Fully-integrated wireless microfluidic immunosensing system for portable detection of staphylococcus aureus

Abstract

The invention discloses a fully-integrated wireless microfluidic immunosensing system which is used for portable detection of staphylococcus aureus. The fully-integrated wireless microfluidic immunosensing system comprises a bendable biological test strip and an electronic module, wherein the biological test strip comprises a microfluidic module and an immunosensing module; the immune sensing module comprises a substrate and an immune sensing layer arranged on the substrate, and a working electrode, a reference electrode and a counter electrode are arranged on the immune sensing layer; the micro-fluidic module comprises a sealing layer, a spacing layer for storing a liquid sample and a pump-free layer for transporting liquid; a fluid inlet and a fluid outlet are formed in the sealing layer; the micro-fluidic module is compounded on the immune sensing module, and an insulating layer is arranged between the pump-free layer and the immune sensing layer. The method shows huge potential and attraction to food and environment safety monitoring.

Description

TECHNICAL FIELD

[0001] The application relates to a full-integrated wireless microfluidic immunosensor system for portable detection of Staphylococcus aureus. BACKGROUND

[0002] Designing and manufacturing a device that is full-integrated and can independently operate without additional accessories is considered as the highest goal of modern device development. Compared with previously reported devices that mainly focus on one module (for example, only a sensing module or only a microfluidic module), the main goal of constructing a full-integrated device is to manufacture various single modules and seamlessly assemble the single modules, so that the full-integrated device can exhibit functions that cannot be achieved by the single modules.

[0003] An electrochemical sensor has the characteristics of high sensitivity, fast detection speed, high cost performance, convenient operation and easy miniaturization, and has been widely used in research fields such as environmental detection, gene sequencing and wearable monitoring. The introduction of full-integrated electrochemical sensing technology into portable Staphylococcus aureus detection can construct a full-integrated portable electrochemical immunosensor system. Compared with traditional electrochemical sensors that mainly focus on testing in the laboratory, the full-integrated portable electrochemical immunosensor system can facilitate on-site analysis in the field environment. S. aureus , Staphylococcus aureus ) detection, a full-integrated portable electrochemical S. aureus immunosensor system can be constructed. Compared with traditional electrochemical S. aureus sensors that mainly focus on testing in the laboratory, the full-integrated portable electrochemical S. aureus immunosensor system can facilitate on-site analysis in the field environment. S. aureus SUMMARY

[0004] The application aims to provide a full-integrated wireless microfluidic immunosensor system for portable detection of Staphylococcus aureus, which uses a PI film with a patterned superhydrophobic and superhydrophilic coating as a pump-free layer of a microfluidic module, can effectively promote the transportation of a liquid sample. At the same time, laser-engraved graphene (LEG) with a porous three-dimensional (3D, three-dimensional) structure is used as an electrode material in a sensing module, which can realize relatively high-performance detection.

[0005] The application provides a bendable biological test strip, which comprises a microfluidic module and an immunosensor module. The immunosensor module comprises a substrate and an immunosensor layer arranged on the substrate, and the immunosensor layer is provided with a working electrode, a reference electrode and a counter electrode. The working electrode is obtained by sequentially modifying EDC / NHS, Staphylococcus aureus antibodies and bovine serum albumin on a laser-printed graphene electrode. The counter electrode is a laser-printed graphene electrode.​ The microfluidic module comprises a sealing layer, a spacer layer for storing a liquid sample and a pump-free layer based on a super-hydrophobic coating and a super-hydrophilic coating for liquid transportation; The sealing layer is provided with a fluid inlet and a fluid outlet, and the fluid inlet and the fluid outlet correspond to the hydrophilic layer; The microfluidic module is combined on the upper surface of the immunosensor module, and an insulating layer is arranged between the pump-free layer and the immunosensor layer.

[0006] In the flexible biological test paper strip, the laser-printed graphene electrode is obtained by laser etching a PI film, and the conditions can be: Under the conditions of room temperature and air environment, using a CO2 laser system, laser power is 9-15 W, laser speed is 500-700 mm / s -1 Under the conditions of room temperature and air environment, using a CO2 laser system, laser power is 9-15 W, laser speed is 500-700 mm / s

[0007] The pump-free layer is formed on the insulating layer; The sealing layer and the insulating layer are made of a PI film; The spacer layer is made of a double-sided adhesive PET film; The substrate is made of a PI film; The reference electrode is an Ag / AgCl electrode.

[0008] In the flexible biological test paper strip, the EDC / NHS solution is added dropwise to the laser-printed graphene electrode; after drying, the solution of Staphylococcus aureus antibody is added dropwise; after incubation, the solution of bovine serum albumin is continuously added dropwise; The EDC / NHS solution is prepared from a MES buffer; The solution of Staphylococcus aureus antibody is prepared from PBS; The incubation temperature is 0-5°C, and the time is 10-20h; The solution of bovine serum albumin is prepared from PBS.

[0009] In the flexible biological test paper strip, the super-hydrophilic coating is formed by a super-hydrophilic nano coating agent; The super-hydrophilic nano coating can be a super-hydrophilic nano material (model: ATF202) produced by Piaqi Nanotechnology Co., Ltd.; The super-hydrophobic coating is formed by a super-hydrophobic coating solution; The superhydrophobic coating solution is obtained based on the surface modification co-reaction between hydrophobic silica particles and fluoroalkylsilane: hydrophobic silica powder and 1H,1H,2H,2H-perfluorooctyltriethoxysilane are added to an ethanol solution, and then the reaction is carried out under stirring. Finally, the solution is ultrasonically treated to obtain a superhydrophobic silica suspension.

[0010] The pump-free layer can be prepared according to the following method: First, a superhydrophilic nano-coating agent is drop-coated onto a PI layer with a patterned mask layer and a LEG sensor, and then dried at room temperature. After removing the mask layer, a patterned superhydrophilic layer is obtained. A superhydrophobic coating solution is then cast onto the PI layer with the patterned mask layer, the superhydrophilic layer, and the LEG sensor, and then dried under ambient conditions. After removing the mask layer, the pump-free layer is obtained.

[0011] Based on the flexible biological test strip, the present invention also provides a fully integrated wireless microfluidic immunosensing system, including the flexible biological test strip and an electronic module. The electronic module is a reusable printed circuit board that integrates various electronic components and is capable of providing signal transduction and processing as well as wireless transmission and control.

[0012] The present invention also provides a method for detecting Staphylococcus aureus, comprising the following steps: The solution to be tested is added to the flexible biological test strip through the fluid inlet. After incubation, a PBS buffer solution of hydroquinone and hydrogen peroxide is added to the flexible biological test strip. After cleaning, apply HRP-anti- S. aureus Add to the flexible biological test strip and place at 37 °C for 30 minutes.

[0013] use i-t Technical testing S. aureus Evaluation based on the cathode current provided by the fully integrated wireless microfluidic immune sensing system S. aureus The concentration.

[0014] The present invention has the following beneficial technical effects: The present application is a fully integrated wireless microfluidic immunosensing system, which is composed of a microfluidic module, an immunosensing module and an electronic control module. It can be used for pump-free, wireless and portable detection of S. aureus in real samples. As an integrated device, the present fully integrated wireless microfluidic immunosensing system exhibits many outstanding features: the microfluidic module uses patterned superhydrophobic and superhydrophilic coatings as pump-free layers, which can effectively transport pump-free liquid samples; the immunosensing module uses LEG with highly porous 3D structure as electrode material, which can detect S. aureus in real samples; the customized electronic module specially matches the microfluidic module and the immunosensing module, which facilitates the whole system to conduct portable and wireless detection of S. aureus. The present fully integrated wireless microfluidic immunosensing system shows great potential and attraction for food and environmental safety monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of the construction of the present application of the bendable biological test strip.

[0016] Figure 2 is a schematic diagram of the preparation process of the sealing layer in the microfluidic module of the present application.

[0017] Figure 3 is a schematic diagram of the preparation process of the spacer layer in the microfluidic module of the present application.

[0018] Figure 4 is a schematic diagram of the preparation process of the PI layer.

[0019] Figure 5 is a schematic diagram of the preparation process of the insulating layer.

[0020] Figure 6 is a digital photo of the present application of the fully integrated wireless microfluidic immunosensing system for detecting S. aureus .

[0021] Figure 7 is a schematic diagram of different layers and corresponding functions in the present application of the bendable biological test strip.

[0022] Figure 8 is a schematic diagram of the present application of the bendable biological test strip.

[0023] Figure 9 is a digital photo of the present application of the microfluidic module for droplet transportation; scale: 1.0 cm, droplet: 20 mM methyl violet aqueous solution.

[0024] Figure 10 is a SEM image of the top of LEG in the present application; scale: 8 μm.

[0025] Figure 11is the N2 adsorption-desorption isotherm graph (A) and the pore size distribution graph (B) of LEG in the present application.

[0026] Figure 12 is the XPS spectrum of LEG in the present application.

[0027] Figure 13 is the result of detection by the method of the present application for the full integration wireless microfluidic immunosensor system. i-t S. aureus

[0028] Figure 14 is the calibration curve of detection by the method of the present application for the sensing module. i-t S. aureus DETAILED DESCRIPTION

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0030] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] Example 1, Preparation of a bendable biological test strip The preparation flow chart is shown in Figure 1 .

[0032] 1. Preparation of microfluidic module 1) Preparation of sealing layer and spacer layer PI film (2.10 cm x 1.30 cm x 50.00 μm, and double-sided adhesive PET film (2.10 cm x 1.30 cm x 175.00 μm, were precisely cut by a digital cutter to obtain the sealing layer and the spacer layer. Figure 2 Figure 3

[0033] 2) Preparation of super-hydrophobic coating solution The hydrophobic coating solution is based on the surface modification co-reaction between hydrophobic silica particles and fluoroalkylsilane. First, 0.3 g of hydrophobic silica powder and 0.4 g of 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane were sequentially added to a 25 mL ethanol solution. Subsequently, the mixture was magnetically stirred at a speed of 650 rpm for about 12 hours. Next, it was ultrasonically treated at ambient temperature for 30 minutes. Finally, a translucent super-hydrophobic silica suspension was obtained.

[0034] 2) Preparation of pump-free layer ​​​​​​First, 20 μL of superhydrophilic nanocoating solution was dropped onto the PI layer with a patterned mask layer and LIG sensor and left to dry at room temperature. After removing the mask layer, a patterned superhydrophilic layer was obtained Figure 1 ). 50 μL of superhydrophobic coating solution was cast onto the PI layer with a patterned mask layer, superhydrophilic layer, and laser-engraved graphene (LEG) sensor and left to dry under ambient conditions. After removing the mask layer, a transport layer was obtained Figure 1 ).

[0035] 3) Assembly of microfluidic module As shown in Figure 1 , the microfluidic module was composed of three parts, including a sealing layer, a spacer layer, and a pump-free layer. The three parts were assembled together to construct the microfluidic module.

[0036] 2) Construction of sensing module 1) Preparation of polyimide (PI) layer After cutting the PI film (2.40 cm x 1.30 cm x 150.00 μm) using a digital cutter, a PI layer was obtained Figure 4 ).

[0037] 2) Preparation of LEG sensor The PI film was ultrasonically cleaned in ethanol and ultrapure water, respectively, to remove surface impurities, and then dried. Under room temperature and air ambient conditions, a CO2 laser system was used to laser etch the PI film at a laser power of 12 W and a laser speed of 600 mm s -1 . Laser irradiation on the surface of the PI film (orange) converted it into LEG (black). Next, using a commercial manual screen printing machine and a custom-made template, silver ink was printed on the PI layer by screen printing technology. After curing in an oven at 93 °C for 15 minutes, a PI layer with a silver layer pattern and LEG was obtained. After pasting an insulating layer Figure 5 , a PI layer with an insulating layer, silver layer, and LEG was obtained. Then, 0.5 μL of 0.1 M FeCl3 solution was dropped onto part of the silver layer for 6 h, and the remaining FeCl3 was washed with water. Finally, it was dried under infrared light to obtain a LEG sensor Figure 1 ).

[0038] 3) Preparation of sensing module First, 4 μL of EDC / NHS (3:1) in pH 6.0 MES was dropped on the working electrode and left for 1 h. The electrode was washed twice with 0.01 M pH 7.4 PBS to remove unreacted EDC / NHS. After drying at room temperature, 1.2 μL of anti- S. aureus (10 μg mL -1 in 0.01 M pH 7.4 PBS) was dropped on the electrode, and the resulting electrode was incubated at 4 °C overnight to immobilize the anti- S. aureus Then, the resulting electrode was washed twice with 0.01 M pH 7.4 PBST and 0.01 M pH 7.4 PBS, respectively, to remove unimmobilized anti- S. aureus After drying at ambient conditions, the anti- S. aureus / LEG was obtained. 1.0 μL of BSA solution (5 mg mL -1 in 0.01 M pH 7.4 PBS) was dropped on the anti- S. aureus / LEG to eliminate non-specific binding effects and block the remaining active groups, and it was left to dry at ambient conditions for 2 h. After washing twice with 0.01 M pH 7.4 PBST and 0.01 M pH 7.4 PBS, respectively, the immunosensor in the immunosensing module (i.e., BSA / anti- S. aureus / LEG) was obtained. Figure 1

[0039] 3. Preparation of a flexible bioassay strip The microfluidic module and the sensing module were assembled together to obtain a flexible bioassay strip. Figure 1

[0040] Example 2. Immunoassay for bacterial detection For bacterial detection, a solution containing bacteria was added to the flexible bioassay strip. The resulting electrode was then incubated at 37 °C for 30 min, followed by washing twice with 0.01 M pH 7.4 PBST and 0.01 M pH 7.4 PBS, respectively, to eliminate unimmobilized S. aureus After washing, 6.5 μL of HRP-anti- S. aureus (2.0 μg mL -1 ​​The solution (0.01 M pH 7.4 PBS) was added to a flexible biological test strip and incubated at 37 °C for 30 minutes. Then, the strip was washed twice with 0.01 M pH 7.4 PBS and four times with 0.01 M pH 7.4 PBS. S. aureus The detection was performed in 0.1 M pH 7.0 PBS containing 2.0 mM hydroquinone (HQ) and 1.5 mM H2O2.

[0041] Example 3: Design and Characterization of a Fully Integrated Wireless Microfluidic Immunosensing System The fully integrated wireless microfluidic immunosensing system of this invention consists of a disposable, flexible biological test strip (composed of a microfluidic module and a sensing module) and an electronic module. Figure 6 ), enabling the analysis of actual samples S. aureus Perform real-time analysis.

[0042] The microfluidic module comprises three layers: a PI membrane-based sealing layer for encapsulating the microfluidic module, a spacer layer based on a polyethylene terephthalate (PET) membrane for liquid sample storage, and a pump-free layer based on a PI membrane coated with superhydrophobic and superhydrophilic coatings for liquid transport. Figure 7 ).

[0043] The immune sensing module consists of three layers: for S. aureus The detection of the immunosensing layer based on laser-engraved graphene (LEG), the insulating layer based on a PI film for insulation, and the PI layer for fixing the microfluidic module and the immunosensing module. Figure 7 The immunosensor within the sensing module consists of a LEG-based working electrode, a LEG counter electrode, and an Ag / AgCl reference electrode. Figure 8 ).

[0044] An electronic module is a reusable printed circuit board that integrates various electronic components, providing signal transduction and processing as well as wireless transmission and control. Figure 6 ).

[0045] use i-t Technical testing S. aureus Evaluation based on the cathode current provided by the fully integrated wireless microfluidic immune sensing system S. aureus concentration ( Figure 6 ).

[0046] Example 4: Design and Characterization of Microfluidic Module When the droplet on the tip of the nozzle, which has already dispensed a droplet, comes into contact with the hydrophilic region at the inlet of the microfluidic chip, the liquid will spontaneously flow along the transport layer towards the fluid outlet, with a transport time / fill time of ~0.75 s. Figure 9 This indicates that the microfluidic chip achieves efficient, spontaneous delivery of liquids without a peristaltic pump.

[0047] Example 5: Design and Characterization of the Sensing Module In order to improve S. aureus To improve detection performance, LEG was used as the electrode material for the sensing module. LEG has a highly porous 3D structure ( Figure 10 High specific surface area (331.15 m²) 2 g -1 )(like Figure 11 (As shown in Figure A) and a unique microporous-mesoporous structure, with pore sizes centered at 0.76, 1.50, 4.84, and 48.53 nm. Figure 11 (Figure B in the middle) This may facilitate the high loading of antibodies on the sensing module. This nanostructure can also provide an uninterrupted species transport pathway, thereby improving electron transfer and enhancing overall performance. XPS spectra show that the C, O, and N contents of LEG are 94.8 at.%, 3.9 at.%, and 1.3 at.%, respectively. Figure 12 This indicates that LEG has achieved a high degree of carbonization and is a carbon-based material. Therefore, LEG's unique nanostructure makes it an ideal choice for electrode materials in constructing sensing modules.

[0048] With the help of i-t technology, S. aureus As the concentration increases, the cathode current response also increases. Figure 13 ), current and S. aureus There is a broad linear relationship between concentrations. Figure 14 ).

Claims

1. A flexible biological test strip, comprising a microfluidic module and an immunosensing module; The immune sensing module includes a substrate and an immune sensing layer disposed on the substrate, wherein the immune sensing layer is provided with a working electrode, a reference electrode and a counter electrode; The working electrode is obtained by sequentially modifying EDC / NHS, Staphylococcus aureus antibody, and bovine serum albumin onto a laser-printed graphene electrode; The counter electrode is a laser-printed graphene electrode; The microfluidic module includes a sealing layer, a spacer layer for liquid sample storage, and a pump-free layer based on superhydrophobic and superhydrophilic coatings for liquid transport. The sealing layer is provided with a fluid inlet and a fluid outlet, both of which correspond to the hydrophilic layer; The microfluidic module is composited on top of the immune sensing module, and an insulating layer is provided between the pump-free layer and the immune sensing layer.

2. The flexible biological test strip according to claim 1, characterized in that: The laser-printed graphene electrode is obtained by laser etching of a PI film; The pump-free layer is formed on the insulating layer; The sealing layer and the insulating layer are made of PI film; The spacer layer is made of double-sided adhesive PET film; The substrate is made of a PI film; The reference electrode is an Ag / AgCl electrode.

3. The flexible biological test strip according to claim 1 or 2, characterized in that: The EDC / NHS solution was dropped onto the laser-printed graphene electrode; after drying, the Staphylococcus aureus antibody solution was dropped on; after incubation, the bovine serum albumin solution was dropped on.

4. The flexible biological test strip according to claim 3, characterized in that: The EDC / NHS solution was prepared using MES buffer solution; The Staphylococcus aureus antibody solution was prepared using PBS; The incubation temperature is 0~5°C, and the time is 10~20h; The bovine serum albumin solution was prepared using PBS.

5. The flexible biological test strip according to any one of claims 1-4, characterized in that: The superhydrophilic coating is formed by a superhydrophilic nano-coating agent; The superhydrophobic coating is formed from a superhydrophobic coating solution.

6. The flexible biological test strip according to claim 5, characterized in that: The superhydrophobic coating solution is obtained based on the surface modification co-reaction between hydrophobic silica particles and fluoroalkylsilane: hydrophobic silica powder and 1H,1H,2H,2H-perfluorooctyltriethoxysilane are added to an ethanol solution, and then the reaction is carried out under stirring. Finally, the solution is ultrasonically treated to obtain a superhydrophobic silica suspension.

7. A fully integrated wireless microfluidic immunosensing system, comprising the flexible biological test strip and electronic module as described in any one of claims 1-6; The electronic module provides signal transduction and processing, as well as wireless transmission and control.

8. The application of the flexible biological test strip according to any one of claims 1-6 and the fully integrated wireless microfluidic immunosensing system according to claim 8 in the detection of Staphylococcus aureus.

9. A method for detecting Staphylococcus aureus, comprising the following steps: The solution to be tested is added through a fluid inlet to the flexible biological test strip of any one of claims 1-6 or the flexible biological test strip of the fully integrated wireless microfluidic immunosensing system of claim 8. After incubation, a PBS buffer solution containing hydroquinone and hydrogen peroxide is added to the flexible biological test strip. After cleaning, apply HRP-anti- S. aureus Place it into the flexible biological test strip.

10. The method according to claim 9, characterized in that: use it The method for detecting the concentration of Staphylococcus aureus.

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