Microfluidic electrochemical immunosensor and application thereof

Abstract

The invention discloses a microfluidic electrochemical immunosensor and application thereof.The microfluidic electrochemical immunosensor comprises a PDMS chip and a gold nano electrode slice, a flow channel system is arranged in the PDMS chip and comprises a plurality of liquid inlet flow channels, a plurality of first flow channels, a transition cavity, a second flow channel, a third flow channel and a liquid outlet flow channel, and the first flow channels, the transition cavity, the second flow channel, the third flow channel and the liquid outlet flow channel are communicated in sequence; a liquid inlet of the liquid inlet flow channel and a liquid outlet of the liquid outlet flow channel penetrate out of the upper surface of the PDMS chip; the transition cavity is obliquely arranged downwards from an inlet to an outlet of the transition cavity in the liquid flowing direction, the third flow channel is provided with a bent part bent in an S shape, and the bottom face of the third flow channel is open; the gold nano electrode plate can be inserted into the PDMS chip and seals the bottom surface of the third flow channel to form a reaction detection area; the surface of a working electrode of the gold nano electrode plate is sequentially modified with undeca-mercaptoundecanoic acid, NHS / EDC, streptavidin and a biotin-labeled allergy marker antibody; the high-sensitivity detection on the food allergy marker can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and in particular to a microfluidic electrochemical immunosensor and applications thereof. Background Art

[0002] Food allergy is a global health problem. Surveys and studies have found that about 8% of children and 4% of adults have allergic symptoms to one or more foods. In order to better prevent and control the occurrence of food allergies, food allergenicity risk assessment has become a research hotspot in the field of food allergies.

[0003] At present, the methods used by various countries and international organizations to evaluate the sensitization of allergenic proteins basically include bioinformatics, serological screening, digestive stability, animal models, and cellular immunology models. The occurrence and severity of food-induced allergies depend on the extent of the release of mediators in the particles after the interaction with the corresponding allergens bound to the surface of basophils and mast cells and the responsiveness of organs to these mediators. The cellular immunology method of the in vitro cell model mediator release experiment can directly reflect the sensitization activity of food allergens.

[0004] In recent years, various biomarker immunoassay technologies for cell in vitro sensitization have been reported at home and abroad, such as enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (ECL), surface plasmon resonance (SPR), fluorescence, electrochemical method, etc. Compared with other immunoassay technologies that require professional operation and centralized laboratory equipment, electrochemical immunosensors have attracted much attention due to their advantages such as rapid analysis, high sensitivity, simple operation, and low cost. However, the current electrochemical immunosensors are affected by external interference, especially the influence of human operation, which makes impedance detection unstable. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a microfluidic electrochemical immunosensor and its application. Based on a microfluidic chip, gold nanoparticles, a nanomaterial with excellent electrical properties, are used to construct gold nanoelectrodes on the electrode surface of the microfluidic chip. On this basis, an electrochemical immunosensor with high sensitivity and good selectivity is prepared, realizing controllable preparation within the microfluidic chip and high-sensitivity detection of food allergy markers.

[0006] In order to achieve the above objectives, an embodiment of the present invention provides a microfluidic electrochemical immunosensor in a first aspect, which comprises:

[0007] A PDMS chip, wherein a flow channel system is arranged inside the PDMS chip, wherein the flow channel system comprises a plurality of liquid inlet flow channels, a plurality of first flow channels connected to the plurality of liquid inlet flow channels in one-to-one correspondence, a transition cavity whose inlet is connected to the plurality of first flow channels, a second flow channel connected to the outlet of the transition cavity, a third flow channel connected to the second flow channel, and a liquid outlet flow channel connected to the third flow channel; the liquid inlet of the liquid inlet flow channel and the liquid outlet of the liquid outlet flow channel both pass through the upper surface of the PDMS chip; the transition cavity is arranged to be inclined downward along the liquid flow direction from its inlet to its outlet; the third flow channel has a curved portion with an S-shaped curve, and the bottom surface of the third flow channel is arranged to be open;

[0008] A gold nanoelectrode sheet, which can be inserted into the PDMS chip and seal the bottom surface of the third flow channel to form a reaction detection area; the working electrode surface of the gold nanoelectrode sheet is sequentially modified with undecaprylondecanoic acid, N HS / EDC, streptavidin and biotin-labeled allergy marker antibodies;

[0009] When used, the gold nanoelectrode sheet is connected to an electrochemical workstation.

[0010] According to the microfluidic electrochemical immunosensor of the present invention, firstly, the PDMS chip is structurally designed with independent liquid flow channels, inclined transition chambers to prevent backflow, S-shaped insertable electrode sheet flow channels, and independent outlets; the independent flow channel design allows multiple experiments to be carried out simultaneously, reduces experimental time, and improves efficiency, and different flow channel layouts can be designed according to experimental needs, thereby increasing the flexibility of experimental design; the inclined surface design can effectively prevent the liquid from flowing back during the flow process, and can more accurately control the liquid flow rate and flow rate, thereby controlling the reaction time and concentration gradient; the S-shaped flow channel design increases the contact area between the liquid and the electrode, thereby improving the efficiency of the electrochemical reaction; the insertable electrode sheet design makes the replacement and maintenance of the electrode more convenient, and adapts to different experimental needs; secondly, the electrochemical immunosensor is integrated in the microfluidic chip, and the undecylthiophene acid, EDC / NHS, and streptavidin are used to measure the reaction time and concentration gradient of the liquid. The coating makes it resistant to flow, and the integrated function of the chip avoids the influence of manual operation on the experimental results; the microfluidic chip has good biocompatibility, so that the biological molecules will not be inactivated in the chip and cannot be detected, which can ensure the accuracy of the detection; gold nanoparticles with good catalytic effect are used to achieve signal amplification; using exactly the same nanomaterials and modification methods, and utilizing the specific binding of antigens and antibodies, high-sensitivity and specific detection of different food allergy markers can be achieved by simply changing the type of food allergy markers. The operation is simple and the detection speed is fast. A large number of samples can be determined in a short time, which is conducive to the commercialization of food allergy marker sensors; there is no need to use enzymes in the markers of the detection antibodies, which avoids detection errors caused by enzyme inactivation and leakage, simplifies the preparation steps of the detection antibody markers, and significantly improves the reproducibility and stability of the electrochemical immunosensor.

[0011] Optionally, a K3Fe(CN)6 solution is further included, and the K3Fe(CN)6 solution is used as a detection electrolyte. After the sample to be tested is passed into the third flow channel from the outlet of the outlet flow channel and incubated, PBS and the K3Fe(CN)6 solution are passed in sequence. Thus, K3Fe(CN)6 is used as an electroactive substance to convert the electron transfer kinetics into electrode impedance changes, thereby achieving dual amplification of electrochemical signals and further improving the sensitivity of the sensor.

[0012] Optionally, there are a plurality of the third flow channels, and a gold nanoelectrode sheet is inserted into the bottom surface of each of the third flow channels.

[0013] Furthermore, the outflow end of the second flow channel is a forked flow channel, and the number of branches of the forked flow channel corresponds to the number of the third flow channels.

[0014] Optionally, the transition chamber has an inclination angle of 30°, and the plane where the first flow channel is located is higher than those of the second flow channel and the third flow channel.

[0015] Optionally, the gold nanoelectrode sheet is a three-electrode system, including a working electrode, an auxiliary electrode and a reference electrode, the working electrode is coated with gold nanoparticles, and the reference electrode is silver / silver chloride.

[0016] Optionally, the seven liquid inlet channels are used to respectively inlet PBS or ethanol solution, sulfuric acid solution or K3Fe(CN)6 solution, undecaprylundecanoic acid, EDC / NHS solution, streptavidin, biotin-labeled allergy marker antibody and blocking solution.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned microfluidic electrochemical immunosensor, comprising the following steps:

[0018] (1) preparing the PDMS chip: using a photolithography method to engrave a flow channel system pattern on the surface of a quartz glass sheet as a mold; then casting PDMS onto the surface of the mold, and removing the PDMS casting layer from the mold after solidification to obtain a PDMS upper layer having the flow channel system inside; then providing a flat PDMS lower layer; connecting the PDMS upper layer and the PDMS lower layer by plasma bonding, and leaving a gap at the position where the gold nanoelectrode sheet is inserted;

[0019] (2) preparing the gold nanoparticle electrode sheet: providing an electrode sheet of a three-electrode system, wherein the base material of the electrode sheet is polyethylene terephthalate, the working electrode and the auxiliary electrode materials are carbon, and the reference electrode material is silver / silver chloride; applying 6 μL of the gold nanoparticle solution on the surface of the working electrode, drying, rinsing with ultrapure water, and drying;

[0020] (3) inserting the gold nanoelectrode sheet into the reserved gap of the PDMS chip, and fastening the PDMS upper layer, the gold nanoelectrode sheet and the PDMS lower layer by a clamp to obtain a microfluidic chip;

[0021] (4) Functionalizing the microfluidic chip: PBS, sulfuric acid, undecylmercaptodecanoic acid, EDC / NHS solution, streptavidin, biotin-labeled allergy marker antibody, and blocking solution are respectively introduced into the flow channel system through injection pumps to sequentially modify the working electrode surface of the gold nanoelectrode sheet with undecylmercaptodecanoic acid, NHS / EDC, streptavidin, and biotin-labeled allergy marker antibody to obtain a microfluidic electrochemical immunosensor.

[0022] In a third aspect, the present invention proposes the application of the above-mentioned microfluidic electrochemical immunosensor or the microfluidic electrochemical immunosensor prepared by the above-mentioned preparation method in the field of allergy marker detection.

[0023] According to the application of the present invention, the design of the microfluidic chip adopts an inclined anti-backflow and a special S-shaped flow channel, which can ensure the accuracy of the detection and reduce the system error of the chip. The electrochemical sensor is based on a gold nanoelectrode, and 11-mercaptoundecanoic acid, NHS / EDC, and streptavidin are deposited on the gold nanobase in sequence to make it resistant to flow on the microfluidic chip; then the biotin-labeled allergy marker antibody can be introduced, and when a sample containing a biomarker, such as an antigen-containing sample, is introduced and incubated, PBS and K3Fe(CN)6 solution are introduced in sequence. The change in the kinetics of electron migration is detected by the electrochemical immunosensor and converted into a change in electrode impedance. The allergy marker can be detected by the impedance size. The microfluidic chip is combined with the electrochemical sensor, which can achieve the advantages of strong specificity, low detection line, high sensitivity, strong universality, and good regeneration while being resistant to flow. It can be used in conjunction with the organ chip later to form a complete detection system of organ chip-electrochemical sensor chip-electrochemical workstation; and it provides ideas for the detection of other biomarkers of electrochemical sensors on microfluidic chips.

[0024] Optionally, the microfluidic electrochemical immunosensor is connected to an electrochemical workstation to test the electrode modification by cyclic voltammetry, with an initial voltage of 0 V, a high voltage of 0.6 V, a low voltage of -0.6 V, a scan rate of 0.075, an interval sampling of 0.001, and a sensitivity of 10 -0.004 ; The modification process of the electrode was tested by AC impedance method, with a high frequency of 100000, a low frequency of 0.1, and an amplitude of 0.05; PBS was used for cleaning before each test, and potassium ferrocyanide was used as the electrolyte solution;

[0025] The sample to be tested is passed into the third flow channel from the liquid outlet of the liquid outlet flow channel and incubated for 1 hour.

[0026] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic structural diagram of a microfluidic electrochemical immunosensor according to an embodiment of the present invention;

[0028] Figure 2 is a perspective view of a microfluidic electrochemical immunosensor according to an embodiment of the present invention;

[0029] Figure 3 is a side perspective view of a PDMS chip according to an embodiment of the present invention;

[0030] Figure 4 is a top perspective view of a PDMS chip according to an embodiment of the present invention;

[0031] Figure 5 is a schematic structural diagram of a gold nanoelectrode sheet according to an embodiment of the present invention;

[0032] Figure 6 This is a real picture of a microfluidic electrochemical immunosensor without a gold nanoelectrode sheet inserted according to an embodiment of the present invention;

[0033] Figure 7 is a schematic diagram of the electrochemical immunosensor according to an embodiment of the present invention;

[0034] Figure 8 is a material characterization diagram of an electrochemical immunosensor according to an embodiment of the present invention;

[0035] Fig. 9 is an electrochemical characterization diagram of a carbon electrode and a gold nanoelectrode according to an embodiment of the present invention;

[0036] Fig.10 is an electrochemical characterization diagram of the electrochemical immunosensor at the functionalization stage according to an embodiment of the present invention;

[0037] Fig.11 1 is an electrochemical characterization diagram of the electrochemical immunosensor in the regeneration stage according to an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0039] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0040] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0041] Example 1

[0042] According to an embodiment of the present invention, a microfluidic electrochemical immunosensor is provided. Figure 1-Figure 6 As shown, the microfluidic electrochemical immunosensor includes a PDMS chip 1 and a gold nanoelectrode sheet.

[0043] Specifically, a flow channel system 100 is provided inside the PDMS chip 1, and the flow channel system 100 includes a plurality of liquid inlet channels 101, a plurality of first flow channels 102 connected to the plurality of liquid inlet channels 101 in one-to-one correspondence, a transition cavity 103 whose inlet is connected to the plurality of first flow channels 102, a second flow channel 104 connected to the outlet of the transition cavity 103, a third flow channel 105 connected to the second flow channel 104, and a liquid outlet channel 106 connected to the third flow channel 105; the liquid inlet of the liquid inlet channel 101 and the liquid outlet of the liquid outlet channel 106 both pass through the PDMS chip 1. The upper surface; the transition chamber 103 is arranged to be inclined downward along the liquid flow direction from its inlet to its outlet; the third flow channel 105 has an S-shaped curved portion, and the bottom surface of the third flow channel 105 is open; the gold nanoelectrode sheet 2 can be inserted into the PDMS chip 1 and close the bottom surface of the third flow channel 105 to form a reaction detection area; the surface of the working electrode 21 of the gold nanoelectrode sheet 2 is modified with undecylmercaptoundecanoic acid, NHS / EDC, streptavidin and biotin-labeled allergy marker antibodies in sequence; when used, the gold nanoelectrode sheet 2 is connected to the electrochemical workstation.

[0044] Therefore, according to the microfluidic electrochemical immunosensor of the embodiment of the present invention, its PDMS chip 1 (microfluidic chip) is structurally designed with independent liquid flow channels (multiple liquid inlet flow channels 101, multiple first flow channels 102, etc.), an inclined transition cavity 103 to prevent backflow, an S-shaped insertable electrode sheet flow channel (i.e., the third flow channel 105), and an independent outlet (liquid outlet flow channel 106); the independent flow channel design allows multiple experiments to be carried out simultaneously, reducing the experimental time and improving the efficiency, and different flow channel layouts can be designed according to the experimental needs, thereby increasing the flexibility of the experimental design; the inclined surface design can effectively prevent the liquid from flowing back during the flow process, and can more accurately control the liquid flow rate and flow rate, thereby controlling the reaction time and concentration gradient; the S-shaped flow channel design increases the contact area between the liquid and the electrode, thereby improving the efficiency of the electrochemical reaction; the insertable electrode sheet design makes the replacement and maintenance of the electrode more convenient, and adapts to different experimental needs; secondly, the electrochemical immunosensor is integrated in the microfluidic In the chip, the coating of undecaprylundecanoic acid, EDC / NHS and streptavidin makes it flow-resistant. Due to the integrated function of the chip, the influence of manual operation on the experimental results is avoided. The microfluidic chip has good biocompatibility, so that the biological molecules will not be inactivated in the chip and cannot be detected, which can ensure the accuracy of the detection. Gold nanoparticles with good catalytic effect are used to achieve signal amplification. Using exactly the same nanomaterials and modification methods, the specific binding of antigens and antibodies is utilized, and high sensitivity and specificity of different food allergy markers can be achieved by changing the type of food allergy markers. The operation is simple and the detection speed is fast. A large number of samples can be determined in a short time, which is conducive to the commercialization of food allergy marker sensors. It is not necessary to use enzymes in the markers of the detection antibodies, which avoids the detection errors caused by enzyme inactivation and leakage, simplifies the preparation steps of the detection antibody markers, and significantly improves the reproducibility and stability of the electrochemical immunosensor.

[0045] In a specific example, a K3Fe(CN)6 solution is also included, and the K3Fe(CN)6 solution is used as a detection electrolyte. After the sample to be tested is passed into the third flow channel 105 from the outlet of the liquid outlet flow channel 106 and incubated, PBS and K3Fe(CN)6 solution are passed in sequence. Thus, K3Fe(CN)6 is used as an electroactive substance to convert the electron transfer kinetics into electrode impedance changes, thereby achieving dual amplification of electrochemical signals and further improving the sensitivity of the sensor.

[0046] In a specific example, there are multiple third flow channels 105, and a gold nanoelectrode sheet 2 is inserted on the bottom surface of each third flow channel 105. For example, as shown in the figure, there are three third flow channels 105, and three gold nanoelectrode sheets 2 are inserted on the bottom surface of the third flow channel 105 in a one-to-one correspondence; at the same time, there are also three liquid outlet channels 106 connected to the third flow channel 105. In this way, different samples to be tested can be detected at the same time. The liquid outlet channel 106 is connected to the waste liquid pump during the sensor manufacturing process. After the sensor is manufactured, these outlets can be used as sample detection inlets, and three different samples can be detected simultaneously by connecting a syringe pump. By using the precise control of the flow rate of the syringe pump, only a trace amount of sample is needed to achieve simultaneous detection of three samples.

[0047] More specifically, the outflow end of the second flow channel 104 is a bifurcated flow channel, and the number of bifurcations of the bifurcated flow channel corresponds to the plurality of third flow channels 105. It can be understood that the end of the second flow channel 104 connected to the transition chamber 103 is a single flow channel, and the end of the second flow channel 104 connected to the third flow channel 105 is a bifurcated flow channel, so that the liquid in the transition chamber 103 can flow into different third flow channels 105 respectively.

[0048] In a specific example, the transition chamber 103 has an inclination angle of 30°, and the plane where the first flow channel 102 is located is higher than the second flow channel 104 and the third flow channel 105. The transition chamber 103 is an inclined plate as a whole, and the inclined upper end of the inclined plate is connected to the first flow channel 102, and the inclined lower end of the inclined plate is connected to the second flow channel 104, so that the liquid can be prevented from flowing back, thereby avoiding cross contamination between samples and interference with the test results, enhancing the stability and controllability of the flow, ensuring the purity of the sample for each test, and thus improving the accuracy and repeatability of the test results.

[0049] In a specific example, there are seven liquid inlet channels 101, and the seven liquid inlet channels 101 are used to feed PBS or ethanol solution, sulfuric acid solution or K3Fe(CN)6 solution, undecylmercaptodecanoic acid, EDC / NHS solution, streptavidin, biotin-labeled allergy marker antibody and blocking solution, respectively. More specifically, liquid inlet channel 101 No. 1 is used to feed PBS or ethanol solution, liquid inlet channel 101 No. 2 is used to feed sulfuric acid solution or K3Fe(CN)6 solution, liquid inlet channel 101 No. 3 is used to feed undecylmercaptodecanoic acid, liquid inlet channel 101 No. 4 is used to feed EDC / NHS solution, liquid inlet channel 101 No. 5 is used to feed streptavidin, liquid inlet channel 101 No. 6 is used to feed biotin-labeled allergy marker antibody, and liquid inlet channel 101 No. 7 is used to feed blocking solution.

[0050] More specifically, the overall height of the PDMS chip 1 is 6 mm, the length is 55 mm, the width is 52 mm, the height of the inlet of the inlet channel 101 is 3 mm, the height of the outlet of the outlet channel 106 is 5 mm, the height of the slope of the transition cavity 103 is 2 mm; the slope inclination angle is 30°, the diameter of each channel of the channel system 100 is 0.5 mm, the inlet channel (the channel from the inlet to the slope device, Figure 3 102) length is 20mm, the outlet flow channel (from the inclined device to the outlet, Figure 3 104 and 105) are 27mm long and 20mm long, respectively; the outer dimensions of the S-shaped flow channel can be designed according to the size of the electrode sheet; the length range of the S-shaped flow channel is 3mm-5mm-7mm-9mm-7mm-5mm-3mm. Among them, the tortuous path of the S-shaped flow channel can effectively reduce the flow rate of the liquid, so that the liquid has enough time in the flow channel to contact the sensor surface or reaction area, thereby improving the detection sensitivity and reaction efficiency; and the tortuous flow channel design helps to reduce the formation of bubbles during the flow of the liquid. The presence of bubbles will affect the stability of the flow and the accuracy of the detection results; furthermore, the S-shaped flow channel can make the liquid more evenly distributed during the flow process, thereby reducing the errors caused by different flow path or liquid volume; in addition, the design of the S-shaped flow channel increases the contact area and mixing time of the liquid in the flow channel, which helps to fully mix the sample and reagent and improve the detection effect.

[0051] In addition, the gold nanoelectrode sheet 2 can be inserted into the gap with a width of 0.5 mm and a length of 16 mm.

[0052] Example 2

[0053] The present invention provides a method for preparing a microfluidic electrochemical immunosensor described in the scheme of Example 1, wherein the preparation method includes: designing and making a microfluidic chip (the making of the microfluidic chip includes the design and making of the upper flow channel, the design and making of the lower substrate, and the bonding of the upper flow channel and the lower substrate), designing and making a gold nanoelectrode sheet 2, assembling the gold nanoelectrode sheet 2 and the microfluidic chip, and making an electrochemical immunosensor (sensor functionalization of the gold nanoelectrode sheet 2 and the microfluidic chip). Wherein, in the sensor functionalization stage, the order of adding reagents is: PBS, sulfuric acid, undecylmercaptoundecanoic acid, EDC / NHS solution, streptavidin, biotinylated antibody, and blocking solution. Each reagent is connected to the corresponding liquid inlet in sequence through a syringe pump, and the waste liquid is discharged into a waste liquid bottle through three liquid outlets. In the sample detection stage, the syringe pump can be connected to the three liquid outlets, and different samples to be tested are injected respectively. After the incubation is completed, potassium ferrocyanide solution is injected through the No. 2 inlet as a detection electrolyte, and the electrode sheet is connected to the electrochemical workstation for detection. After the test is completed, PBS solution is injected through inlet 1 to wash the potassium ferrocyanide solution on the electrode sheet. During the chip regeneration stage, PBS can be injected through inlet 1 to clean the electrode sheet, and then sulfuric acid solution can be injected through inlet 2 for secondary cleaning.

[0054] Specifically:

[0055] (1) preparing a PDMS chip 1;

[0056] Prepare SU8 molds with different patterns (for example, the template for the PDMS substrate used to process the upper channel mentioned above is a microfabrication mold with a reverse channel design made of photoresist).

[0057] PDMS pretreatment: Use a disposable paper cup and an electronic scale to weigh the PDMS glue. The mass ratio of glue A to glue B is 10:1. Use a stirring rod (or chopsticks) to stir and mix. Degassing (vacuum degassing) Put the mixed PDMS glue into a vacuum degassing device and draw a vacuum to remove the bubbles in the mixture for later use.

[0058] PDMS pouring: Place the mold for the chip to be prepared in the pouring tray. Slowly pour the degassed PDMS into the tray, the thickness is determined according to the requirements, and let it stand for 10 minutes.

[0059] PDMS curing: Place the casting tray in an oven for heat drying, usually at 70°C to 80°C for half an hour, and take it out after the PDMS is cured.

[0060] PDMS separation: Open the buckles on both sides of the PDMS pourer, remove the upper part of the pourer, first take out the cured PDMS and mold as a whole, and then carefully separate the PDMS and mold.

[0061] Cutting and hole preparation: Use a cutter to cut the PDMS chip as needed to prepare a chip of the required size. Use a puncher to make the required holes on the chip for fluid inlet and outlet. Thus, an upper flow channel with a flow channel system 100 is made; and then a lower base is provided.

[0062] PDMS chip bonding: Use tape to remove dust from the surface of the prepared PDMS chip; clean the upper and lower substrates (upper flow channel and lower base) to ensure that the surface is clean and dust-free; place the upper and lower layers of the processed PDMS chip on a tray and put it into plasma for surface treatment. If the PDMS chip needs to align the upper and lower layers, it is necessary to use the alignment platform treatment in advance for pre-alignment (but do not fit), and then take the completed PDMS chip out of the alignment platform together with the tray and put it into plasma for surface treatment. Quickly fit the upper and lower layers of the treated PDMS chip, put it in an oven at 85°C for 30 minutes, and take it out. In this way, a PDMS chip 1 with an overall rectangular shape is produced by plasma bonding; a gap is reserved for the PDMS chip 1 at the position where the electrode sheet is inserted.

[0063] (2) preparing a gold nanoelectrode sheet 2;

[0064] Gold nanoelectrodes are made by drop-coating gold nanoparticles on a carbon electrode; a three-electrode system based on a carbon electrode, such as Figure 5 As shown, it includes a working electrode (WE) 21, an auxiliary electrode (CE) 22, and a reference electrode (RE) 23. Among them, the working electrode 21 is used to carry out electrochemical reactions, the reference electrode 23 provides a stable reference potential, and the auxiliary electrode 22 ensures that the current is evenly distributed. This configuration allows for more accurate control and analysis of electrochemical processes. Specifically, the base material is polyethylene terephthalate (PET); the materials of the working electrode 21 and the auxiliary electrode 22 are carbon, and the material of the reference electrode 23 is silver / silver chloride; the size of the electrode sheet 2 is 12×34mm; the thickness of the electrode sheet 2 is 0.3mm; and the diameter of the working electrode 21 is 5mm.

[0065] Add 4.1 mL of 1% chloroauric acid to 95.8 mL of ultrapure water, add 10 mL of 38.8 mmol / L sodium citrate while stirring, reflux at 100°C for 15 min, centrifuge, discard the precipitate, and the evenly dispersed wine-red supernatant is the gold nanoparticle solution; drop 6 μL of the gold nanoparticle solution on the surface of the working electrode, dry, rinse with ultrapure water, and dry to obtain the gold nanoelectrode sheet 2.

[0066] (3) preparing a solution;

[0067] Self-assembled monolayer SAM solution: Dissolve 109.18 mg MUA (11-mercaptoundecanoic acid) in 50 mL ethanol to obtain a 10 mM SAM solution. Seal the lid with parafilm to avoid evaporation and store the solution at 4°C. SAM can be stored at 4°C for about 2 months.

[0068] 50mM Citric Acid Buffer Solution: Dissolve 0.335g sodium citrate and 0.261g citric acid in 400mL distilled water. Adjust the solution to pH 4.5 using 1M hydrochloric acid or 1M sodium hydroxide and fill the solution to a final volume of 500mL to obtain Citric Acid Buffer. Citric Acid Buffer can be stored at 4°C for at least 6 months.

[0069] Equimolar (25 mM) EDC / NHS solution: Dissolve 5.75 mg NHS in 1 mL 50 mM citrate buffer to prepare a 50 mM NHS solution. Then dissolve 9.55 mg EDC in 1 mL 50 mM citrate buffer to prepare a 50 mM EDC solution; mix the two solutions in a 1:1 ratio to obtain an EDC / NHS solution. Prepare the EDC / NHS solution freshly before the experiment.

[0070] 50mM K3Fe(CN)6 solution: Dissolve 823.15mg of K3Fe(CN)6 in 50mL of deionized water. K3Fe(CN)6 solution can be stored in the dark at room temperature for 1 to 2 months. K3Fe(CN)6 is a photosensitive substance and decomposes slowly when exposed to light.

[0071] 10 μg mL -1 SPV stock solution: Dissolve 1 mg of SPV (streptomycin and penicillin) into 1 mg mL -1 Then, 140 μL of SPV solution was added to 13.88 mL of PBS to dilute to 10 μg mL -1 It can be stored at -20℃ for 3 months, avoid repeated freezing and thawing.

[0072] 10mM sulfuric acid stock solution: Mix 500μL of 1M sulfuric acid solution with 49.5mL of distilled water. The sulfuric acid solution can be stored in a chemical cabinet at room temperature (18-24℃) for at least 6 months.

[0073] 10 μg mL -1 Biotinylated antibody solution (biotin-labeled allergy marker antibody): dilute the stock solution to 10 μg mL with PBS -1, the preparation of antibodies is based on the preparation of biotinylation kit, specifically: dissolve the biotin lyophilized powder in DMF; take the antibody to be labeled and add it to the ultrafiltration tube, and add an appropriate volume of Labeling buffer to a total volume of 0.5mL, and centrifuge at 12000×g for 10 minutes; add an appropriate amount of dissolved biotin (the recommended mass ratio of antibody: biotin is 10:1), such as 0.5mL 2mg / mL antibody (MW 150000), add 10μL of biotin with a concentration of 10mg / mL, and gently blow to mix, and put it in a 37℃ constant temperature box incubated in the dark for 30 minutes; after the incubation, centrifuge at 12000×g for 10 minutes; washing: add an appropriate amount of Labeling buffer to the above ultrafiltration tube to make a final volume of 0.5mL, gently blow to mix, centrifuge at 12000×g for 10 minutes, and repeat this operation once; recovery: after the centrifugation, take 0.2mL Labeling Add buffer to the above ultrafiltration tube, blow gently, turn the filter upside down and place it in another clean centrifuge tube, centrifuge at 6000×g for 10 minutes; fix volume: collect the solution in the centrifuge tube, which is the antibody labeled with biotin. Add Labeling buffer as needed to adjust the concentration, then add the same volume of protective agent and store at -20℃; the biotinylated antibody solution should be freshly prepared before the experiment.

[0074] (4) inserting three gold nanoelectrode sheets 2 into the PDMS chip 1 and clamping them with a clamp 3 to fix the PDMS chip 1 and the electrode sheets;

[0075] (5) An electrochemical immunosensor is fabricated on the PDMS chip 1 assembled with three gold nanoelectrode sheets 2. The principle is as follows: Figure 7 As shown, the surface of the working electrode 21 of the gold nanoelectrode sheet 2 is first coated with 11-mercaptodecanoic acid (11-MUA), and then combined with a 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinker solution to activate the carboxyl group of 11-MUA and couple streptavidin (SPV). After creating a uniform SPV molecular pattern on the surface of the working electrode 21, the biotinylated capture probe is fixed through a strong biotin-SPV interaction, making the bioreceptor surface resistant to flow. The nonspecific binding on the biosensor surface is eliminated by a bovine serum albumin (BSA) blocking solution, which needs to be prepared and used immediately. K3Fe(CN)6 is a common redox mediator whose electrons can be quickly transferred at the interface between the substrate and the electrode, and it is low in cost. It can be used to make signal labels, and a sample containing a biomarker, such as a sample containing an antigen, is passed through and incubated, and then PBS and K3Fe(CN)6 solutions are passed through in sequence. When the antibody or aptamer captures the antigen, [Fe(CN)6] 4- With [Fe(CN)6] 3-Interconversion occurs, and changes in electron migration kinetics are detected by the immunosensor and converted into changes in electrode impedance. When the captured antigen on the electrode surface is saturated, the antigen will shield electron migration and require regeneration for further measurement. The electrode is regenerated by a two-step wash with sulfuric acid and PBS, and the CV and EIS of the regenerated electrode are measured by K3Fe(CN)6. Once regenerated, the functionalization of microelectrodes for biomarker detection can be completed by repeating the above steps.

[0076] The following washing is performed by connecting different inlet channels 101 of the channel system 100 through injection pumps, so that the reagent flows from the inlet channel 101 through the first channel 102, the transition chamber 103, the second channel 104, the third channel 105 and the outlet channel 106 in sequence, and the three outlet channels 106 are connected to the waste liquid bottle.

[0077] Specifically as follows:

[0078] ① Washing of the gold nanoelectrode sheet 2 in the PDMS chip 1:

[0079] First, PBS was stirred at 5.0 mL·h -1 Then, 10 mM sulfuric acid solution was added at 5.0 mL·h -1 The solution was flowed for 6 min under CV potential scanning, and then flowed for an additional 6 min under CV potential scanning. The CV scanning parameters were: high voltage 1.8 V, low voltage 0 V, scan rate 0.1, sampling interval 0.001, sensitivity 1×10 -0.002 ;

[0080] The second step of EC cleaning was carried out at 5.0 mL·h -1 A 50 mM K3Fe(CN)6 solution was flowed for 6 min under CV potential scanning, and then flowed for an additional 6 min under CV potential scanning. The CV parameters were: initial voltage 1.0 V, high and low voltages 1.2 V and -1.2 V, respectively, scan rate 0.2, sampling interval 0.002, sensitivity 1×10 -0.004 .

[0081] ②Measure the bare electrode signal through CV and EIS:

[0082] After washing with K3Fe(CN)6 solution, the bare electrode parameters were measured by CV and EIS. The CV measurement parameters were: initial voltage was 0 V, high voltage and low voltage were 0.6 V and -0.6 V, respectively, scan rate was 0.075, sampling interval was 0.001, and sensitivity was 1×10 -0.004 The EIS measurement parameters are: initial voltage 0V, high frequency 100000V, low frequency 0.1V, sensitivity 1×10 -0.004 .

[0083] ③ Incubation of SAM solution:

[0084] First, at 5.0 mL·h -1 Anhydrous ethanol was flowed at 5.0 mL·h -1 Flow 10 mM SAM solution for 6 min to ensure that the detection chamber is filled with SAM only and there is no possible contamination by other reagents. Incubate the SAM solution at room temperature for 1 hour.

[0085] ④Measure the signal after modification of 11-MUA (SAM solution) by CV and EIS:

[0086] First, at 5.0 mL·h -1 PBS was flowed for 6 min under the conditions and then at 5.0 mL·h -1 50 mM K3Fe(CN)6 solution was flowed downward for 6 min; the measurement parameters were the same as above ②.

[0087] ⑤ Incubation of streptavidin (SPV):

[0088] First, at 5.0 mL·h -1 Anhydrous ethanol was flowed for 6 min, and then at 5 mL·h -1 PBS was flowed for 6 min under the conditions; then at 5.0 mL·h -1 25 mM equimolar EDC / NHS solution was flowed for 6 min under the conditions, and the EDC / NHS solution was incubated at room temperature for 30 min; finally, the mixture was incubated at 5.0 mL·h -1 10 μg mL-1 SPV was flowed for 6 min. SPV was incubated at room temperature for 1 hour.

[0089] ⑥Measure the signal after SPV modification by CV and EIS

[0090] First, at 5.0 mL·h -1 PBS was flowed for 6 min under the conditions and then at 5.0 mL·h -1 50 mM K3Fe(CN )6 Solution for 6 minutes; measurement parameters are the same as ②.

[0091] ⑦Incubation of biotinylated antibody

[0092] 10 μg·mL -1 The biotinylated antibody solution was heated at 5.0 mL·h -1 The biotinylated antibody was incubated at room temperature for 1 hour.

[0093] ⑧Measure the signal after biotinylated antibody modification by CV and EIS

[0094] First, at 5.0 mL·h-1 PBS was flowed for 6 min under the conditions and then at 5.0 mL·h -1 50 mM K3Fe(CN)6 solution was flowed downward for 6 min; the measurement parameters were the same as ②.

[0095] Next, the microfluidic electrochemical immunosensor prepared above was used to detect allergy markers:

[0096] The allergy marker detected in this example is the food allergy marker OX40L, which is cultured by culturing mouse intestinal epithelial cells (CMT-93) in the upper layer of the Transwell chamber; culturing dendritic cells (DC2.4) in the lower layer of the Transwell chamber; adding a certain concentration of allergen OVA to the upper layer of the chamber to stimulate for 24-48 hours; collecting the supernatant of the cells in the lower layer of the chamber, centrifuging and storing, and obtaining the sample to be tested. Specifically, the two cells are co-cultured under Transwell conditions, and a 24-well Transwell chamber is taken. After the cells reach 80-100% confluence, the cells in the culture dish are passaged and digested into a single cell suspension. 200 μL, 1×10 5 cells / mL of CMT-93 were inoculated on the upper layer of the chamber; 500 μL, 1×10 5 DC2.4 cells with a concentration of 100 μg / mL were inoculated in the lower layer of the chamber, and the chamber was placed in a CO2 incubator for sterile culture for 3 days (the medium can be changed on the second day of culture). Then, 100 μg / ml of the allergen OVA was added to the upper side of the chamber. After stimulation for 24 hours, 100 μL of culture medium was collected from the lower layer of the chamber, centrifuged to obtain the supernatant, and stored at -20°C as the sample to be tested.

[0097] Testing of samples to be tested:

[0098] First, at 5.0 mL·h -1 The blocking solution was flowed for 6 min under the conditions. The blocking solution was a PBS solution containing 5% BSA (bovine serum albumin) to prevent nonspecific adsorption. Then, the solution was stirred at 5.0 mL·h -1 Flow the sample for 6 min; incubate the sample at room temperature for 1 h.

[0099] Measuring the signal after cell culture medium modification by CV and EIS:

[0100] First, at 5.0 mL·h -1 PBS was flowed for 6 min under the conditions and then at 5.0 mL·h -1 50 mM K3Fe(CN)6 solution was flowed downward for 6 min; the measurement parameters were the same as above ②.

[0101] like Figure 8As shown in the figure, the changes in the electrode surface morphology during the electrode modification process were characterized by SEM. The morphological characteristics of the carbon electrode, gold nanoelectrode, modified gold nanoelectrode and regenerated gold nanoelectrode were studied by SEM. Figure 8 (a) SEM image of a carbon electrode showing a rough surface with an extensive 3D network structure, and defects in the carbon electrode such as microcracks, pores, and impurities can be observed; these defects may affect the electrochemical performance and service life of the electrode. Figure 8 In (b), gold nanoparticles can be observed to be uniformly deposited on the surface of the carbon electrode. At a magnification of 500 nm, gold nanoparticles with a diameter of about 100-200 nm can be clearly observed. This AuNFs-modified carbon electrode with a highly rough surface area can enhance electron transfer ability, high chemical reaction rate and stability, and is the basis for the production of electrochemical sensors. Figure 8 (c) shows the modified gold nanoparticles with obvious flocculent precipitation, indicating that the biomarker modification process on the sensor based on microfluidics has been successful. Figure 8 (d) shows that the biomarkers on the electrochemical sensor after one regeneration have been washed away and Figure 8 (b) The comparison shows that the roughness of gold nanoparticles increases and the density of gold nanoparticles decreases. In subsequent experiments, the thickness of gold nanoparticle deposition can be increased according to the required number of regenerations to achieve multiple regeneration effects.

[0102] like Fig. 9 As shown in the figure, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to study the effects of different modified electrodes on [Fe(CN)6] 3- / 4- Electrochemical behavior in solution (5 mM). Cyclic voltammetry (CV) was used to test the electrochemical performance of carbon electrode and gold nanoelectrode in [Fe(CN)6] 3- / 4- The redox peaks in the solution. The redox response current of the gold nanoelectrode was greatly enhanced, with the oxidation current intensity increasing by 341.7% and the reduction current intensity increasing by 343.3% (the oxidation peak of the gold nanoelectrode was 9.988×10 -4 A, reduction peak is 7.887×10 -4 A; The oxidation peak of carbon electrode is 2.261×10 -4 A; reduction peak is 1.779×10 -4 A); The peak area of ​​the gold nanoelectrode is significantly larger than that of the carbon electrode, indicating that [Fe(CN)6] 3- / 4- The amount of total charge transferred in the solution is greater in the gold nanoelectrode; this indicates that the introduction of gold nanoparticles promotes the transfer of electrons, can improve the response performance of the electrode, and is more suitable for the preparation of electrochemical sensors.

[0103] like Fig.10As shown in the figure, the bare electrode (Bare), 11-MUA (SAM), streptavidin (SPV), biotinylated antibody (Bio-antibody), and sample (Medium) test process of gold nanoelectrode were characterized by CV and EIS. (a) The CV graph shows that as the modification process proceeds, [Fe(CN)6] 3- / 4- The redox peak of the bare electrode gradually decreased, and the response current of the oxidation peak increased from 9.988×10 -4 A is reduced to 1.808×10 -5 A; In the electrochemical AC impedance (b) spectrum, the diameter of the Nyquist curve is approximately equal to the charge transfer resistance Rct. It shows that as the modification process proceeds, the impedance of the electrode surface gradually increases. The bare gold nanoelectrode without any material modification has a smaller impedance value, which is measured to be about 960.6Ω; Due to the certain insulation effect of 11-MUA and SPV modification, the impedance value of the electrode gradually increases after self-assembly; With the fixation of biotinylated antibodies and antigens (biomarkers to be tested) on the electrode surface, the thickness of the electrode surface film gradually increases, and the resistance to electron transfer in the redox reaction on the electrode surface also gradually increases, causing the radius of the Nyquist curve to gradually increase, and finally the electrode impedance value increases to the maximum value, which is about 15571.5Ω. The above characterization results show that the microfluidic electrochemical immunosensor is successfully constructed, and the trend of impedance change is consistent with the peak current change law in cyclic voltammetry. Combined with the SEM image, it also effectively proves that the modification of the electrode surface material is successful.

[0104] like Fig.11 As shown in Figure 1, the resistance change of the bare gold electrode and the regenerated gold electrode was characterized by EIS. Regeneration increased the roughness of the electrode. The Nyquist plot showed that the impedance of the regenerated gold electrode increased relatively, from 1280Ω of the bare gold electrode to 1382.4Ω of the first regenerated electrode, and then to 1664Ω of the second regenerated electrode. The increase in the impedance of the regenerated electrode may be because the regeneration process (such as electrochemical cleaning, physical wear or chemical treatment) causes the electrode surface to become rougher. The increase in surface roughness affects the charge transfer path on the electrode surface, making the charge transfer process more complicated, thereby increasing the impedance; and the regeneration process may change the chemical composition or state of the electrode surface. For example, an oxide layer or other chemical substances may be formed on the electrode surface, which may affect the electrochemical activity of the electrode, thereby increasing the impedance. The impedance increase of the second regenerated electrode relative to the bare electrode is significant, and the number of electrode regenerations can be increased by increasing the thickness of the gold nanoparticle deposition, optimizing the experimental conditions, and so on.

[0105] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0106] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A microfluidic electrochemical immunosensor, characterized in that: include: A PDMS chip, wherein a flow channel system is arranged inside the PDMS chip, wherein the flow channel system comprises a plurality of liquid inlet flow channels, a plurality of first flow channels connected to the plurality of liquid inlet flow channels in one-to-one correspondence, a transition cavity whose inlet is connected to the plurality of first flow channels, a second flow channel connected to the outlet of the transition cavity, a third flow channel connected to the second flow channel, and a liquid outlet flow channel connected to the third flow channel; the liquid inlet of the liquid inlet flow channel and the liquid outlet of the liquid outlet flow channel both pass through the upper surface of the PDMS chip; the transition cavity is arranged to be inclined downward along the liquid flow direction from its inlet to its outlet; the third flow channel has a curved portion with an S-shaped curve, and the bottom surface of the third flow channel is arranged to be open; A gold nanoelectrode sheet, which can be inserted into the PDMS chip and seal the bottom surface of the third flow channel to form a reaction detection area; the working electrode surface of the gold nanoelectrode sheet is sequentially modified with undecylmercaptoundecanoic acid, NHS / EDC, streptavidin and biotin-labeled allergy marker antibodies; When used, the gold nanoelectrode sheet is connected to an electrochemical workstation.

2. The microfluidic electrochemical immunosensor according to claim 1, characterized in that: It also includes a K3Fe(CN)6 solution, which is a detection electrolyte. After the sample to be tested is passed into the third flow channel from the outlet of the outlet flow channel and incubated, PBS and the K3Fe(CN)6 solution are passed in sequence.

3. The microfluidic electrochemical immunosensor according to claim 1, characterized in that: There are a plurality of the third flow channels, and a gold nano-electrode sheet is inserted on the bottom surface of each of the third flow channels.

4. The microfluidic electrochemical immunosensor according to claim 3, characterized in that: The outflow end of the second flow channel is a forked flow channel, and the number of forks of the forked flow channel corresponds to the number of the third flow channels.

5. The microfluidic electrochemical immunosensor according to claim 1, characterized in that: The transition chamber has an inclination angle of 30°, and the plane where the first flow channel is located is higher than those of the second flow channel and the third flow channel.

6. The microfluidic electrochemical immunosensor according to claim 1, characterized in that: The gold nano-electrode sheet is a three-electrode system, including a working electrode, an auxiliary electrode and a reference electrode. The working electrode is coated with gold nano-particles, and the reference electrode is silver / silver chloride.

7. The microfluidic electrochemical immunosensor according to claim 1, characterized in that: The seven liquid inlet flow channels are respectively used to inlet PBS or ethanol solution, sulfuric acid solution or K3Fe(CN)6 solution, undecaprylundecanoic acid, EDC / NHS solution, streptavidin, biotin-labeled allergy marker antibody and blocking solution.

8. The method for preparing a microfluidic electrochemical immunosensor according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) preparing the PDMS chip: using a photolithography method to engrave a flow channel system pattern on the surface of a quartz glass sheet as a mold; then casting PDMS onto the surface of the mold, and removing the PDMS casting layer from the mold after solidification to obtain a PDMS upper layer having the flow channel system inside; then providing a flat PDMS lower layer; connecting the PDMS upper layer and the PDMS lower layer by plasma bonding, and leaving a gap at the position where the gold nanoelectrode sheet is inserted; (2) preparing the gold nanoparticle electrode sheet: providing an electrode sheet of a three-electrode system, wherein the base material of the electrode sheet is polyethylene terephthalate, the working electrode and the auxiliary electrode materials are carbon, and the reference electrode material is silver / silver chloride; applying 6 μL of the gold nanoparticle solution on the surface of the working electrode, drying, rinsing with ultrapure water, and drying; (3) inserting the gold nanoelectrode sheet into the reserved gap of the PDMS chip, and fastening the PDMS upper layer, the gold nanoelectrode sheet and the PDMS lower layer by a clamp to obtain a microfluidic chip; (4) Functionalizing the microfluidic chip: PBS, sulfuric acid, undecylmercaptodecanoic acid, EDC / NHS solution, streptavidin, biotin-labeled allergy marker antibody, and blocking solution are respectively introduced into the flow channel system through injection pumps to sequentially modify the working electrode surface of the gold nanoelectrode sheet with undecylmercaptodecanoic acid, NHS / EDC, streptavidin, and biotin-labeled allergy marker antibody to obtain a microfluidic electrochemical immunosensor.

9. Application of the microfluidic electrochemical immunosensor according to any one of claims 1 to 7 or the microfluidic electrochemical immunosensor prepared by the preparation method according to claim 8 in the field of allergy marker detection.

10. The use as claimed in claim 9, characterized in that The microfluidic electrochemical immunosensor was connected to an electrochemical workstation and the electrode modification was tested by cyclic voltammetry with an initial voltage of 0 V, a high voltage of 0.6 V, a low voltage of -0.6 V, a scan rate of 0.075, an interval sampling of 0.001, and a sensitivity of 10 -0.004 ; The modification process of the electrode was tested by AC impedance method, with a high frequency of 100000, a low frequency of 0.1, and an amplitude of 0.05; PBS was used for cleaning before each test, and K3Fe(CN)6 was used as the electrolyte solution; The sample to be tested is passed into the third flow channel from the liquid outlet of the liquid outlet flow channel and incubated for 1 hour.

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