Paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes

Abstract

The present invention relates to a paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes, and belongs to the technical field of disease detection sensors. The sensor comprises a microfluidic pre-treatment module and an electrochemical detection module. The microfluidic module uses cellulose chromatography filter paper as a substrate, and uses a wax jet printer to construct a three-dimensional liquid flow channel; the electrochemical detection module uses a polyimide film as a substrate, and uses a laser engraving machine to make double-sided graphene electrodes. The two modules are pressed and assembled by a clamp. It has a five-layer structure, namely, a sample addition layer, a reagent layer, a detection layer, an electrode layer and an absorption layer. After the sample is added from the sample layer, it is combined with the reagent in the reagent layer, and the laser-induced graphene electrode contact reaction between the detection layer and the electrode layer is obtained by an electrochemical workstation. The quantitative detection result is finally absorbed in the absorption layer. The present invention combines a paper chip with laser-induced graphene to produce a new type of biosensor that is efficient, sensitive, green and environmentally friendly, simple in process and low in cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease detection sensors and relates to a paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes. The present invention combines the microfluidic paper chip technology prepared based on fluid mechanics principles and wax jet printing, laser-induced graphene technology and electrochemical sensor technology, and designs a general structure of a three-dimensional microfluidic biosensor that can simultaneously detect multiple disease markers using a conventional electrochemical workstation. Background Art

[0002] Paper-based microfluidic biosensors are used to detect disease markers (including proteins, nucleic acids, polysaccharides, etc.). Through the affinity recognition of biomolecules, the markers are combined with modified nanomaterials (such as nanoparticles, nanowires, superlattices, etc.) and modified graphene electrodes to generate electrical signal changes, which are then measured and analyzed using an electrochemical workstation. Existing biosensors have complex manufacturing processes, long cycles, high costs, and low detection efficiency, and cannot achieve efficient, rapid, and sensitive detection. For example, the gold electrode method using magnetron sputtering technology has a long cycle and complex operations; the microelectrode preparation using screen printing technology cannot arbitrarily design the shape, and the manufacturing process is complex. Current paper chip sensors are mostly single-layer structures, and colloidal gold methods are usually used, which have the disadvantages of low detection efficiency and poor accuracy. Summary of the invention

[0003] In order to solve the above problems, the present invention proposes a general structure of a new type of biosensor including a microfluidic pretreatment module and an electrochemical detection module. By spraying wax on the filter paper substrate to print the microfluidic channel, the flow rate and direction of the fluid are controlled, so that it can achieve parallel pretreatment of multiple markers in the sample; laser-induced graphene is engraved on both sides of the polyimide film to produce graphene electrodes with excellent electrical and chemical properties; the paper chip assembly and detection efficiency are improved, and the cost is effectively reduced. The present invention uses cellulose chromatography filter paper as the substrate, uses a wax jet printer to construct a microfluidic channel, and engraves laser-induced graphene electrodes on the polyimide film. After assembly, the electrochemical method is used to perform rapid parallel detection of the sample.

[0004] The present invention combines a paper-based microfluidic module with electrochemical detection, while having the advantages of low cost and easy shaping of paper chips and high precision and good sensitivity of electrochemical detection. At the same time, it innovatively designs a three-dimensional microfluidic processing module and uses laser-induced graphene technology to prepare electrodes, which improves detection efficiency and simplifies the production process.

[0005] The technical solution of the present invention is as follows:

[0006] A paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes comprises a microfluidic pretreatment module and an electrochemical detection module; the microfluidic pretreatment module comprises a three-layer structure, which comprises a sample loading layer 1, a reagent layer 2, a detection layer 3 and an absorption layer 5 from top to bottom; the electrochemical detection module comprises an electrode layer 4; the sample loading layer 1, the reagent layer 2 and the detection layer 3 are interconnected as a whole, and a three-dimensional structure is formed by folding along a dividing line, and the three layers are connected through overlapping hydrophilic areas; the electrode layer 4 and the absorption layer 5 are independent, and each layer is aligned and superimposed and assembled using a clamp.

[0007] The electrode layer 4 is double-sidedly engraved, with the front side being the working electrode 4-1 and the back side being the auxiliary electrode and the reference electrode 4-4. The auxiliary electrode and the reference electrode 4-4 are partial circular ring structures, wherein the auxiliary electrode is in the shape of a 270° circular ring and the reference electrode is in the shape of a 30° circular ring. The symmetry center of the working electrode 4-1 coincides with the center of the auxiliary electrode and the reference electrode 4-4. The working electrode 4-1 is entirely located in the detection zone 3-1 of the detection layer 3, and the auxiliary electrode and the reference electrode 4-4 are entirely located in the absorption zone 5-1 of the absorption layer 5. A central hole 4-2 and a plurality of surrounding holes 4-3 are provided on the electrode layer 4. The working electrode 4-1 is tangent to the central hole 4-2 and is evenly distributed. The surrounding holes 4-3 are tangent to the detection zone 3-1, and the surrounding holes 4-3 are centrally symmetrically arranged relative to the center of the central hole 4-2. The detection zone 3-1 is connected to the absorption zone 5-1 through the central hole 4-2 and the surrounding holes 4-3.

[0008] The reagent layer 2, wherein the reagent area 2-1 comprises four mutually unconnected circular hydrophilic areas, can be used for the simultaneous detection of multiple analytes.

[0009] The base material of the microfluidic pretreatment module is cellulose chromatography filter paper, and the hydrophobic barrier material is paraffin; the base material of the electrochemical detection module is polyimide, and the electrode material is laser-induced graphene; each electrode wire part is encapsulated by hot stamping of thermoplastic polyurethane film.

[0010] Four reagents that can specifically bind to different test proteins are added to the four circular areas of the reagent zone, and recognition units (peptides or antibodies) that can specifically bind to the test proteins are modified on the working electrodes in the working zone, so that multiple test objects can be detected simultaneously.

[0011] When in use, the sample is added from the sample layer and combined with the reagent in the reagent layer. It is captured by the molecular recognition unit on the surface of the laser-induced graphene electrode in the electrode layer in the detection layer. The quantitative detection result is obtained through the electrochemical workstation and finally absorbed in the absorption layer.

[0012] Beneficial effects of the present invention: The present invention uses cellulose chromatography filter paper as a substrate, prepares a three-dimensional microfluidic paper channel of a specific shape based on the principle of fluid mechanics and wax jet printing, carves laser-induced graphene electrodes on a polyimide film, and uses an electrochemical method for rapid parallel detection. It can realize the processing and parallel detection of multiple markers in a sample, simplify the production process, shorten the cycle, reduce the cost, and increase the detection efficiency, thereby realizing efficient, rapid, and sensitive detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the structure of each layer of the microfluidic pre-treatment module in the present invention;

[0014] Figure 2 It is a schematic diagram of the front and back structures of the electrochemical detection module of the present invention;

[0015] Figure 3 It is a schematic diagram of the assembly of the present invention;

[0016] Figure 4 It is the fitting curve of the protein detection by the biosensor in the embodiment.

[0017] In the figure: 1 sample adding layer; 2 reagent layer; 3 detection layer; 4 electrode layer; 5 absorption layer; 1-1 sample adding area; 2-1 reagent area; 3-1 detection area; 5-1 absorption area; 4-1 working electrode; 4-2 central hole; 4-3 surrounding holes; 4-4 auxiliary electrode and reference electrode. DETAILED DESCRIPTION

[0018] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0019] like Figure 1-Figure 3 As shown, a paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes includes a microfluidic pretreatment module and an electrochemical detection module. The microfluidic pretreatment module includes a sample addition layer 1, a reagent layer 2, a detection layer 3 and an absorption layer 5, each layer is respectively provided with a sample addition area 1-1, a reagent area 2-1, a detection area 3-1 and an absorption area 5-1, and the layers are arranged in order from top to bottom. The electrochemical detection module includes an electrode layer 4, the front of the electrode layer 4 is a working electrode 4-1, and the back is an auxiliary electrode and a reference electrode 4-2.

[0020] Furthermore, the substrate of the three-dimensional microfluidic channel is cellulose chromatography filter paper, the hydrophobic barrier material is paraffin, the electrode layer base material is polyimide, and the electrode material is laser-induced graphene.

[0021] Furthermore, the polyimide film of the electrode layer contains a central hole 4-2 and peripheral holes 4-3 to connect the detection area 3-1 with the water absorption area 5-1.

[0022] Furthermore, the pattern of the graphene working electrode 4-1 can be freely designed by a laser engraving machine, preferably a circular structure, each working electrode is tangent to the central hole 4-2 and is evenly distributed; the surrounding holes 4-3 are tangent to the hydrophilic area 3-1 of the detection layer, arranged symmetrically with the center of the center, and the center of symmetry is the center of the central hole; the auxiliary electrode and reference electrode 4-4 are partial circular ring structures.

[0023] Furthermore, the graphene electrode of the electrode layer is double-sided engraved, and after registration, the symmetry center of the front working electrode coincides with the center of the back auxiliary electrode. Each electrode wire part is encapsulated by hot stamping of thermoplastic polyurethane film.

[0024] Furthermore, the sample addition layer 1, reagent layer 2, and detection layer 3 are interconnected as a whole and folded along the dividing line to form a three-dimensional structure. The electrode layer 4 and the absorption layer 5 are independent of each other, and each layer is aligned and superimposed and assembled with a fixture.

[0025] In this embodiment, the specific parameters are as follows:

[0026] In the microfluidic pre-treatment module, each layer is a square with a side length of 16 mm.

[0027] The radius of the sample addition area 1-1 is 2mm, the center of the circle coincides with the center of the square, the four trapezoidal sample addition channels are distributed along the diagonal of the square, the short side is 1.6mm, the long side connected to the circle is 1.8mm, and the circular radius of the hydrophobic area is 6mm; the radius of the four circles in the reagent area 2-1 is 1.6mm, and the circular radius of the hydrophobic area is 6mm; the radius of the detection area 3-1 is 4mm, the center of the circle coincides with the center of the square, the four trapezoidal sample addition channels are distributed along the diagonal of the square, the long side is 4.6mm, the short side connected to the circle is 2.8mm, and the circular radius of the hydrophobic area is 7.5mm; the radius of the absorption area 5-1 is 4mm, and the center of the circle coincides with the center of the square.

[0028] like Figure 2As shown, a general structure of a paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes, the electrode layer 4 is 20 mm in length and width, and the detection electrode structure includes a working electrode 4-1, a central hole 4-2, surrounding holes 4-3, an auxiliary electrode and a reference electrode 4-4. The working electrode 4-1 has a radius of 1mm, is tangent to the central hole 4-2, and is arranged symmetrically with the center of the central hole. The width of the wire end is 3mm, the length is 6mm, the upper end is 2.5mm away from the upper edge of the electrode layer 4, and the central angle of the intersection line of the electrode quadrilateral part and the circular part is 60°; the radius of the central hole 4-2 is 1mm, the center of the circle is 8mm away from the upper edge of the electrode layer 4, and 10mm from the left edge; the radius of the surrounding holes 4-3 is 1mm, the center of the circle is on a circle with a radius of 3mm with the central hole as the center, and they are arranged symmetrically; the auxiliary electrode and the reference electrode 4-4 are circular ring structures with radii of 1.5mm and 3mm, the auxiliary electrode angle is 270°, the reference electrode angle is 30°, the wire end width is 3mm, the length is 7mm, the left edge of the electrode is 4.5mm away from the left edge of the electrode layer 4, and they are arranged symmetrically along the center line of the electrode layer.

[0029] Figure 3 It is an assembly diagram of the present invention. After the three-dimensional microfluidic partial structure of the present invention is drawn using Auto CAD software, the channel structure is printed on the cellulose chromatography filter paper using a wax jet printer and cut, so that the sample layer 1, the reagent layer 2, and the detection layer 3 are connected, and the absorption layer 5 is independent; then it is placed on a heating plate and heated at 80°C for 60s until the wax melts to form a hydrophobic barrier, and then the reagent that can identify the protein to be tested and generate an electrochemical signal is added dropwise to the reagent area 2-1, and placed in a 4°C environment to dry. The reagent used in this embodiment is a nanosilver particle with a surface modified antibody against the protein to be tested.

[0030] Laser-induced graphene electrodes were engraved on both sides of the polyimide film using a laser engraving machine. The maximum laser power of the semiconductor laser selected in this embodiment can reach 10W. The engraving power used in the embodiment is 16% for the front working electrode and 14% for the back. Copper tape was pasted on the electrode as a pin, and a thermoplastic polyurethane film was stamped with an electric soldering iron to partially encapsulate the graphene wire. Silver chloride paste was applied to the back reference electrode and dried in the shade at room temperature.

[0031] Dissolve 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) powder in dimethylformamide to prepare a 10mM PBASE solution. Take 10μl and drop it on the surface of the working electrode to cover it. Wait for the solution to dry and rinse with water to complete the modification. PBASE is used as a linker to connect the recognition unit for the protein to be tested to the electrode surface.

[0032] The recognition unit (peptide or antibody) that can specifically bind to the protein to be tested is dissolved in water to make a solution, which is then dripped onto the surface of the working electrode to cover it. After the solution dries, it is rinsed with clean water to complete the modification.

[0033] Dissolve bovine serum albumin (BSA) in phosphate buffered saline (PBS) to make a 3 mg / ml BSA solution. Take 10 μl of the BSA solution and drip it onto the surface of the working electrode to cover it. Wait for the solution to dry and rinse with water to complete the modification. BSA acts as a blocker for nonspecific protein binding sites on the electrode surface.

[0034] like Figure 3 As shown in the assembly diagram of the present invention, after each layer is processed, the sample loading layer 1, the binding layer 2, and the detection layer 3 are folded along the edge line to form a vertical multi-layer structure, and are aligned with the electrode layer 4 and the absorption layer 5, and then pressed and assembled using an acrylic clamp.

[0035] During measurement, different concentrations of the protein solution to be tested are dripped from the top sample well, combined with the modified nanosilver particles through the microfluidic pre-treatment module, and further flowed to the electrode layer to contact the working electrode. The modified working electrode can bind to the protein to be tested and the modified nanosilver particles.

[0036] The electrode is connected to the electrochemical workstation and detected using the differential voltammetric pulse method. The nanosilver particles on the surface of the working electrode will undergo an electrochemical reaction to generate a current signal, and the signal size is positively correlated with the concentration of the protein to be tested.

[0037] Figure 4 The figure shows the response current and fitting curve of the biosensor for different concentrations of the test protein. The test results show that the detection limit of the sensor is as low as 10fg / ml, and it has a linear response in the concentration range of 10fg / ml to 1ng / ml, with a fitting degree R 2 =0.99782.

Claims

1. A paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes, characterized in that: The paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes comprises a microfluidic pretreatment module and an electrochemical detection module; the microfluidic pretreatment module comprises a three-layer structure, which comprises a sample loading layer (1), a reagent layer (2), a detection layer (3) and an absorption layer (5) from top to bottom; the electrochemical detection module comprises an electrode layer (4); the sample loading layer (1), the reagent layer (2) and the detection layer (3) are interconnected as a whole, and a three-dimensional structure is formed by folding along a dividing line, and the three layers are connected through overlapping hydrophilic regions; the electrode layer (4) and the absorption layer (5) are independent of each other, and each layer is aligned and superimposed and assembled using a clamp; The electrode layer (4) is double-sidedly engraved, with the front side being the working electrode (4-1) and the back side being the auxiliary electrode and the reference electrode (4-4), wherein the auxiliary electrode and the reference electrode (4-4) are partial circular ring structures, wherein the auxiliary electrode is in the shape of a 270° circular ring and the reference electrode is in the shape of a 30° circular ring; the symmetry center of the working electrode (4-1) coincides with the center of the auxiliary electrode and the reference electrode (4-4), the working electrode (4-1) is entirely in the detection area (3-1) of the detection layer (3), and the auxiliary electrode and the reference electrode (4-4) are in the detection area (3-1) of the detection layer (3). The electrodes (4-4) are all located in the absorption area (5-1) of the absorption layer (5); a central hole 4-2 and a plurality of peripheral holes (4-3) are provided on the electrode layer (4); the working electrode (4-1) is tangent to the central hole (4-2) and is evenly distributed; the peripheral holes (4-3) are tangent to the detection area (3-1); the peripheral holes (4-3) are arranged in a central symmetric manner relative to the center of the central hole (4-2); the detection area (3-1) and the absorption area (5-1) are connected through the central hole 4-2 and the peripheral holes (4-3).

2. The paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrodes according to claim 1, characterized in that: The base material of the microfluidic pretreatment module is cellulose chromatography filter paper, and the hydrophobic barrier material is paraffin; the base material of the electrochemical detection module is polyimide, and the electrode material is laser-induced graphene; each electrode wire part is encapsulated by hot stamping of thermoplastic polyurethane film.

3. The paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrode according to claim 1 or 2, characterized in that: The reagent layer (2), wherein the reagent area (2-1) comprises four mutually unconnected circular hydrophilic areas, can be used for the simultaneous detection of multiple analytes.

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