A Pt-Ni@erGO electrochemical probe, its preparation method and application

Abstract

This invention discloses a Pt-Ni@erGO electrochemical probe, its preparation method, and its applications. The Pt-Ni@erGO electrochemical probe comprises an erGO matrix and Pt-Ni nanoparticles supported on the erGO matrix. The Pt-Ni@erGO electrochemical probe of this invention uses erGO as a matrix to grow Pt-Ni alloy nanoparticles in situ. erGO simultaneously exhibits two functions to enhance electrochemical detection sensitivity: firstly, it acts as a carrier to load high-density Pt-Ni alloy nanoparticles, reducing the shielding effect of biorecognition elements on the catalytic sites of nanozymes; secondly, it acts as a biorecognition element to specifically bind to *E. coli*. Through the rational combination of erGO and Pt-Ni nanoparticles, the obtained Pt-Ni@erGO possesses high antibody-like activity and electrocatalytic activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a Pt-Ni@erGO electrochemical probe, its preparation method, and its application. Background Technology

[0002] Pathogenic bacterial infections (such as E. coli) caused by contaminated food, drinking water, and air pose an increasingly serious threat to human life and health, causing more than 15 million deaths worldwide each year. There is growing interest in developing rapid and sensitive bioanalytical techniques for detecting E. coli, including enzyme-linked immunosorbent assays (ELISA), polymerase chain reactions (PCR), and electrochemical biosensors. Among these techniques, electrochemical biosensors have become an attractive method for detecting E. coli, bodily fluids (such as urine), and object surfaces due to their low cost, simplicity, speed, and sensitivity.

[0003] Electrochemical biosensors are combinations of sensitive signal transducer elements and specific biorecognition elements that convert chemical energy into electrical energy through electrodes and electrolyte solutions. Widely used signal sensor elements in electrochemical biosensors include horseradish peroxidase (HRP), iron-based nanozymes (e.g., Pluton nanoparticles), carbon-based nanozymes (e.g., metal-organic frameworks), and noble metal-based nanozymes (e.g., platinum and nickel nanoparticles). Among these various signal sensor elements, platinum (Pt) and nickel (Ni) nanozyme-based electrochemical biosensors have attracted increasing attention in the development of point-of-care testing (POCT) bioanalytical technologies due to their high stability, excellent electronic properties, and high catalytic activity.

[0004] However, the direct chemical modification or adsorption of biorecognition elements (such as antibodies and aptamers) on the surface of nanozymes creates a shielding effect on the catalytic sites of nanozymes, leading to a sharp decrease in the catalytic activity of Pt and Ni nanozymes. Furthermore, this shielding effect becomes more pronounced with increasing concentration of the biorecognition element. Therefore, reducing the shielding effect of biorecognition elements on the catalytic sites of nanozymes has become an urgent problem to be solved. Summary of the Invention

[0005] The main objective of this invention is to provide a Pt-Ni@erGO electrochemical probe, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0007] According to a first aspect of the present invention, a Pt-Ni@erGO electrochemical probe is provided, comprising an erGO matrix and Pt-Ni nanoparticles loaded on the erGO matrix.

[0008] According to a second aspect of the present invention, a method for preparing a Pt-Ni@erGO electrochemical probe is provided, comprising the following steps:

[0009] S1. Dissolve graphite powder and NaNO3 completely in H2SO4 solution to obtain erGO aqueous solution;

[0010] S2. NiCl2·6H2O and sodium borohydride were added to the erGO aqueous solution obtained in step S1. After stirring, PtCl4·5H2O was added. Then, the mixture was centrifuged and the precipitate was collected. The precipitate was washed to obtain the Pt-Ni@erGO multifunctional electrochemical probe.

[0011] Furthermore, step S1 includes fully dissolving NaNO3 and KMnO4 in H2SO4 solution, then adding graphite powder at regular intervals, obtaining a mixed solution after the graphite powder has been completely added, adding H2O2 to the mixed solution, and stirring to obtain an erGO aqueous solution.

[0012] Furthermore, in step S1, the mass ratio of graphite powder to NaNO3 is 1:1.

[0013] Furthermore, H2O2 was added to the mixed solution and stirred at 500 rpm for 10 minutes to obtain an erGO aqueous solution.

[0014] Further, step S2 includes adding NiCl2·6H2O and sodium borohydride to the erGO aqueous solution obtained in step S1, stirring and refluxing for 30 min, then adding 5 mL of 20 mM PtCl4·5H2O, stirring for 5 min, cooling to 25 °C and stirring for 5 h, and finally centrifuging at 9000 rpm for 10 min to collect the precipitate.

[0015] Furthermore, the density of the erGO aqueous solution is 1 mg / mL, the concentration of NiCl2·6H2O is 0.02 M, the concentration of sodium borohydride is 150 mM, and the concentration of PtCl4·5H2O is 20 mM.

[0016] Furthermore, step S2 includes storing the obtained Pt-Ni@erGO multifunctional electrochemical probe at a temperature of 4°C.

[0017] According to a third aspect of the present invention, a method for fabricating an electrochemical biosensor is provided, comprising the following steps:

[0018] ① Polish the electrode with alumina paste, then treat it multiple times with piranha solution, and then wash it with deionized water to obtain a clean electrode;

[0019] ② Add SWCNTs to a clean electrode surface, dry it, and then immerse it in HAuCl4 solution. React at the deposition potential to obtain an electroplated AuNP layer and obtain an AuNP-SWCNTs modified electrode.

[0020] ③ Add E. coli capture antibody to AuNP-SWCNTs modified electrode, incubate, add BSA solution to block unbound sites, incubate and wash to obtain working electrode for later use;

[0021] ④ Add different concentrations of Escherichia coli to the working electrode, incubate, and wash repeatedly with buffer solution. Then add the above Pt-Ni@erGO electrochemical probe to the working electrode, incubate, and wash away the unreacted Pt-Ni@erGO electrochemical probe with PBS buffer to obtain the electrochemical biosensor.

[0022] According to a fourth aspect of the present invention, an application of a Pt-Ni@erGO electrochemical probe in the detection of Escherichia coli is provided.

[0023] Compared with the prior art, the advantages of the present invention include:

[0024] This invention designs a Pt-Ni@erGO electrochemical probe, its preparation method, and its applications. The Pt-Ni@erGO electrochemical probe uses erGO as a matrix to grow Pt-Ni alloy nanoparticles in situ. erGO simultaneously exhibits two functions to enhance electrochemical detection sensitivity: firstly, it acts as a carrier to load high-density Pt-Ni alloy nanoparticles, reducing the shielding effect of biorecognition elements on the catalytic sites of nanozymes; secondly, it acts as a biorecognition element to specifically bind to *E. coli*. Through a rational combination of erGO and Pt-Ni nanoparticles, the resulting Pt-Ni@erGO possesses high antibody-like activity and electrocatalytic activity. Attached Figure Description

[0025] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0026] Figure 1 This is a schematic diagram illustrating the detection principle of the Pt-Ni@erGO-labeled electrochemical biosensor of the present invention for sensitive detection of Escherichia coli.

[0027] Figure 2a A schematic diagram of the Pt-Ni@erGO integrated route;

[0028] Figure 2b SEM images of erGO;

[0029] Figure 2c TEM image of erGO;

[0030] Figure 2d SEM image of Pt-Ni@erGO;

[0031] Figure 3a Schematic diagram of Pt-Ni@erGO binding with Escherichia coli;

[0032] Figure 3b and 3c SEM images of Pt-Ni@erGO binding to Escherichia coli;

[0033] Figure 3d and 3e Scanning electron microscopy images of Pt-Ni@erGO bound to Staphylococcus aureus;

[0034] Figure 4a This is a schematic diagram of the layer-by-layer self-assembly of bare electrodes;

[0035] Figure 4b and 4c This is a schematic diagram showing the changes in CV and EIS signals after continuous modification of the a: bare electrode with b: SWCNTs, c: AuNPs, d: capture antibody and e: BSA;

[0036] Figure 4d This is a schematic diagram illustrating the changes in the CV signal after continuously modifying a: the working electrode (b:E).

[0037] Figure 4e This is a schematic diagram showing the changes in the it signal of an electrochemical biosensor under different concentrations of E. coli.

[0038] Figure 5a The response of the biosensor to the electrochemical signal intensity of different concentrations of Escherichia coli;

[0039] Figure 5b This is a graph showing the linear range for E. coli detection.

[0040] Figure 5c and 5d The specificity evaluation diagram was obtained by monitoring the changes in electrochemical signals after reactions with Staphylococcus aureus, Staphylococcus aureus-1, MRSA, and Escherichia coli. Detailed Implementation

[0041] In view of the shortcomings of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. The following will further explain and illustrate this technical solution, its implementation process, and its principles.

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] One aspect of this invention provides a Pt-Ni@erGO electrochemical probe comprising an erGO matrix and Pt-Ni nanoparticles loaded on the erGO matrix.

[0045] According to a second aspect of the present invention, a method for preparing a Pt-Ni@erGO electrochemical probe is provided, comprising the following steps:

[0046] S1. Dissolve graphite powder and NaNO3 completely in H2SO4 solution to obtain erGO aqueous solution;

[0047] In a typical embodiment, 0.5 g of graphite powder and 0.5 g of NaNO3 are fully dissolved in 46 mL of H2SO4 to obtain an erGO aqueous solution; specifically, 0.5 g of NaNO3 and 3 g of KMnO4 are fully dissolved in 46 mL of H2SO4. Then, 4 mL of oxidant solution is added to the graphite powder solution every 20 minutes until all is added. 40 mL of H2O2 is added to the mixed solution. After stirring at 500 rpm for 10 minutes, the product is successfully prepared.

[0048] S2. NiCl2·6H2O and sodium borohydride were added to the erGO aqueous solution obtained in step S1. After stirring, PtCl4·5H2O was added. Then, the mixture was centrifuged and the precipitate was collected. The precipitate was washed to obtain the Pt-Ni@erGO multifunctional electrochemical probe.

[0049] In a typical embodiment, 5 mL of 0.02 M NiCl2·6H2O and 15 mL of sodium borohydride (150 mM) can be added to an erGO aqueous solution (1 mg / mL). After vigorous stirring and reflux heating for 30 min, 5 mL of 20 mM PtCl4·5H2O is added to the above reaction solution, stirred for 5 min, cooled to 25 °C, stirred for 5 h, and finally collected by centrifugation at 9000 rpm for 10 min. The product is washed three times with deionized water and stored at 4 °C.

[0050] According to a third aspect of the present invention, a method for fabricating an electrochemical biosensor is provided, comprising the following steps:

[0051] ① Polish the electrode with alumina paste, then treat it multiple times with piranha solution, and then wash it with deionized water to obtain a clean electrode;

[0052] ② Add SWCNTs to a clean electrode surface, dry it, and then immerse it in HAuCl4 solution. React at the deposition potential to obtain an electroplated AuNP layer and obtain an AuNP-SWCNTs modified electrode.

[0053] ③ Add E. coli capture antibody to AuNP-SWCNTs modified electrode, incubate, add BSA solution to block unbound sites, incubate and wash to obtain working electrode for later use;

[0054] ④ Add different concentrations of Escherichia coli to the working electrode, incubate, and wash repeatedly with buffer solution. Then add the above Pt-Ni@erGO electrochemical probe to the working electrode, incubate, and wash away the unreacted Pt-Ni@erGO electrochemical probe with PBS buffer to obtain the electrochemical biosensor.

[0055] In a typical embodiment, the electrode was first polished with 0.05 μm alumina paste for 5 min. The polished electrode was then treated three times with piranha solution (H2SO4:H2O2 = 3:1) and washed with deionized water to obtain a clean electrode. Then, 9 μL of SWCNTs (1 mg / mL) was added to the electrode surface and dried at 37 °C. The SWCNT-modified electrode was further immersed in 1% HAuCl4 solution and reacted at a deposition potential of 0.2 V for 50 s to obtain an electroplated AuNP layer. Next, 10 μL of E. coli capture antibody (10 μg / mL) was added to the AuNP-SWCNTs-modified electrode and incubated at 4 °C for 12 h. After the reaction, 8 μL of LBSA solution (10 mg / mL) was added to block unbound sites, and the electrode was incubated at 37 °C for 0.5 h. The electrode was then washed three times with PBS buffer and stored at 4 °C for later use. Subsequently, different concentrations of E. coli were added to the working electrode and incubated at 37 °C for 90 min. After washing three times with PBS buffer, Pt-Ni@erGO was added to the working electrode and incubated at 37°C for 60 minutes. Unreacted Pt-Ni@erGO was removed by washing with PBS buffer.

[0056] The present invention will be described in detail below with reference to specific embodiments:

[0057] like Figure 1The diagram illustrates the detection principle of the Pt-Ni@erGO-labeled electrochemical biosensor for sensitive detection of *E. coli*. The electrochemical biosensor uses a gold electrode as the base electrode. SWCNTs, AuNPs, and a capture antibody are then sequentially modified onto the gold electrode surface. Upon addition of *E. coli*, it is captured by the capture antibody on the working electrode surface. Subsequently, the prepared Pt-Ni@erGO is mixed with the working electrode. After the reaction, the antibody-like Pt-Ni@erGO forms a sandwich detection pattern with *E. coli* and the capture antibody. Finally, the reaction electrode is immersed in H2O2 solution, and the electrochemical signal of the electrochemical biosensor is amplified by Pt-Ni@erGO-based enzyme catalysis. The reader records the it signal.

[0058] Experiment 1

[0059] Synthesis of epoxy-rich graphene oxide

[0060] Preparation of graphene solution: Dissolve 0.5g graphite powder and 0.5g NaNO3 completely in 46mL H2SO4. Preparation of oxidant solution: Dissolve 0.5g NaNO3 and 3g KMnO4 completely in 46mL H2SO4. Then, add 4mL of oxidant solution to the graphite powder solution every 20 minutes until all is added. Add 40mL H2O2 to the mixed solution. After stirring at 500 rpm for 10 minutes, the product was successfully prepared.

[0061] Synthesis of Pt-Ni nanoparticle-modified erGO (Pt-Ni@erGO)

[0062] Add 5 mL of 0.02 M NiCl2·6H2O and 15 mL of sodium borohydride (150 mM) to an erGO aqueous solution (1 mg / mL). After vigorous stirring and reflux heating for 30 min, add 5 mL of 20 mM PtCl4·5H2O to the above reaction solution, stir for 5 min, cool to 25 °C, stir for 5 h, and finally centrifuge at 9000 rpm for 10 min to collect the product. Wash three times with deionized water and store at 4 °C.

[0063] Bacterial culture

[0064] Escherichia coli, Staphylococcus aureus, Staphylococcus aureus-1, and MRSA strains were cultured in LB gravy at 37°C for 6 hours. Bacterial counts were performed using the plate count method, and the colony concentration was adjusted to 1.5 × 10⁻⁶. 8CFU / mL. Collect bacteria in 1.5 mL Eppendorf tubes, centrifuge at 5000 rpm for 5 minutes, and then wash three times with 0.01 M PBS buffer. Finally, dilute the bacteria to different concentrations with 0.01 M PBS buffer.

[0065] Fabrication of electrochemical biosensors

[0066] First, the electrode was polished with 0.05 μm alumina paste for 5 min. The polished electrode was then treated three times with piranha solution (H₂SO₄:H₂O₂ = 3:1) and washed with deionized water to obtain a clean electrode. Next, 9 μL of SWCNTs (1 mg / mL) was added to the electrode surface and dried at 37 °C. The SWCNT-modified electrode was further immersed in 1% HAuCl₄ solution and reacted at a deposition potential of 0.2 V for 50 s to obtain an electroplated AuNP layer. Then, 10 μL of E. coli capture antibody (10 μg / mL) was added to the AuNP-SWCNTs-modified electrode and incubated at 4 °C for 12 h. After the reaction, 8 μL of BSA solution (10 mg / mL) was added to block unbound sites, and the electrode was incubated at 37 °C for 0.5 h. The electrode was washed three times with PBS buffer and stored at 4 °C for later use. Subsequently, different concentrations of E. coli were added to the working electrode and incubated at 37 °C for 90 min. After washing three times with PBS buffer, Pt-Ni@erGO was added to the working electrode and incubated at 37°C for 60 minutes. Unreacted Pt-Ni@erGO was removed by washing with PBS buffer.

[0067] The synthesis and characterization results of erGO and Pt-Ni@erGO are as follows: Figure 2a Schematic diagram of the Pt-Ni@erGO synthesis route. SEM image of 2b erGO. TEM image of 2c erGO. SEM image of 2d Pt-Ni@erGO.

[0068] The characterization results of the binding efficiency of Pt-Ni@erGO to Escherichia coli and Staphylococcus aureus are as follows: 3a Schematic diagram of Pt-Ni@erGO binding to Escherichia coli. 3b and 3c SEM images of Pt-Ni@erGO binding to Escherichia coli. 3d and 3e Scanning electron microscopy images of Pt-Ni@erGO binding to Staphylococcus aureus.

[0069] The electrocatalytic activity characterization results of Pt-Ni@erGO are as follows: 4a Schematic diagram of layer-by-layer self-assembly of the bare electrode. 4b and 4c Changes in CV and EIS signals after continuous modification of the bare electrode with b: SWCNTs, c: AuNPs, d: capture antibody, and e: BSA. 4d Changes in CV signal after continuous modification of the working electrode with b: E. coli and c: Pt-Ni@erGO. 4e Changes in the it signal of the electrochemical biosensor under different concentrations of E. coli. The working potential is -0.4V. The concentrations of E. coli are 0, 1.5 × 10⁵ CFU / mL, and 1.5 × 10⁷ CFU / mL, respectively.

[0070] Under optimal experimental conditions, a Pt-Ni@erGO-labeled electrochemical biosensor was used to study different concentrations (1.5 × 10⁻⁶). 2 -1.5×10 7 E. coli in CFU / mL PBS buffer solution was monitored. The it signal value was selected as the signal output and recorded using a CHI660D electrochemical workstation. The limit of detection (LOD) was defined as the mean it signal value of the blank sample plus three times the standard deviation. As shown in the figure, the it signal value is related to the concentration of E. coli (1.5 × 10⁻⁶ CFU / mL). 2 -1.5×10 7 The CFU / mL ratio showed a good correlation, with the equation I = 19.77lgC - 1.976.

[0071] By comparing several common pathogenic bacteria such as MRSA and Staphylococcus aureus at 1.5 × 10⁻⁶ 6 The specificity of the developed Pt-Ni@erGO-labeled electrochemical biosensor was investigated at a concentration of 1.5 × 10⁻⁶ CFU / mL. Additionally, the specificity was further investigated at a concentration of 1.5 × 10⁻⁶ CFU / mL. 6 CFU / mL of Escherichia coli served as a positive control. Compared to other non-target pathogens, the Pt-Ni@erGO-labeled electrochemical biosensor exhibited a significant signal response to Escherichia coli, indicating its high selectivity for the specific detection of Escherichia coli against other pathogens.

[0072] The analytical performance of the Pt-Ni@erGO-labeled electrochemical biosensor for detecting *E. coli* is as follows: The 5a biosensor's response to the electrochemical signal intensity of different concentrations of *E. coli*. The *E. coli* concentrations were 0 and 1.5 × 10⁻⁶. 2 1.5×10 3 1.5×10 4 1.5×10 5 1.5×10 6 and 1.5×10 7CFU / mL. 5b Linear range graph for E. coli detection. Error bars represent the standard deviations of three independent experiments. 5c and 5d Specificity was evaluated by monitoring changes in electrochemical signals after the reaction for Staphylococcus aureus, Staphylococcus aureus-1, MRSA, and E. coli. The specificity analysis bacterial concentration was 1.5 × 10⁻⁶. 6 CFU / mL.

[0073] Example 1:

[0074] The Pt-Ni@erGO-labeled electrochemical biosensor constructed in this invention was used to detect low concentrations of Escherichia coli in urine (calibrated concentration: 1.5 × 10² CFU / mL). The measured concentration was 1.483 × 10² CFU / mL, and the recovery rate was 98.85%.

[0075] Example 2:

[0076] The Pt-Ni@erGO-labeled electrochemical biosensor constructed in this invention was used to detect medium-concentration Escherichia coli in urine (calibrated concentration: 1.5 × 10³ CFU / mL). The measured concentration was 1.451 × 10³ CFU / mL, and the recovery rate was 96.73%.

[0077] Example 3:

[0078] The Pt-Ni@erGO-labeled electrochemical biosensor constructed in this invention was used to detect low concentrations of Escherichia coli in urine (calibrated concentration: 1.5 × 10⁴ CFU / mL). The measured concentration was 1.474 × 10⁴ CFU / mL, and the recovery rate was 98.29%.

[0079] The above descriptions are merely some embodiments of the present invention. It should be noted that those skilled in the art can make other modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for fabricating an electrochemical biosensor, characterized in that, Includes the following steps: ① Polish the electrode with alumina paste, then treat it multiple times with piranha solution, and then wash it with deionized water to obtain a clean electrode; ② Add SWCNTs to a clean electrode surface, dry it, and then immerse it in HAuCl4 solution. React at the deposition potential to obtain an electroplated AuNP layer and obtain an AuNP-SWCNTs modified electrode. ③ Add E. coli capture antibody to AuNP-SWCNTs modified electrode, incubate, add BSA solution to block unbound sites, incubate and wash to obtain working electrode for later use; ④ Add different concentrations of Escherichia coli to the working electrode, incubate and wash repeatedly with buffer solution, add Pt-Ni@erGO electrochemical probe to the working electrode, incubate, and wash away unreacted Pt-Ni@erGO electrochemical probe with PBS buffer solution to obtain the electrochemical biosensor. The Pt-Ni@erGO electrochemical probe comprises an erGO matrix and Pt-Ni nanoparticles loaded on the erGO matrix. The preparation method of the Pt-Ni@erGO electrochemical probe includes the following steps: S1. Dissolve graphite powder and NaNO3 completely in H2SO4 solution to obtain erGO aqueous solution; S2. NiCl2·6H2O and sodium borohydride were added to the erGO aqueous solution obtained in step S1. After stirring, PtCl4·5H2O was added. Then, the mixture was centrifuged and the precipitate was collected. The precipitate was washed to obtain the Pt-Ni@erGO multifunctional electrochemical probe.

2. The method for fabricating the electrochemical biosensor according to claim 1, characterized in that: Step S1 includes fully dissolving NaNO3 and KMnO4 in H2SO4 solution, then adding graphite powder at regular intervals until all the graphite powder is added to obtain a mixed solution, adding H2O2 to the mixed solution, and stirring to obtain an erGO aqueous solution.

3. The method for fabricating the electrochemical biosensor according to claim 2, characterized in that: In step S1, the mass ratio of graphite powder to NaNO3 is 1:

1.

4. The method for fabricating the electrochemical biosensor according to claim 2, characterized in that: After adding H2O2 to the mixed solution, stir at 500 rpm for 10 minutes to obtain an erGO aqueous solution.

5. The method for fabricating the electrochemical biosensor according to claim 1, characterized in that: Step S2 includes adding NiCl2·6H2O and sodium borohydride to the erGO aqueous solution obtained in step S1, stirring and refluxing for 30 min, then adding 5 mL of 20 mM PtCl4·5H2O, stirring for 5 min, cooling to 25°C, stirring for 5 h, and finally collecting the precipitate by centrifugation at 9000 rpm for 10 min.

6. The method for fabricating the electrochemical biosensor according to claim 5, characterized in that: The density of the erGO aqueous solution is 1 mg / mL, the concentration of NiCl2·6H2O is 0.02 M, the concentration of sodium borohydride is 150 mM, and the concentration of PtCl4·5H2O is 20 mM.

7. The method for fabricating the electrochemical biosensor according to claim 1, characterized in that: Step S2 includes storing the obtained Pt-Ni@erGO multifunctional electrochemical probe at a temperature of 4°C.

8. The application of an electrochemical biosensor fabricated using the method described in claim 1 in the detection of Escherichia coli.

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