CN115290712A - Paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrode - Google Patents

Paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrode Download PDF

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CN115290712A
CN115290712A CN202210793352.1A CN202210793352A CN115290712A CN 115290712 A CN115290712 A CN 115290712A CN 202210793352 A CN202210793352 A CN 202210793352A CN 115290712 A CN115290712 A CN 115290712A
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layer
electrode
detection
laser
microfluidic
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张航与
吴斌
刘波
吕莹
李娜
万雪
张郑瑶
侯娜
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China Inspection And Certification Group Liaoning Co ltd
Dalian University of Technology
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China Inspection And Certification Group Liaoning Co ltd
Dalian University of Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure

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Abstract

The invention relates to a paper-based three-dimensional microfluidic biosensor based on a laser-induced graphene electrode, and belongs to the technical field of disease detection sensors. The sensor comprises a microfluidic pretreatment module and an electrochemical detection module. The microfluidic module takes cellulose chromatographic filter paper as a base material, and a three-dimensional fluid flow channel is constructed by using a wax-jet printer; the electrochemical detection module takes a polyimide film as a base material, and a laser engraving machine is utilized to manufacture double-sided graphene electrodes. The two modules are assembled by pressing through a clamp. Has a five-layer structure, namely a sample adding layer, a reagent layer, a detection layer, an electrode layer and an absorption layer. The sample is added from the sample layer and then combined with the reagent of the reagent layer, the laser-induced graphene electrode contact reaction is carried out on the detection layer and the electrode layer, and a quantitative detection result is obtained through an electrochemical workstation and is finally absorbed in the absorption layer. According to the invention, the paper chip and the laser-induced graphene are combined to manufacture the novel biosensor which is efficient, sensitive, environment-friendly, simple in process and low in cost.

Description

Paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrode
Technical Field
The invention belongs to the technical field of disease detection sensors, and relates to a paper-based three-dimensional microfluidic biosensor based on a laser-induced graphene electrode, which is a universal structure of the three-dimensional microfluidic biosensor for simultaneously detecting multiple disease markers by using a conventional electrochemical workstation by combining a technology for preparing a microfluidic paper chip based on a hydrodynamics principle and wax-spraying printing, a technology for preparing a laser-induced graphene chip and an electrochemical sensor technology.
Background
The paper-based microfluidic biosensor is used for detecting disease markers (including protein, nucleic acid, polysaccharide and the like), the markers are combined with modified nano materials (such as nano particles, nano wires, superlattice and the like) and modified graphene electrodes through affinity identification of biomolecules to generate electric signal change, and measurement and analysis are completed through an electrochemical workstation. The existing biosensor has the disadvantages of complex manufacturing process, long period, high cost, low detection efficiency and incapability of realizing efficient, rapid and sensitive detection. For example, the magnetron sputtering technology is used for preparing the gold electrode, the period is long, and the operation is complex; the microelectrode prepared by the screen printing technology cannot be designed at will, and the manufacturing process is complex. Most of the existing paper chip sensors are of single-layer structures, and a colloidal gold method is generally used, so that the defects of low detection efficiency, poor precision and the like are overcome.
Disclosure of Invention
In order to solve the problems, the invention provides a general structure of a novel biosensor comprising a microfluidic pretreatment module and an electrochemical detection module. The method comprises the following steps of printing a microfluidic channel by spraying wax on a filter paper substrate, and controlling the flow speed and the flow direction of fluid to realize parallel pretreatment of multiple markers in a sample; carving laser-induced graphene on two sides of a polyimide film to manufacture a graphene electrode with excellent electrical and chemical properties; the paper chip assembling and detecting efficiency is improved, and the cost is effectively reduced. The method takes cellulose chromatographic filter paper as a base material, utilizes a wax-spraying printer to construct a microfluidic channel, engraves a laser-induced graphene electrode on a polyimide film, and utilizes an electrochemical method to perform rapid parallel detection on a sample after assembly.
The paper-based microfluidic control module is combined with electrochemical detection, the paper-based microfluidic control module has the advantages of low cost, easiness in shaping, high electrochemical detection precision, good sensitivity and the like, meanwhile, the three-dimensional microfluidic control processing module is innovatively designed, the laser-induced graphene technology is used for preparing the electrode, the detection efficiency is improved, and the manufacturing process is simplified.
The technical scheme of the invention is as follows:
the paper-based three-dimensional microfluidic biosensor based on the laser-induced graphene electrode comprises a microfluidic pretreatment module and an electrochemical detection module; the microfluidic pretreatment module comprises a three-layer structure, namely a sample adding layer 1, a reagent layer 2, a detection layer 3 and an absorption layer 5 from top to bottom in sequence; the electrochemical detection module comprises an electrode layer 4; the sample adding layer 1, the reagent layer 2 and the detection layer 3 are connected into a whole, a three-dimensional structure is formed by folding along a boundary, and the three layers are communicated through overlapped hydrophilic regions; the electrode layer 4 and the absorption layer 5 are independent respectively, and the layers are assembled by a clamp after registration and superposition.
The electrode layer 4 is carved on two sides, the front side of the electrode layer is a working electrode 4-1, the back side of the electrode layer is an auxiliary electrode and a reference electrode 4-4, the auxiliary electrode and the reference electrode 4-4 are of partial circular ring structures, the auxiliary electrode is 270-degree circular ring shape, and the reference electrode is 30-degree circular ring shape; the symmetrical center of the working electrode 4-1 is coincided with the circle centers of the auxiliary electrode and the reference electrode 4-4, the working electrode 4-1 is completely positioned in the detection area 3-1 of the detection layer 3, and the auxiliary electrode and the reference electrode 4-4 are completely positioned in the absorption area 5-1 of the absorption layer 5; the electrode layer 4 is provided with a central hole 4-2 and a plurality of peripheral holes 4-3, the working electrode 4-1 is tangent to the central hole 4-2 and is uniformly distributed, the peripheral holes 4-3 are tangent to the detection area 3-1, and the peripheral holes 4-3 are arranged in central symmetry relative to the center of circle of the central hole 4-2; the detection area 3-1 is communicated with the absorption area 5-1 through the central hole 4-2 and the peripheral holes 4-3.
The reagent zone 2-1 of the reagent layer 2 contains four round hydrophilic areas which are not communicated with each other, and can be used for synchronous detection of various objects to be detected.
The base material of the vertical microfluidic pretreatment module is cellulose chromatographic 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 lead part is hot stamped and packaged by a thermoplastic polyurethane film.
Four reagents capable of specifically binding different proteins to be detected are respectively added into four circular areas of the reagent area, and a recognition unit (polypeptide or antibody) capable of specifically binding the proteins to be detected is modified on the working electrode of the working area, so that various substances to be detected can be detected simultaneously.
When the detection device is used, a sample is added from a sample layer and then combined with a reagent of a reagent layer, the sample is captured by a molecule recognition unit on the surface of a laser-induced graphene electrode of an electrode layer on a detection layer, a quantitative detection result is obtained through an electrochemical workstation, and the sample is finally absorbed in an absorption layer.
The invention has the beneficial effects that: the method takes cellulose chromatographic filter paper as a base material, prepares a three-dimensional microfluidic paper channel with a specific shape based on the hydrodynamics principle and wax-spraying printing, engraves a laser-induced graphene electrode on a polyimide film, and utilizes an electrochemical method to carry out rapid parallel detection. The method can realize the treatment and parallel detection of various markers in the sample, simplifies the manufacturing process, has short period, low cost and high detection efficiency, and realizes efficient, rapid and sensitive detection.
Drawings
FIG. 1 is a schematic diagram of the structure of each layer of the microfluidic pretreatment module according to the present invention;
FIG. 2 is a schematic diagram showing the front and back side structures of a part of an electrochemical detection module according to the present invention;
FIG. 3 is an assembly schematic of the present invention;
FIG. 4 is a fitting curve of protein detection to be detected of the biosensor in the example.
In the figure: 1 a sample adding layer; 2, a reagent layer; 3, detecting the layer; 4 electrode layers; 5 an absorption layer; 1-1 sample addition zone; 2-1 reagent zone; 3-1 detection area; 5-1 absorption zone; 4-1 working electrode; 4-2 central holes; 4-3 holes around; 4-4 auxiliary electrode and reference electrode.
Detailed Description
The following further describes the specific embodiments of the present invention with reference to the drawings and technical solutions.
As shown in fig. 1-3, a paper-based three-dimensional microfluidic biosensor based on a laser-induced graphene electrode comprises a microfluidic pretreatment module and an electrochemical detection module. The microfluidic pretreatment module comprises a sample adding layer 1, a reagent layer 2, a detection layer 3 and an absorption layer 5, wherein each layer is correspondingly provided with a sample adding area 1-1, a reagent area 2-1, a detection area 3-1 and an absorption area 5-1 respectively, the layers are arranged from top to bottom in sequence, the electrochemical detection module comprises an electrode layer 4, the front side of the electrode layer 4 is a working electrode 4-1, and the back side of the electrode layer 4 is an auxiliary electrode and a reference electrode 4-2.
Further, the substrate of the three-dimensional microfluidic channel is cellulose chromatographic filter paper, the hydrophobic barrier material is paraffin, the electrode layer substrate material is polyimide, and the electrode material is laser-induced graphene.
Furthermore, the electrode layer polyimide film contains a central hole 4-2 and peripheral holes 4-3 which communicate the detection area 3-1 with the water absorption area 5-1.
Furthermore, the patterns of the graphene working electrodes 4-1 can be freely designed through a laser engraving machine, preferably are in a circular structure, and all the working electrodes are tangent to the central hole 4-2 and are uniformly distributed; the peripheral holes 4-3 are tangent to the hydrophilic region 3-1 of the detection layer and are arranged in a central symmetry mode, and the center of symmetry is the center of the center hole; the auxiliary electrode and the reference electrode 4-4 are in a partial circular ring structure.
Further, the graphene electrode of the electrode layer is engraved on two sides, and after registration, the center of symmetry of the front working electrode coincides with the center of the back auxiliary electrode. Each electrode lead part is hot stamped and packaged by a thermoplastic polyurethane film.
Furthermore, the sample adding layer 1, the reagent layer 2 and the detection layer 3 are connected into a whole, a three-dimensional structure is formed by folding along a boundary, the electrode layer 4 and the absorption layer 5 are respectively independent, and the layers are assembled by a clamp after being registered and superposed.
In this embodiment, the specific parameters are as follows:
and each layer of the microfluidic pretreatment module is a square with the side length of 16 mm.
The radius of the sample adding area 1-1 is 2mm, the center of the circle coincides with the center of the square, four trapezoidal sample adding channels are distributed along the diagonal line of the square, the length of the short side is 1.6mm, the length of the long side connected with the circle is 1.8mm, and the radius of the circle in the hydrophobic area is 6mm; the radius of four circles in the reagent zone 2-1 is 1.6mm, and the radius of a circle in the hydrophobic zone is 6mm; the radius of the detection area 3-1 is 4mm, the center of the circle coincides with the center of the square, four trapezoidal sample adding channels are distributed along the diagonal line of the square, the length of the long side is 4.6mm, the length of the short side connected with the circle is 2.8mm, and the radius of the circle in the hydrophobic area is 7.5mm; the radius of the absorption area 5-1 is 4mm, and the circle center is coincided with the center of the square.
As shown in figure 2, the paper-based three-dimensional microfluidic biosensor universal structure based on the laser-induced graphene electrode is characterized in that the length and the width of an electrode layer 4 are both 20mm, and the detection electrode part structure comprises a working electrode 4-1, a central hole 4-2, peripheral holes 4-3, an auxiliary electrode and a reference electrode 4-4. The radius of the working electrode 4-1 is 1mm, the working electrode is tangent to the central hole 4-2 and is arranged in a central symmetry mode by taking the center of the central hole as a center, the tail end of the lead is 3mm in width and 6mm in length, the upper end of the lead is 2.5mm away from the upper edge of the electrode layer 4, and the central angle of the intersecting secant of the quadrilateral part and the circular part of the electrode is 60 degrees; the radius of the central hole is 4-2 mm, the center of the central hole is 8mm away from the upper edge of the electrode layer 4, and the center of the central hole is 10mm away from the left edge; the radius of the holes at the periphery is 1mm, the circle center is on a circle with the radius of 3mm and the hole at the center is arranged in a central symmetry way; the auxiliary electrode and the reference electrode 4-4 are circular ring partial structures with the radius of 1.5mm and 3mm, the included angle of the auxiliary electrode is 270 degrees, the included angle of the reference electrode is 30 degrees, the width of the tail end of the lead is 3mm, the length of the lead is 7mm, and the distance between the left edge of the electrode and the left edge of the electrode layer 4 is 4.5mm and the auxiliary electrode and the reference electrode are symmetrically arranged along the central line of the electrode layer.
FIG. 3 is an assembly diagram of the present invention, after the Auto CAD software is used to draw the three-dimensional microfluidic part structure of the present invention, a wax-jet printer is used to print out the channel structure on the cellulose chromatographic filter paper and cut it, so that the sample-adding layer 1, the reagent layer 2 and the detection layer 3 are connected, and the absorption layer 5 is independent; and then placing the solution on a heating plate, heating the solution for 60s at 80 ℃ until the wax is melted to form a hydrophobic barrier, then dropwise adding a reagent which can identify the protein to be detected and can generate an electrochemical signal into the reagent area 2-1, and placing the reagent area in an environment at 4 ℃ for drying. The reagent used in this example is a silver nanoparticle with a surface modified with an antibody against the protein to be detected.
The laser engraving machine is used for engraving the laser-induced graphene electrodes on the two sides of the polyimide film, the maximum laser power of the selected semiconductor laser in the embodiment can reach 10W, the engraving power used in the embodiment is 16% of the front working electrode, and 14% of the back working electrode. And adhering a copper tape on the electrode to serve as a pin, partially packaging the graphene conductor by using an electric soldering iron hot stamping thermoplastic polyurethane film, smearing silver chloride slurry on the back reference electrode, and drying in the shade at normal temperature.
1-pyrenebutanoic acid N-hydroxysuccinimide ester (PBASE) powder was dissolved in dimethylformamide to prepare a 10mM PBASE solution. And (3) dripping 10 mu l of the solution on the surface of the working electrode to cover the working electrode, and washing the solution with clear water after the solution is dried to finish the modification. PBASE acts as a linker for the attachment of the recognition unit for the protein to be detected to the electrode surface.
And (3) dissolving the recognition unit (polypeptide or antibody) capable of being specifically combined with the protein to be detected in water to prepare a solution, dropwise adding the solution on the surface of the working electrode to cover the working electrode, and after the solution is dried, washing with clear water to finish modification.
Bovine Serum Albumin (BSA) was dissolved in Phosphate Buffered Saline (PBS) to prepare a BSA solution of 3 mg/ml. And (3) dropwise adding 10 mu l of BSA solution on the surface of the working electrode to cover the surface of the working electrode, and after the solution is dried, washing with clear water to finish modification. BSA served as a blocker of nonspecific protein binding sites on the electrode surface.
As shown in fig. 3, the assembly diagram of the present invention is that after each layer is processed, the sample-adding 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 registered with the electrode layer 4 and the absorption layer 5, and then pressed and assembled by using an acrylic material clamp.
During measurement, protein solutions to be measured with different concentrations are dripped from the top layer sample adding hole, combined with the modified nano silver particles through the microfluidic pretreatment module, and further flowed to the electrode layer to be contacted with the working electrode, and the modified working electrode can be combined with the protein to be measured and the modified nano silver particles.
The electrode is connected to an electrochemical workstation, a differential volt-ampere pulse method is used for detection, the nano silver particles on the surface of the working electrode can generate electrochemical reaction to generate a current signal, and the signal size is positively correlated with the concentration of the protein to be detected.
FIG. 4 shows the response current and the corresponding fitted curve obtained by the biosensor for detecting different concentrations of the protein to be detected. The test result shows that the lower limit of detection of the sensor is as low as 10fg/ml, the sensor has linear response in the concentration range of 10fg/ml to 1ng/ml, and the fitting degree R 2 ＝0.99782。

Claims (3)

1. The paper-based three-dimensional microfluidic biosensor based on the laser-induced graphene electrode is characterized by comprising a microfluidic pretreatment module and an electrochemical detection module; the microfluidic pretreatment module comprises a three-layer structure, and comprises a sample adding layer (1), a reagent layer (2), a detection layer (3) and an absorption layer (5) from top to bottom in sequence; the electrochemical detection module comprises an electrode layer (4); the sample adding layer (1), the reagent layer (2) and the detection layer (3) are connected into a whole, a three-dimensional structure is formed by folding along a boundary, and the three layers are communicated through overlapped hydrophilic regions; the electrode layer (4) and the absorption layer (5) are independent respectively, and the layers are assembled by a clamp after registration and superposition;
the electrode layer (4) is carved on two sides, the front side of the electrode layer is a working electrode (4-1), the back side of the electrode layer is an auxiliary electrode and a reference electrode (4-4), the auxiliary electrode and the reference electrode (4-4) are of partial circular ring structures, the auxiliary electrode is in a 270-degree circular ring shape, and the reference electrode is in a 30-degree circular ring shape; the symmetrical center of the working electrode (4-1) is coincided with the circle centers of the auxiliary electrode and the reference electrode (4-4), the working electrode (4-1) is completely positioned in the detection area (3-1) of the detection layer (3), and the auxiliary electrode and the reference electrode (4-4) are completely positioned in the absorption area (5-1) of the absorption layer (5); the electrode layer (4) is provided with a central hole (4-2) and a plurality of peripheral holes (4-3), the working electrode (4-1) is tangent to the central hole (4-2) and is uniformly distributed, the peripheral holes (4-3) are tangent to the detection area (3-1), and the peripheral holes (4-3) are arranged in central symmetry relative to the circle center of the central hole (4-2); the detection area (3-1) is communicated with the absorption area (5-1) through the central hole (4-2) and the peripheral holes (4-3).
2. The paper-based three-dimensional microfluidic biosensor based on the laser-induced graphene electrode according to claim 1, wherein a base material of the vertical microfluidic pretreatment module is cellulose chromatographic filter paper, and a hydrophobic barrier material is paraffin; the base material of the electrochemical detection module is polyimide, and the electrode material is laser-induced graphene; and each electrode lead part is hot stamped and packaged by a thermoplastic polyurethane film.
3. The paper-based three-dimensional microfluidic biosensor based on the laser-induced graphene electrode according to claim 1 or 2, wherein the reagent region (2-1) of the reagent layer (2) comprises four round hydrophilic regions which are not communicated with each other, and can be used for synchronous detection of multiple analytes.
CN202210793352.1A 2022-07-07 2022-07-07 Paper-based three-dimensional microfluidic biosensor based on laser-induced graphene electrode Pending CN115290712A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116814414A (en) * 2023-08-30 2023-09-29 北京芯畅科技有限公司 Laser-induced graphene PCR detection device and method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116814414A (en) * 2023-08-30 2023-09-29 北京芯畅科技有限公司 Laser-induced graphene PCR detection device and method
CN116814414B (en) * 2023-08-30 2023-12-12 北京芯畅科技有限公司 Laser-induced graphene PCR detection device and method

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