Electrochemical biosensor for rapidly capturing microbial pathogens and preparation method of electrochemical biosensor

Abstract

The invention discloses an electrochemical biosensor for rapidly capturing microbial pathogens and a preparation method thereof, and belongs to the technical field of electrochemical sensors, the electrochemical biosensor comprises a support base, a working electrode mounting plate and a support footstock; a micro vibrator is arranged on the top surface of the supporting base; the working electrode mounting plate is correspondingly mounted at the top end of the miniature vibrator, and a working electrode groove for mounting a WE electrode is formed in the top surface of the working electrode mounting plate; the supporting top seat is correspondingly buckled on the working electrode mounting plate, an RE electrode and a CE electrode are respectively embedded on the top surface of the supporting top seat, the oxidation-reduction probe is connected with the WE electrode, and the RE electrode and the CE electrode form a three-electrode path. According to the electrochemical biosensor, the working electrode with the 3D net-shaped structure is connected with the micro vibrator structure, and the collision probability and the binding rate of a sample containing pathogenic microorganisms in unit time are improved by providing proper vibration frequency, so that the detection efficiency and the performance of the electrochemical biosensor are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and in particular to an electrochemical biosensor for rapidly capturing microbial pathogens and a preparation method thereof. Background Art

[0002] Pathogenic microorganisms are important invasive factors that cause diseases. At the molecular level, they involve fungi, protozoa, bacteria, viruses, and prions. Widely existing pathogenic microorganisms enter the human body through food, water, air, etc., causing nearly 15 million deaths worldwide each year. In the case of its increasingly serious incidence, Staphylococcus aureus, as one of the most common highly pathogenic bacteria, has a significant proportion of foodborne diseases. It can tolerate high temperatures and its infection rate is second only to Escherichia coli. Highly pathogenic Staphylococcus aureus (S. aureus) can cause pneumonia, pseudomembranous colitis and pericarditis, and even systemic infections such as sepsis and septicemia by producing enterotoxins, which can also affect human health. In recent years, with the abuse of antibiotics, pathogenic bacteria with drug-resistant mutations have been widely spread with the large-scale flow of biological tissues, food, equipment, etc. It has spread all over the world, posing a serious threat to the economy and society, especially in densely populated countries such as remote areas and developing countries with limited resources. The evolution of bacteria has proved that even low doses can cause severe or even fatal clinical symptoms and diseases. Therefore, to reduce the risk of food poisoning, early identification and rapid detection of specific viral species and associated quantitative signals are essential.

[0003] How to achieve fast, accurate, automated, and real-time analysis is the development direction pursued by modern analytical equipment. Electrochemical detection has been proven to be an effective and fast method for direct analysis of pathogenic microorganisms. Electrochemical technology uses electrochemical workstations, which provide advantages such as low cost, high sensitivity, and portability for routine analysis. Most of the sensors currently presented use planar electrodes such as glassy carbon and carbon screen printing, that is, pathogenic microorganisms can only be detected when they move in a single direction to the electrode surface. However, in actual detection, the movement of pathogenic microorganisms is disordered and multi-directional, so traditional planar electrodes limit the performance of biosensor detection. Summary of the invention

[0004] Technical problem to be solved: In response to the problems existing in the prior art, the present invention provides an electrochemical biosensor for rapidly capturing microbial pathogens and a method for preparing the same. The working electrode of the 3D mesh structure is connected to a micro-vibrator structure. By providing a suitable vibration frequency, the collision probability and binding rate of samples containing pathogenic microorganisms per unit time are increased, thereby greatly improving the detection efficiency and performance of the electrochemical biosensor.

[0005] Technical solution: The present invention discloses an electrochemical biosensor for rapidly capturing microbial pathogens, the electrochemical biosensor comprising a support base, a working electrode mounting plate and a support top seat; The top surface of the support base is provided with a mounting slot and a micro vibrator embedded in the mounting slot; The working electrode mounting plate is correspondingly mounted on the top of the micro vibrator, and the top surface of the working electrode mounting plate is provided with a working electrode groove for mounting the WE electrode; The support top seat is correspondingly buckled on the working electrode mounting plate, and the top surface thereof is respectively embedded with an RE electrode and a CE electrode, and a through hole connected to the working electrode groove is provided between the RE electrode and the CE electrode. The redox probe is dripped into the through hole and connects the WE electrode, the RE electrode and the CE electrode to form a three-electrode path.

[0006] The present invention discloses a method for preparing an electrochemical biosensor, which comprises the following steps: Step 1: Use Solidworks 3D software to design a support base that can accommodate the installation of the micro vibrator, a working electrode mounting plate connected to the working electrode, and a support top seat embedded with the RE electrode and the CE electrode; Step 2: Use 3D printing technology to print out the support base, working electrode mounting plate and support top seat respectively, and install the micro vibrator on the support base accordingly; use non-conductive glue to fix the silver wire as RE electrode and the platinum wire as CE electrode on the support top seat, and use matching fixing clips to assemble the support base, working electrode mounting plate and support top seat; Step 3: Prepare a Bare / HCL / Au / L-Cys / Ab / BSA / Ag-modified WE electrode, and insert the WE electrode into the working electrode groove accordingly. Use a syringe to drip the redox probe into the through hole of the self-supporting top seat so that the WE electrode, RE electrode and CE electrode form a loop.

[0007] Preferably, the specific preparation steps of the WE electrode are: Step S 1 : The SSWM was cut into sheets with a single electrode length of 20 mm and a width of 10 mm, and then ultrasonically cleaned in acetone, ethanol and double distilled water for 15 min in sequence, and dried at 60 °C to obtain the SSWM conductive substrate; Step S 2 :Use plastic sealing paper with holes on one side to seal the SSWM conductive substrate to a fixed area to provide an effective detection area; Step S 3: Prepare 0.5M hydrochloric acid solution to etch the surface of the SSWM conductive substrate with a fixed area for 10s, and immediately put it into a container filled with deionized water, then wash it repeatedly three times with a washing bottle, and then dry it naturally at room temperature; Step S 4 : Use deionized water to prepare 10 mM chloroauric acid solution, select CHT660D device to build a three-electrode device, and use cyclic voltammetry CV to set the process parameters as follows: origin E: -0.6, high E: 0.2, low E: -0.6, scan rate: 0.1v / s, sweep segment: 10, and deposit it on the surface of the SSWM electrode after hydrochloric acid treatment; Step S 5 : Prepare 2.2 mg / ml L-Cys and 11.42 mg / ml TCEP HCL solutions respectively, and mix them in a 1:1 ratio for 20 min. Take 20ul of the mixed solution and incubate it on the surface of the SSWM electrode for 2h at room temperature. Then rinse the dry well with deionized water, and use an ear washing bulb to wash and blow the active molecules on the surface of the SSWM electrode. Step S 6 : Use PBS at pH = 5 to prepare 11.5 mg / 2ml EDC solution and 7 mg / 2ml NHS solution respectively, take 10ul of EDC solution and NHS solution and place them in 80ul PBS solution at pH = 5 to make a 30 mM mixed solution, take 20ul of the mixed solution and incubate it on the SSWM electrode surface at room temperature for 1h; Step S 7 : The aptamer with a diluted concentration of 2 μmol / L was dropped on the surface of the SSWM electrode, placed in a 4°C refrigerator for 22 h, and then rinsed; Step S 8 : Under moisturizing conditions, take 20ul of 1%wt BSA and drop it on the surface of the SSWM electrode, incubate it at 37℃ for 1h, rinse it and dry it naturally; Step S 9 : The ATCC25923 Staphylococcus aureus standard strain was subcultured and counted, and a 1×103 CFU / mL SA sample was obtained by dilution. 20ul of the SA sample was dropped on the surface of the SSWM electrode and reacted for 30 minutes; Step S 10 : Insert the treated working electrode into the working electrode groove, drop the redox probe into the WE electrode, RE electrode and CE electrode to form a three-electrode path; Step S 11 : Turn on a power supply with a voltage of 2V and a current of 0.049A to compare the electrochemical responses of the WE electrode under static and vibrating conditions.

[0008] Compared with the prior art, the present invention has at least the following outstanding advantages: 1. The electrochemical biosensor of the present invention uses a stainless steel 3D mesh structure working electrode, which has excellent conductivity and low cost advantages, greatly reducing the production cost of using glassy carbon, gold, platinum and other materials to make working electrodes; the working electrode does not require complex modification, the production process is simple, and can be processed and produced in high-efficiency batches; 2. The electrochemical biosensor connects the working electrode with the micro-vibrator, automates the original manual operation in the chemical analysis, and can strictly control the sample volume during sample placement, which can not only reduce sample loss, but also reduce errors caused by human factors. By providing a suitable vibration frequency, the collision probability and binding rate of samples containing pathogenic microorganisms per unit time are increased, greatly improving the detection efficiency and performance of the electrochemical biosensor; 3. The electrochemical biosensor adopts a mesh-structured SSWM electrode with a high surface area and excellent catalytic performance, which increases the contact area between the working electrode and the sample to be tested. The working electrode can fully contact the sample, thereby improving the detection efficiency of pathogenic microorganisms; 4. The electrochemical biosensor has a microstructure as a whole, is easy to carry, and is easy to assemble. The detection results are real and effective. The sensor has excellent stability, reproducibility and reusability, which is conducive to long-term use in actual analysis and detection. 5. The prepared sensor has excellent performance. The minimum detection number of Staphylococcus aureus can be 15 CFU / mL in one injection, which reduces the difficulty of sample preparation; 6. The sensor has good biocompatibility, high sensitivity and selectivity, and can realize online monitoring of pathogenic microorganisms, especially suitable for online monitoring of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a schematic diagram of the structure of the electrochemical biosensor of the present invention; Figure 2 for Figure 1 The three-electrode path diagram formed by the redox probe dripping into the groove of the middle working electrode; Figure 3 The schematic diagram of the preparation principle of the working electrode (CE) of the present invention and its vibration state and electrochemical response of static comparison; Figure 4 The SEM and EDX images of the working electrode (CE) of the present invention; Figure 5 To prepare the working electrode (CE), HAuCl was electrochemically deposited 4 DPV curves obtained from each step of experimental modification of the solution before; Figure 6 To prepare the working electrode (CE), HAuCl was electrochemically deposited 4 EIS curves obtained from each step of experimental modification of the solution before; Figure 7 CV curves of Au in 2 mM FcMeOH at different scan rates (2.5-100 mV / s) when preparing the working electrode (CE); Figure 8 for Figure 7 Linear relationship between peak intensity and square root of scan rate in CV curve; Fig. 9 DPV curves of Bare / HCL / Au / L-Cys, Bare / HCL / Au / L-Cys / Ab, Bare / HCL / Au / L-Cys / Ab / BSA, and Bare / HCL / Au / L-Cys / Ab / BSA / Ag in the presence of 2 mM FcMeOH when preparing working electrodes (CE); Fig.10 EIS curves of Bare / HCL / Au / L-Cys, Bare / HCL / Au / L-Cys / Ab, Bare / HCL / Au / L-Cys / Ab / BSA, and Bare / HCL / Au / L-Cys / Ab / BSA / Ag in the presence of 2 mM FcMeOH when preparing the working electrode (CE); Fig.11 The current ΔI of the working electrode (CE) varies with time (SA=10 3 CFU / mL)(A: static state; B: vibration state); Fig.12 For the working electrode (CE) at different concentrations of Staphylococcus aureus (from lg10 1 CFU / mL to lg10 6 The current response relationship diagram of Bare / HCL / Au / L-Cys / Apt / BSA / SA in the presence of 4% (CFU / mL) (A: static state; B: vibration state); Fig.13 For the working electrode (CE) at different concentrations of Staphylococcus aureus (from lg10 1 CFU / mL to lg10 6 The current ΔI relationship diagram of Bare / HCL / Au / L-Cys / Apt / BSA / SA in the presence of 4% (CFU / mL) (A: static; B: vibration state); Fig.14 For the working electrode (CE) at different concentrations of Staphylococcus aureus (from lg10 1 CFU / mL to lg106 Calibration curves of Bare / HCL / Au / L-Cys / Apt / BSA / SA for Staphylococcus aureus detection in the presence of 500 μg / mL (CFU / mL) (A: static; B: vibrating), error bars represent the standard deviation of triplicate measurements; Fig.15 The working electrode (CE) was used for the blank sample, Bacillus sample, Escherichia coli sample, and low concentration Staphylococcus aureus sample (SA=10 3 CFU / mL and higher concentrations of Staphylococcus aureus (SA=10 4 CFU / mL) selectivity; Fig.16 The stability of the Bare / HCL / Au / L-Cys / Apt / BSA / SA electrode incubated for more than 7 days under vibration conditions (SA=10 3 CFU / mL).

[0010] Figure numerals: 100, electrochemical biosensor; 1, support base; 2, mounting slot; 3, vibrator; 4, connecting wires; 5, working electrode mounting plate; 6, working electrode groove; 7, WE electrode; 8, support top seat; 9, RE electrode; 10, CE electrode; 11, redox probe. DETAILED DESCRIPTION

[0011] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the attached Figures 1 to 16 The technical solutions of the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0012] Example 1: Figure 1~2As shown, the present invention discloses an electrochemical biosensor for rapidly capturing microbial pathogens. The electrochemical biosensor 100 includes a support base 1, a working electrode mounting plate 5, and a support top seat 8. The top surface of the support base 1 is provided with a mounting slot 2 and a micro vibrator 3 embedded in the mounting slot 2. The specific size of the mounting slot 2 can be selected to be 31 mm long, 34 mm wide, and 14 mm high. The bottom diameter of the micro vibrator is 10 mm and the top diameter is 6 mm. The micro vibrator 3 is correspondingly mounted in the mounting slot 2. The micro vibrator 3 is provided with a connecting wire 4 connected to an external power supply. The vibration parameters of the micro vibration are set to: voltage 2V, current 0.49A. The working electrode mounting plate 5 is correspondingly mounted on the top of the micro vibrator 3, and the top surface of the working electrode mounting plate 5 is provided with a working electrode groove 6 for installing a WE electrode 7. The WE electrode 7 is the working electrode, which can be conveniently inserted into the working electrode groove 6. After the WE electrode 7 is prepared, it is correspondingly inserted into the working electrode groove 6 to perform subsequent detection work. The support top seat 8 is correspondingly buckled on the working electrode mounting plate 5, and the top surface thereof is respectively embedded with the RE electrode 9 and the CE electrode 10, and a through hole connected to the working electrode groove 6 is provided between the RE electrode 9 and the CE electrode 10, and the redox probe 11 is dripped from the through hole and connects the WE electrode 7, the RE electrode 9 and the CE electrode 10 to form a three-electrode path. The RE electrode 9 is a reference electrode, and is fixed to the corresponding position of the support top seat 8 using a 0.5mm diameter Ag / AgCl wire using AB glue; the CE electrode 10 is a comparative electrode, and is fixed to the corresponding position of the support top seat 8 using a 0.5mm diameter platinum wire using AB glue; two layers of silica gel layers are correspondingly provided on the working electrode groove 6 and the bottom surface of the support top seat 8, and the WE electrode 7 can be correspondingly fixed between the two layers of silica gel layers.

[0013] The electrochemical biosensor of the present invention connects the working electrode with the micro-vibrator, automates the original manual operation in the chemical analysis, and can strictly control the sample volume when sampling, which can not only reduce the loss of the sample, but also reduce the error caused by human factors. By providing a suitable vibration frequency, the collision probability and binding rate of samples containing pathogenic microorganisms per unit time are increased, thereby greatly improving the detection efficiency and performance of the electrochemical biosensor.

[0014] Example 2: Figure 1~2 As shown, the present invention discloses a method for preparing an electrochemical biosensor, the preparation method comprising the following steps: Step 1: Use Solidworks 3D software to design a support base 1 capable of accommodating the installation of a micro vibrator 3, a working electrode mounting plate 5 connected to the working electrode, and a support top seat 8 embedded with an RE electrode 9 and a CE electrode 10.

[0015] Step 2: Use 3D printing technology to print out the support base 1, working electrode mounting plate 5 and support top seat 8 respectively, and clamp the micro vibrator 3 on the support base 1 accordingly; use non-conductive glue to fix the silver wire as RE electrode and the platinum wire as CE electrode on the support top seat 8, and use matching fixing clips to assemble the support base 1, working electrode mounting plate 5 and support top seat 8. The 3D printing device uses polylactic acid (PLA) filament as the printing material and prints the flow-through electrochemical biosensor through fused deposition modeling (FDM).

[0016] Step 3: Prepare the WE electrode 7 modified with Bare / HCL / Au / L-Cys / Ab / BSA / Ag, and insert the WE electrode 7 into the working electrode groove 6, and use a syringe to drip the redox probe into the through hole of the self-supporting top seat 8 so that the WE electrode 7, RE electrode 9 and CE electrode 10 form a loop. The RE electrode 9 is fixed to the corresponding position of the supporting top seat 8 using a 0.5 mm diameter Ag / AgCl wire using AB glue, and the CE electrode 10 is fixed to the corresponding position of the supporting top seat 8 using a 0.5 mm diameter platinum wire using AB glue. The WE electrode 7 is correspondingly mounted in the working electrode groove 6, and the WE electrode 7, RE electrode 9 and CE electrode 10 can be connected to form a detection loop through the redox probe.

[0017] The electrochemical biosensor of the present invention is a microstructure during detection, which is easy to carry, and the sensor assembly is simple, and the detection result is true and effective; the sensor has excellent stability, reproducibility and reusability, which is conducive to long-term use in actual analysis and detection. The prepared SSWM electrode is used as a working electrode and integrated into the working electrode mounting plate 5, which can effectively reduce the volume of the sample to be tested and improve the detection efficiency of the sample.

[0018] Example 3: Figure 3 As shown, the specific preparation steps of the WE electrode 7 of the electrochemical biosensor of the present invention are: Step S 1 : The 1000-mesh SSWM woven from 304 stainless steel wire was cut into sheets with a single electrode length of 20 mm and a width of 10 mm, and then ultrasonically cleaned in acetone, ethanol and deionized water for 15 min in sequence, and dried at 60°C to obtain the SSWM conductive substrate. Figure 4 (A) is the SEM image of the SSWM conductive substrate, from which it can be clearly seen that the SSWM conductive substrate has abundant pores.

[0019] Step S 2: The SSWM conductive substrate is sealed to a fixed area using a single-sided plastic sealing paper with holes to provide an effective detection area; the size of the plastic sealing paper is A4, the thickness is 125μm, the pore size is 2mm; the detection area diameter is 4mm.

[0020] Step S 3 : Prepare 0.5M hydrochloric acid solution to etch the surface of the SSWM conductive substrate with a fixed area for 10s, and immediately put it into a container filled with deionized water, then repeatedly wash it three times with a washing bottle, and then dry it naturally at room temperature.

[0021] Step S 4 : Use deionized water to prepare 10 mM chloroauric acid solution, select CHT660D device to construct a three-electrode device, and use cyclic voltammetry CV to set the process parameters as follows: origin E: -0.6, high E: 0.2, low E: -0.6, scanrate: 0.1v / s, sweep segment: 10, and deposit it on the surface of the SSWM electrode after hydrochloric acid treatment.

[0022] Step S 5 : Prepare 2.2 mg / ml L-Cys and 11.42 mg / ml TCEP HCL solutions respectively and mix them in a 1:1 ratio for 20 min. Take 20ul of the mixed solution and incubate it on the surface of the SSWM electrode for 2h at room temperature. Then rinse the dry well with deionized water and use an ear washing bulb to wash and blow away the active molecules on the surface of the SSWM electrode.

[0023] Step S 6 : Use PBS at pH = 5 to prepare 11.5 mg / 2ml EDC solution and 7 mg / 2ml NHS solution respectively. Take 10ul of EDC solution and NHS solution and place them in 80ul PBS solution at pH = 5 to make a 30 mM mixed solution. Take 20ul of the mixed solution and incubate it on the SSWM electrode surface at room temperature for 1h.

[0024] Step S 7 : The aptamer with a diluted concentration of 2 μmol / L was dropped on the surface of the SSWM electrode, placed in a 4°C refrigerator for 22 hours, and then rinsed.

[0025] Step S 8 : Under moisturizing conditions, take 20ul of 1%wt BSA and drop it on the surface of the SSWM electrode, incubate it at 37℃ for 1h, rinse it and dry it naturally.

[0026] Step S 9 : The ATCC25923 Staphylococcus aureus standard strain was subcultured and counted to obtain 1×10 3CFU / mL SA sample, take 20ul of SA sample and drop it on the surface of SSWM electrode to react for 30min.

[0027] Step S 10 : Insert the treated working electrode into the working electrode groove 6, drop the redox probe into the WE electrode 7, RE electrode 9 and CE electrode 10 to form a three-electrode path.

[0028] Step S 11 : A power supply with a voltage of 2V and a current of 0.049A was turned on to compare the electrochemical responses of the WE electrode 7 under static and vibrating states.

[0029] In Examples 1 to 3, L-Cys is L-cysteine, and L-Cys forms a gold-sulfur bond with Au NPs to match the functional group bound by Aptamer. Au NPs are gold nanoparticles that can be deposited on the electrode surface by an electrodeposition method, thereby increasing the conductivity of the working electrode, and the high specific surface area increases the electrochemical active area. Aptamer is an aptamer used to specifically connect to Staphylococcus aureus molecules. BSA (Business Software Alliance) is bovine serum albumin, which is used to encapsulate nonspecific sites, thereby blocking nonspecific molecules from being encapsulated on the electrode surface. SA (Staphylococcus aureus) is Staphylococcus aureus, which is the sample to be tested.

[0030] The electrochemical biosensor of the present invention adopts a SSWM electrode with a 3D mesh structure of stainless steel, which has excellent conductive properties and low cost advantages, greatly reducing the production cost of using glassy carbon, gold, platinum and other materials to make working electrodes; the working electrode does not require complex modification, the production process is simple, and can be processed and produced in high-efficiency batches; the electrochemical biosensor adopts a mesh structure with a high surface area and excellent catalytic performance, increases the contact area between the working electrode and the sample to be tested, and the working electrode can fully contact the sample, thereby improving the detection efficiency of pathogenic microorganisms.

[0031] The WE electrode constructed in the present invention is combined with a micro vibrator, which can transfer analytical samples and reagents from traditional test tubes, beakers, and containers into droplets. The sample to be tested and the redox probe complete physical mixing and chemical reaction in the working electrode to meet the market detection needs of scarce samples, expensive reagents, and rapid analysis. Fig.11 The graph shows the relationship between the detection current ΔI and time of the electrochemical sensor under static and vibrating conditions. When pathogenic microorganisms bind to the surface of the modified SSWM electrode, the electrochemical reduction signal can be measured transiently.

[0032] The present invention studies the electrochemical behavior based on SSWM electrode vibration analysis, such as Figures 5 to 8 shown. Figure 5 The DPV (Differential pulse voltammetry) curves of 1000 mesh SSWM electrodes in 2mM FcMeOH solution are shown below: Figure 5 It can be seen that there are significant differences in the electrochemical signal response of the smooth untreated, hydrochloric acid treated, and gold nanoparticle-modified 1000-mesh SSWM working electrode in 2 mM FcMeOH solution. Figure 6 It can be seen that the electrochemical impedance spectrum (EIS) of the gold nanoparticle-modified SSWM working electrode is also smaller than that of the untreated SSWM working electrode. The lower resistance and higher current response indicate that the electron transfer kinetics of the redox species are faster. Figure 7 The CV scan rate of 2.5~100mV / s was used to study the effect of the peak current at the SSWM working electrode. Figure 8 It shows that both the oxidation peak and the reduction peak have a good linear relationship with the square root of the scan rate, indicating that the electron transfer on the SSWM working electrode is a diffusion-controlled process. According to the Randles-Sevcik equation, the electroactive surface area of ​​the SSWM working electrode is estimated to be 0.275 cm 2 , the mesh area is about 0.142cm 2 (circle with a diameter of 4 mm). Figures 9-10 The electrochemical response of the electrode at different stages of immunomodification was analyzed using DPV technology and electrochemical impedance spectroscopy technology. These results show that the SSWM working electrode with a 3D grid microstructure has a high effective surface area, which can promote electron transfer and mass transfer between the electrode and the electrolyte, thereby improving the sensitivity of the sensor. On the basis of the SSWM electrode, L-Cys, aptamer, and BSA were modified in sequence to construct an electrochemical biosensor. The vibrating electrochemical device based on the SSWM electrode has higher mass transfer efficiency and excellent analytical performance, which lays a good foundation for the electrochemical analysis of Staphylococcus aureus.

[0033] The stainless steel wire mesh vibrating electrochemical sensor of the present invention is applied to the detection process of Staphylococcus aureus, such as Fig.11 As shown; Fig.11 A and 11B illustrate the effect of vibration on the electrochemical response time of Staphylococcus aureus. 3The electrical signal of the CFU / ml sample increased with the increase of time during the detection process; under static conditions, the electrochemical signal slowed down and the inflection point did not appear until 40 minutes; under the vibration state, the electrochemical signal slowed down and the inflection point appeared at 2 minutes, which means that the colony is more fully combined with the SSWM electrode base at this concentration, and the electrochemical response change value of 2 minutes under the vibration state is not much different from the electrochemical response change value of 40 minutes under the static state, indicating that the vibration condition can accelerate the kinetic rate of Staphylococcus aureus and improve the detection time of the biosensor.

[0034] The stainless steel wire mesh vibrating electrochemical sensor of the present invention quantitatively measures Staphylococcus aureus of various concentrations by DPV method. Fig.12 The left and right figures are the comparison of the response effects of the constructed electrochemical signal under static and vibration conditions. Fig.13 To record the current change response of different Staphylococcus aureus colony numbers in 20 μL SA sample under optimized experimental conditions, the results show that the current response change value of Staphylococcus aureus increases with the increase of colony number. Fig.14 The calibration curves of Staphylococcus aureus in static and vibrating states show that the current response change value of the number of Staphylococcus aureus colonies is proportional to the cell number within 10 1 ~10 6 The linear relationship is within the range, and the linear equation is ΔI1=3.05x-2.95 (R 2 =0.964, static); the linear equation is ΔI2=3.64x-3.82 (R 2 =0.960, vibration), where I is the peak current height (μA), X is the number of colonies, the vibration detection limit is 15 colonies (S / N=3), and the static detection limit is 20 colonies (S / N=3). The above excellent biosensing capabilities indicate that the sensor can be used to monitor Staphylococcus aureus at relatively low levels.

[0035] Fig.15 To interfere with the experiment, 1% PBS was used as a blank control, and different types of pathogenic microorganisms (Bacillus subtilis and Escherichia coli) were used for titration analysis to observe the specificity of the constructed electrochemical biosensor. Fig.16 To test the stability of the Bare / HCL / Au / L-Cys / Apt / BSA / SA electrode incubated for more than 7 days under vibration conditions, the current response of Staphylococcus aureus remained essentially unchanged after repeated testing for seven days, with an RSD value of 3.07%, indicating that the method has good operational stability and repeatability.

[0036] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An electrochemical biosensor for rapidly capturing microbial pathogens, characterized in that: The electrochemical biosensor (100) comprises a support base (1), a working electrode mounting plate (5) and a support top seat (8); The top surface of the support base (1) is provided with a mounting slot (2) and a micro vibrator (3) embedded in the mounting slot (2); The working electrode mounting plate (5) is mounted correspondingly on the top of the micro vibrator (3), and the top surface of the working electrode mounting plate (5) is provided with a working electrode groove (6) for mounting a WE electrode (7); The support top seat (8) is correspondingly buckled on the working electrode mounting plate (5), and the top surface thereof is respectively embedded with an RE electrode (9) and a CE electrode (10), and a through hole connected to the working electrode groove (6) is provided between the RE electrode (9) and the CE electrode (10), and a redox probe (11) is dripped into the through hole and connects the WE electrode (7), the RE electrode (9) and the CE electrode (10) to form a three-electrode path.

2. A method for preparing an electrochemical biosensor as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Step 1: Use Solidworks 3D software to design a support base (1) capable of accommodating the installation of a micro vibrator (3), a working electrode mounting plate (5) connected to the working electrode, and a support top seat (8) embedded with an RE electrode (9) and a CE electrode (10); Step 2: Use 3D printing technology to print out the support base (1), the working electrode mounting plate (5) and the support top seat (8) respectively, and mount the micro vibrator (3) on the support base (1) accordingly; use non-conductive glue to fix the silver wire as the RE electrode and the platinum wire as the CE electrode on the support top seat (8), and use a matching fixing clip to assemble the support base (1), the working electrode mounting plate (5) and the support top seat (8); Step 3: Prepare a Bare / HCL / Au / L-Cys / Ab / BSA / Ag modified WE electrode (7), insert the WE electrode (7) into the working electrode groove (6), and use a syringe to drip the redox probe into the through hole of the self-supporting top base (8) so that the WE electrode (7), RE electrode (9) and CE electrode (10) form a loop.

3. The method for preparing an electrochemical biosensor according to claim 2, characterized in that: The specific preparation steps of the WE electrode (7) are: Step S1: SSWM was cut into sheets with a single electrode length of 20 mm and a width of 10 mm, and then ultrasonically cleaned in acetone, ethanol and deionized water for 15 min in sequence, and dried at 60°C to obtain a SSWM conductive substrate; Step S2: using plastic sealing paper with holes on one side to plastic seal the SSWM conductive substrate to a fixed area to provide an effective detection area; Step S3: Prepare a 0.5M hydrochloric acid solution to etch the surface of the SSWM conductive substrate with a fixed area for 10 seconds, and immediately put it into a container filled with deionized water, and then repeatedly wash it three times with a washing bottle, and then dry it naturally at room temperature; Step S4: Prepare 10 mM chloroauric acid solution with deionized water, select CHT660D device to construct a three-electrode device, set the process parameters of cyclic voltammetry CV as follows: origin E: -0.6, high E: 0.2, low E: -0.6, scan rate: 0.1 v / s, sweep segment: 10, and deposit it on the surface of the SSWM electrode after hydrochloric acid treatment; Step S5: Prepare 2.2 mg / ml L-Cys and 11.42 mg / ml TCEP HCL solutions respectively, and mix them in a 1:1 ratio for 20 min. Take 20ul of the mixed solution and incubate it on the surface of the SSWM electrode for 2 h at room temperature. Then rinse the dry well with deionized water, and use an ear cleaning bulb to wash and blow the active molecules on the surface of the SSWM electrode. Step S6: Use PBS at pH = 5 to prepare 11.5 mg / 2 ml EDC solution and 7 mg / 2 ml NHS solution respectively, take 10 ul of EDC solution and NHS solution respectively and place them in 80 ul PBS solution at pH = 5 to prepare a 30 mM mixed solution, take 20 ul of the mixed solution and incubate it on the surface of the SSWM electrode at room temperature for 1 hour; Step S7: drop the aptamer with a diluted concentration of 2 μmol / L on the surface of the SSWM electrode, place it in a 4°C refrigerator for 22 h, and then rinse it; Step S8: Under moisturizing conditions, take 20ul of 1%wt BSA and drop it on the surface of the SSWM electrode, incubate it at 37°C for 1h, rinse it and dry it naturally; Step S9: Subculture the ATCC25923 Staphylococcus aureus standard strain and count it to obtain 1×10 3 CFU / mL SA sample, take 20ul of SA sample and drop it on the surface of SSWM electrode to react for 30min; Step S 10 : Insert the treated working electrode into the working electrode groove (6), drop a redox probe into the WE electrode (7), the RE electrode (9) and the CE electrode (10) to form a three-electrode path; Step S 11 : A power supply with a voltage of 2V and a current of 0.049A was turned on to compare the electrochemical responses of the WE electrode (7) under static and vibrating conditions.

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