1 mL of Escherichia coli corresponding to 5 mL of sample. The rest is roughly the same as in Example 2 and will not be repeated here.

[0077] Please see Figure 8 As shown, Examples 3-5 were tested at concentrations of 1*10. 9 CFU / mL, 1*10 7 CFU / mL, 1*10 6 CFU / mL, 1*10 5 CFU / mL, 1*10 4 CFU / mL, 1*10 3 Electrochemical detection results were obtained for different concentrations of bacterial markers in three liquid samples at CFU / mL. The figures show that the electrochemical detection system can effectively detect the metabolic proliferation process of *E. coli* in three common everyday liquid samples. As the concentration of *E. coli* markers increases, the electrochemical response signal also strengthens, indicating that *E. coli* can be detected in these complex real-world samples, and its concentration changes can be monitored in real time.

[0078] Please see Figure 9 As shown, Examples 3-5 tested Escherichia coli (concentration 1*10⁻⁶) at 4°C in a refrigerator and at room temperature. 3The concentration of *E. coli* (CFU / mL) was continuously monitored for 5 days. Electrochemical analysis was performed on three samples (4℃ and room temperature) at the same time each day, resulting in a continuous electrochemical monitoring graph of the added *E. coli* concentration in the three liquid samples. The graph shows no significant visual change in the appearance of the three liquids over time. Electrochemical detection revealed that at 4℃, the reduction peak signals of the three samples were weak and increased slowly over time. At 25℃, the reduction peak signals were initially strong and then gradually stabilized. This result indicates that bacterial proliferation and metabolism require certain nutrients (such as milk), and that metabolic activity is stronger and the electrochemical detection signal is stronger at 25℃. These results demonstrate that this electrochemical system can dynamically monitor the metabolic and proliferative processes of *E. coli* over a long period.

[0079] In summary, this invention provides a method for preparing and applying a multi-stage signal amplification electrochemical dynamic detection platform. First, gold / polydopamine nanoparticles are prepared; then, the gold / polydopamine nanoparticles are modified onto the surface of a gold electrode to obtain a gold electrode with surface-modified gold / polydopamine nanoparticles; finally, the gold electrode with surface-modified gold / polydopamine nanoparticles is used as the working electrode, a platinum electrode as the counter electrode, and a silver or silver chloride electrode as the reference electrode, and assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain a multi-stage signal amplification electrochemical dynamic detection platform. This invention modifies electrode materials with gold / polydopamine nanoparticles (first-level signal amplification: nano-network structure electrode), utilizing a redox multi-electron cycling system mediated by phenolic hydroxyl groups on the polydopamine surface. It introduces two electron transporters with different redox potentials to achieve a cascaded signal method (second-level signal amplification: redox dual-electron cycling system). In application, it targets β-galactosidase specifically secreted by *E. coli*, catalyzing the production of p-aminophenol with the substrate, achieving specific recognition of *E. coli* (third-level signal amplification: β-galactosidase-catalyzed reaction). Furthermore, p-aminophenol participates in the electrochemical cycling system, synergistically working with ruthenium terpyridine to achieve highly efficient electron transfer. This electrochemical dynamic detection platform, through multiple signal amplification effects, possesses advantages such as high sensitivity, strong anti-interference ability, and the ability to achieve specific detection. When used for the electrochemical detection of *E. coli*, it can dynamically monitor *E. coli* in real samples such as food and beverages.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An application of a multi-stage signal amplification electrochemical dynamic detection platform, characterized in that, An electrochemical dynamic detection platform with multi-stage signal amplification was used to perform electrochemical detection on the Escherichia coli detection solution; the Escherichia coli detection solution included Escherichia coli and 4-aminophenyl-β-D-galactopyranoside solution, and the Escherichia coli detection solution also included ruthenium tripyridine solution; The preparation method of the multi-stage signal amplification electrochemical dynamic detection platform includes the following steps: S1. Polydopamine nanoparticles are uniformly dispersed in deionized water to obtain a polydopamine solution. Chloroauric acid solution is added to the polydopamine solution under shaking condition, and then shaken at constant temperature for 20-28 hours. After multiple centrifugation treatments and washing, gold / polydopamine nanoparticles are obtained. S2. The gold / polydopamine nanoparticles obtained in step S1 are modified onto the surface of the gold electrode to obtain a gold electrode with surface-modified gold / polydopamine nanoparticles. S3. Using the gold electrode with surface-modified nano-gold / polydopamine obtained in step S2 as the working electrode, the platinum electrode as the counter electrode, and the silver or silver chloride electrode as the reference electrode, the three are assembled in an electrolytic cell device and connected to an electrochemical workstation to obtain an electrochemical dynamic detection platform with multi-stage signal amplification.

2. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 1, characterized in that, In step S1, the polydopamine nanoparticles have a particle size of 50–200 nm; the polydopamine solution has a concentration of 0.5%–1.5%; and the chloroauric acid solution has a concentration of 0.05%–0.2%.

3. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 2, characterized in that, The volume ratio of the chloroauric acid solution to the polydopamine solution is (10-20):

1.

4. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 1, characterized in that, In step S1, the preparation method of the polydopamine nanoparticles includes the following steps: S11. Dissolve dopamine hydrochloride in deionized water to obtain a dopamine hydrochloride solution with a concentration of 1-2 mg / mL; place the magnetic ball into the dopamine hydrochloride solution and heat and stir to 45-60°C; S12. Add a 0.8-1.5M NaOH solution to the hydrochloric acid dopamine solution being stirred in step S11, continue stirring for 1-3 minutes, let it stand for 4-6 hours after it becomes uniform, and then perform multiple centrifugations and washing to obtain the polydopamine nanoparticles.

5. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 1, characterized in that, In step S2, the method for modifying the gold / polydopamine nanoparticles on the surface of the gold electrode is as follows: the gold / polydopamine nanoparticles are uniformly mixed with a solvent to prepare a gold / polydopamine nanoparticle solution, and the gold / polydopamine nanoparticle solution is uniformly dispersed on the surface of the gold electrode by electrodeposition or dropwise addition. After drying, the gold electrode with surface-modified gold / polydopamine nanoparticles is obtained.

6. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 4, characterized in that, In step S12, the volume ratio of the dopamine hydrochloride solution to the NaOH solution is (200-250):

1.

7. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 1, characterized in that, In step S1, the constant temperature is 25°C; the centrifugation speed is 6000-10000 rpm, and the time is 8-15 min.

8. The application of the multi-stage signal amplification electrochemical dynamic detection platform according to claim 4, characterized in that, In step S12, the centrifugation speed is 8000-12000 rpm and the time is 8-15 min.

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