Paper-based graphene enzyme biosensor and preparation method thereof

Abstract

The application discloses a paper-based graphene enzyme biosensor and a preparation method thereof. The paper-based substrate of the biosensor comprises a sensor region, a measuring liquid drop adding region and an interconnecting microchannel. The measuring liquid drop adding region is connected with each sensor region through the interconnecting microchannel. One end of an auxiliary electrode, a working electrode and a reference electrode of the sensor region is connected with a liquid suction region, and the liquid suction region is connected with the interconnecting microchannel. The auxiliary electrode and the working electrode are graphene nanomaterial electrodes formed by processing on the surface of the paper-based substrate, and the reference electrode is an electrode formed after coating an electrode layer on the surface of the paper-based substrate. The working electrode is provided with specific enzymes for identifying a to-be-measured index, and all the specific enzymes can not be the same. Liquid blocking zones are arranged on the auxiliary electrode, the working electrode and the reference electrode on the side close to the liquid suction region. The application solves the problems of complex introduction of nanomaterials and structures, separation of microchannel electrode manufacturing, low efficiency and single detection target of the current paper-based biomolecular detection chip.

Description

Technical Field

[0001] This invention belongs to the field of sensor manufacturing and biomolecular detection technology, specifically relating to a paper-based graphene enzyme biosensor and its preparation method. Background Technology

[0002] Molecular detection technology is an important tool for health monitoring and disease diagnosis. However, currently used detection methods often rely on complex equipment and devices, requiring professional operation, resulting in high costs and limited application scenarios. Electrochemical biosensors, typically in the form of chips, consist of sensing electrodes and microchannels as basic units. They eliminate the need for large equipment and professional personnel, significantly simplifying operation and making them suitable for various scenarios. They can achieve sensitive and convenient detection of disease biomarkers in samples such as blood, sweat, and urine. In recent years, biomedical sensors using paper as a substrate material have received widespread attention and research. Compared with traditional biosensor substrate materials such as silicon and glass, paper-based biosensors have advantages such as no external pump required, flexibility, and low cost, and are expected to promote the widespread application of molecular detection technology.

[0003] In paper-based biosensors, sensing electrodes perform the detection function, while microfluidic structures support convenient multi-sensor operation. However, in existing paper-based biosensors, the manufacturing processes for sensing electrodes and microfluidic channels are separate and inefficient. Introducing nanomaterials or structures to improve sensing electrode performance is complex, limiting the detection diversity and application flexibility of paper-based sensors. Therefore, it is necessary to provide a manufacturing method that can efficiently produce both the sensing electrodes and microfluidic structures in paper-based biosensors. Summary of the Invention

[0004] To address the problems of separation and low efficiency in the manufacturing process of sensing electrodes / microchannels in current paper-based biosensors, as well as the complexity of introducing nanomaterials and structures, the present invention aims to provide a paper-based graphene enzyme biosensor and its preparation method.

[0005] The technical solution adopted in this invention is as follows: A paper-based graphene enzyme biosensor includes a paper substrate, the paper substrate comprising a sensor region, a liquid addition region, and interconnected microchannels, wherein the liquid addition region is connected to each sensor region via interconnected microchannels; The sensor area includes a liquid absorption area, an auxiliary electrode, a working electrode, and a reference electrode. One end of the auxiliary electrode, one end of the working electrode, and one end of the reference electrode are connected to the liquid absorption area, which is connected to interconnected microchannels. Both the auxiliary electrode and the working electrode are graphene nanomaterial electrodes (also known as graphene nanoelectrodes) formed on the surface of a paper substrate. The reference electrode is an electrode formed after coating an electrode layer on the surface of a paper substrate. The working electrode is equipped with a specific enzyme for identifying the target indicator. The specific enzymes for identifying the target indicator set in the working electrode area of ​​all graphene nanomaterial electrodes are not all the same. The auxiliary electrode, working electrode, and reference electrode have a liquid blocking zone on the side near the liquid absorption area.

[0006] Preferably, the auxiliary electrode, working electrode, and reference electrode all adopt a strip structure. The working electrode is located between the auxiliary electrode and the reference electrode. One end of the working electrode located on the liquid absorption area is set as a circular area with a diameter larger than the width of the working electrode. One end of the auxiliary electrode located on the liquid absorption area is set as an arc segment, which is concentric with the circular area of ​​the working electrode. The end of the arc segment extends to the end of the reference electrode located on the liquid absorption area and leaves a gap between it and the end of the reference electrode. The end of the interconnecting microchannel connected to the liquid absorption area is located between the end of the arc segment of the auxiliary electrode and the end of the reference electrode.

[0007] Preferably, the electrode layer is an Ag / AgCl electrode, the paper substrate is cellulose paper, and the liquid blocking region is located at the edge of the liquid absorption region.

[0008] The method for preparing the paper-based graphene enzyme biosensor described above in this invention includes the following steps: Auxiliary and working electrodes were fabricated on a flame-retardant paper substrate using laser processing. The working electrode is modified to deposit and immobilize a specific enzyme for identifying the target indicator; an electrode layer is coated to form a reference electrode. After the auxiliary electrode, working electrode and reference electrode are processed, a hydrophobic agent is dropped on the side of the auxiliary electrode, working electrode and reference electrode near the liquid absorption area to form a liquid blocking area on the side of the auxiliary electrode, working electrode and reference electrode near the liquid absorption area; Finally, by using laser processing, interconnected microchannels and liquid droplet addition areas were cut out on a flame-retardant paper substrate to obtain a paper-based graphene enzyme biosensor that can be used for multi-index detection.

[0009] Preferably, the flame-retardant treatment process for the paper substrate includes: The flame retardant is evenly sprayed onto the surface of the paper substrate and dried to complete the flame retardant treatment. The flame retardant is an ammonium polyphosphate solution.

[0010] Preferably, the flame retardant is an ammonium polyphosphate solution with a concentration of 0.1~0.2g / ml. After the flame retardant is evenly sprayed onto the surface of the paper substrate, it is dried at room temperature while keeping the surface of the paper substrate flat.

[0011] Preferably, when processing the auxiliary electrode and the working electrode on the flame-retardant paper substrate by laser processing, the process is carried out in the laser grating mode, using a defocused laser scanning method, with the optimized defocus distance set to 1.5~1.7mm, the laser power set to 2.4~3W, the laser scanning rate set to 112.5~137.5mm / s, the number of scans set to 3~5, and the pixel density set to 480~520PPI.

[0012] Preferably, when cutting interconnected microchannels and liquid droplet application areas on a flame-retardant paper substrate using laser processing, the laser process parameters are a laser power of 17.7~18.3W and a scanning speed of 362.5~387.5mm / s.

[0013] Preferably, the hydrophobic agent is polydimethylsiloxane.

[0014] Preferably, when modifying the working electrode to deposit and immobilize specific enzymes for identifying the target indicator, the modification includes the modification of a glucose detection sensor, a lactate detection sensor, and a uric acid detection sensor, wherein: When modifying the glucose detection sensor: deposit Prussian blue solution in the working electrode area and fix 2~4 μl of glucose oxidase-chitosan solution, then let it stand at 2~6℃ for 2~5 hours; When modifying the lactate detection sensor: fix 2~4μl of lactase-chitosan solution in the working electrode area, and then let it stand at 2~6℃ for 2~5 hours; When modifying the uric acid detection sensor: fix 2~4μl of uricase-chitosan solution in the working electrode area, and then let it stand for 2~5 hours at 2~6℃.

[0015] The present invention has the following beneficial effects: This invention relates to a paper-based graphene enzyme biosensor that uses readily available and manufactured paper substrates as raw materials, making it environmentally friendly and biodegradable, significantly reducing detection and processing costs. Furthermore, the capillary action of the paper fibers drives liquid flow, eliminating the need for external pumps, broadening its applicability, and facilitating integrated sample transfer and detection. The paper substrate has several sensor regions employing a three-electrode system, including a liquid absorption region, an auxiliary electrode, a working electrode, and a reference electrode. The working electrode region contains a specific enzyme for identifying the target indicator. Therefore, each graphene nanomaterial electrode in this invention can function as a separate biomolecular detection unit. The specific enzymes for identifying the target indicator in all working electrode regions can be different, allowing the paper-based graphene enzyme biosensor to have two or more different biomolecular detection units, thus supporting convenient detection of multiple target signals. This adapts well to flexible molecular detection applications and efficiently provides richer data references for medical applications. Alternatively, the paper-based graphene enzyme biosensor can have multiple identical biomolecular detection units, supporting multiple detections of the same target signal. In summary, this invention solves the problems of separation and low efficiency in the manufacturing process of sensing electrodes / microchannels in current paper-based biosensors, as well as the complexity of introducing nanomaterials and structures. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the manufacturing principle and process of processing graphene nanomaterial electrodes and microchannels according to the present invention. Figure 2 This is a physical image of the paper-based sensor designed and manufactured according to the present invention, taking the detection of three indicators as an example; Figure 3 The Raman spectral characterization results of laser-induced paper-based graphene of this invention are shown below. Figure 4 This is a cyclic voltammetry curve of the paper-based graphene enzyme-catalyzed molecular detection unit under different laser powers in an embodiment of the present invention; Figure 5(a) shows the response results of the paper-based detection unit to glucose solutions of different concentrations in this embodiment of the invention; Figure 5(b) shows the response results of the paper-based detection unit to lactic acid solutions of different concentrations in this embodiment of the invention; Figure 5(c) shows the response results of the paper-based detection unit to uric acid solutions of different concentrations in this embodiment of the invention. In the figure, 1-auxiliary electrode, 1-1-arc segment, 2-working electrode, 2-1-circular region, 3-reference electrode, 4-liquid droplet addition region, 5-interconnected microchannel, 6-first graphene sensor, 7-second graphene sensor, 8-third graphene sensor, 9-paper substrate, 10-graphene nanomaterial electrode, 11-liquid absorption region. Detailed Implementation

[0017] Exemplary embodiments of this application are described below with reference to the accompanying drawings. It should be understood that these specific descriptions are for teaching those skilled in the art how to implement this application only, and are not intended to exhaustively describe all possible methods of this application, nor to limit the scope of this application.

[0018] The basic approach of this invention is to induce and cut out a sensor integrating multiple structurally controllable graphene nanomaterial sensing electrodes and microfluidics on a paper substrate using laser modulation, thereby rapidly fabricating a paper-based biomolecular detection chip capable of simultaneously detecting multiple signals. Depending on the detection requirements, the type of enzymatic molecules in the target signal can be controlled by selecting specific enzymes. Taking the detection of three different enzymatic molecular indicators as an example, the specific implementation of this invention is as follows: like Figure 2 As shown, the paper-based graphene enzyme biosensor designed and manufactured in this embodiment, which can be used for the detection of three indicators, includes a paper substrate. The paper substrate includes sensor regions (see the structures corresponding to the first graphene sensor 6, the second graphene sensor 7, and the third graphene sensor 8 in the figure), a liquid addition region 4, and interconnected microfluidic channels 5. The liquid addition region 4 is connected to each sensor region through the interconnected microfluidic channels 5. The sensor region includes a liquid absorption region 11, an auxiliary electrode 1, a working electrode 2, and a reference electrode 3. One end of the auxiliary electrode 1, one end of the working electrode 2, and one end of the reference electrode 3 are connected to the liquid absorption region 11, and the liquid absorption region 11 is connected to the interconnected microfluidic channels 5. The auxiliary electrode 1 and the working electrode 2 are both graphene nanomaterial electrodes processed on the surface of the paper substrate. The reference electrode 3 is an electrode formed by coating an electrode layer on the surface of a paper substrate. The working electrode 2 is equipped with a specific enzyme for identifying the target indicator. The specific enzymes for identifying the target indicator in the working electrode areas of all graphene nanomaterial electrodes can be different or the same. In this case, the present invention can realize the detection of multiple indicators at one time (when the specific enzymes are different), or the present invention can realize the detection of the same indicator multiple times at one time (when the specific enzymes are the same). The auxiliary electrode 1, the working electrode 2, and the reference electrode 3 are provided with a liquid blocking region on the side near the liquid absorption region 11. The liquid blocking region is not shown in the figure. It should be as close as possible to the liquid absorption region 11. Its function is to prevent the liquid in the liquid absorption region 11 from penetrating into the auxiliary electrode 1, the working electrode 2, and the reference electrode 3. The graphene nanomaterial electrode is formed by in-situ carbonization of organic materials into a three-dimensional structure containing graphene nanomaterials through moderate pyrolysis by laser, exhibiting a micro-nano scale porous morphology.

[0019] In this invention, the paper-based multi-index molecular detection chip, which can be used for the detection of three indicators, involves placing a droplet of the test liquid in the test liquid application area 4. The test liquid then seeps along the interconnected microchannels 5 into the absorption area 11 of each sensor area. At the absorption area 11, the test liquid... Figure 2Taking the orientation of the third graphene sensor 6 below as an example, the liquid absorption area 11 absorbs the test liquid and wets the left end of the auxiliary electrode 1, working electrode 2, and reference electrode 3 to meet the measurement requirements. In the paper-based graphene enzyme biosensor of this invention, the test liquid addition area 4, the interconnected microfluidic channel 5, and the liquid absorption area 11 in the sensor area are all made of unprocessed paper substrate material. The auxiliary electrode 1 and working electrode 2 are both processed in situ on the paper substrate by laser processing. The reference electrode 3 is formed by coating an electrode layer on the paper substrate. Therefore, the main material of the paper-based graphene enzyme biosensor of this invention is paper, and thus, the paper-based graphene enzyme biosensor of this invention can also be called a paper-based multi-index molecular detection chip. For a preferred embodiment of this invention, see [link to relevant documentation]. Figure 1 and Figure 2 The auxiliary electrode 1, working electrode 2, and reference electrode 3 all adopt a strip structure. The working electrode 2 is located between the auxiliary electrode 1 and the reference electrode 3. One end of the working electrode 2 located on the liquid absorption area 11 is set as a circular area 2-1. The diameter of the circular area 2-1 is larger than the width of the working electrode 2. This circular area 2-1 facilitates the diffusion of the test liquid. One end of the auxiliary electrode 1 located on the liquid absorption area 11 is set as an arc segment 1-1. This arc segment 1-1 is concentric with the circular area 2-1 of the working electrode 2 to maximize the ratio of the area of ​​the auxiliary electrode to the area of ​​the working electrode and reduce the measurement error caused by the polarization of the auxiliary electrode. The end of the arc-shaped segment 1-1 extends to one end of the reference electrode 3 located on the liquid absorption area 11 and is spaced apart from that end of the reference electrode 3. This spacing allows the test liquid guided by the interconnected microfluidic channel 5 to enter between the arc-shaped segment of the auxiliary electrode 1 and the right circular portion of the working electrode 2 on the liquid absorption area 11, thereby ensuring that the right ends of the auxiliary electrode 1, the working electrode 2, and the reference electrode 3 can all make full contact with the test liquid. The end of the interconnected microfluidic channel 5 connected to the liquid absorption area 11 is located between the end of the arc-shaped segment 1-1 of the auxiliary electrode 1 and the end of the reference electrode 3.

[0020] As a preferred embodiment of the present invention, in the above-described embodiment, the electrode layer can be an Ag / AgCl electrode, the paper substrate is cellulose paper (e.g., 3MM cellulose paper can be used), and the liquid blocking region is located at the edge of the liquid absorption region 11.

[0021] See Figure 1 The preparation method of the paper-based graphene enzyme biosensor of the present invention includes the following steps: Step 1: Paper flame retardant treatment Flammable paper is pretreated with an effective flame retardant to prevent it from burning through under laser irradiation. The treated paper is then dried at room temperature. Specifically, in experiments on treating flammable paper using different flame retardant methods, ammonium polyphosphate, which has good induction effect and does not detach from graphene nanomaterial electrodes, was selected for further research. At high temperatures, it can usually achieve flame retardant effects by reacting with organic matter, dehydrating to form a protective layer, and releasing non-flammable gases.

[0022] This invention uses a 0.1~0.2 g / ml ammonium polyphosphate solution, which is evenly sprayed onto cellulose paper and dried at room temperature to avoid paper wrinkling caused by rapid drying.

[0023] Step 2: Fabrication of graphene nanomaterial electrodes The electrode clusters and dimensions of the required paper-based graphene enzyme biosensor are drawn in AutoCAD drawing software and imported into the laser processing equipment. Then, the flame-retardant paper is firmly fixed to a specific position on the laser processing platform using tape to reduce the error caused by the fluctuation of the laser spot height, thereby preventing deformation and displacement during the printing process from affecting the induction of nanomaterials, and preparing for laser printing.

[0024] To optimize the electrical performance of graphene nanomaterial electrodes, a defocused laser scanning method was selected, and the optimized defocus distance was set to 1.5~1.7mm.

[0025] The induction of graphene material electrodes was performed in laser grating mode, and the degree of graphene induction was adjusted by multi-parameter control of the laser. In grating mode, the laser induction of graphene nanomaterial electrodes was performed with a power of 2.4–3.0 W, a scanning rate of 112.5–137.5 mm / s, 3–5 scans, and a pixel density of 480–520 PPI.

[0026] Step 3: Laser cutting of microchannels The microchannel and chip model cutting was performed in vector mode. During cutting, the laser power was set to 17.7~18.3W and the scanning rate was set to 362.5~387.5mm / s in vector mode.

[0027] Step 4: Cutting the microfluidic chip body The interconnected microchannels 5 and the liquid droplet addition area 4 are cut out on paper using laser processing, and then the overall structure of the sensor is cut out to obtain the paper-based graphene enzyme biosensor.

[0028] Step 5: Reference electrode coating and electrode modification The working electrode 2 was modified to deposit and immobilize a specific enzyme for identifying the target indicator; in order to enhance the temperature performance and reliability of the electrochemical sensing, Ag / AgCl was coated on the reserved reference electrode RE position of the sensor and dried at 60°C.

[0029] A hydrophobic agent (such as polydimethylsiloxane PDMS) is dropped onto the side of the auxiliary electrode 1, working electrode 2 and reference electrode 3 near the liquid absorption region 11 to form a liquid blocking region on the side of the auxiliary electrode 1, working electrode 2 and reference electrode 3 near the liquid absorption region 11.

[0030] When modifying the working electrodes of each sensor in the multi-index molecular detection chip, the specific enzymes that identify the target index are deposited and fixed at the corresponding positions, and then left to stand at 2-6℃. The specific enzyme modification process for the paper-based graphene enzyme-catalyzed biosensor can be as follows: 1) Modification of glucose detection sensor: Prussian blue solution was deposited on the working electrode and 2~5μl of glucose oxidase-chitosan solution was fixed and left to stand for 2~5 hours at 2~6℃.

[0031] 2) Modification of lactic acid detection sensor: Fix 2~5μl of lactase-chitosan solution on the working electrode and let it stand for 2~5 hours at 2~6℃.

[0032] 3) Modification of uric acid detection sensor: Fix 2~5μl of uricase-chitosan solution on the working electrode and let it stand for 2~5 hours at 2~6℃.

[0033] In the above method, the mixture should be left to stand for 2 to 5 hours at 2 to 6 degrees Celsius, generally in a refrigerator for at least 2 hours.

[0034] Example like Figure 2 As shown in the figure, this embodiment proposes to use 3MM paper to prepare a paper-based multi-index molecular detection chip that can simultaneously support the detection of three indicators. It mainly consists of three graphene sensors (i.e., the first graphene sensor 6, the second graphene sensor 7 and the third graphene sensor 8) that detect enzyme-catalyzed molecular indicators respectively, a liquid droplet addition region 4 and an interconnected microfluidic channel 5.

[0035] The mechanism of multi-target detection using this paper-based multi-index molecular detection chip is as follows: the sample solution to be tested is added to the droplet area 4 of the test solution. Under the capillary action of the paper base, the sample solution flows along the channel and enters the modified working electrodes in the three sensor areas corresponding to the three indicators. The electrochemical workstation can capture the electrical changes generated under the principle of enzyme-catalyzed redox reaction in real time, thereby realizing the multi-concentration detection of glucose, lactic acid and uric acid in the test solution.

[0036] To demonstrate the feasibility of the paper-based multi-index molecular detection chip, specifically, this embodiment fabricated and modified independent sensor regions for the detection of glucose, lactic acid, and uric acid. Each independent sensor region can be referred to as a paper-based graphene enzymatic molecular detection unit, which is used to demonstrate the detection response of the independent sensor region to different concentrations of test solutions.

[0037] like Figure 3 As shown, the Raman spectroscopy characterization results show relatively clear D peaks, G peaks, and 2D peaks, indicating the induced formation of graphene on paper-based materials.

[0038] like Figure 4 As shown, the electrochemical performance of a paper-based graphene enzymatic molecular detection unit fabricated in the power range of 2.4~3W was characterized using cyclic voltammetry. The paper-based graphene enzymatic molecular detection unit is an independent sensor region, with a power rating of 100×10⁻⁶. –3 5×10 M KCl solution prepared –3 The M K3[Fe(CN)6] / K4[Fe(CN)6] solution was used as the electrolyte. Although the paper-based graphene sensing electrode exhibits better electrochemical properties at a laser power of 3.0W, its mechanical properties are poor, making it prone to breakage during use. Therefore, in this embodiment, the induction parameters for the graphene nanomaterial electrode of the paper-based graphene enzyme-catalyzed molecular detection unit were selected as follows: power 2.7W, speed 125mm / s, and scan count 4.

[0039] In this embodiment, the modification process of the working electrode surface of the paper-based graphene enzyme-catalyzed molecular detection unit includes: 1) Modification method of working electrode of paper-based graphene glucose detection unit: Prussian blue solution prepared by cyclic voltammetry, consisting of 2.5 mM K3Fe(CN)6, 2.5 mM FeCl3, 100 mM KCl, and 100 mM HCl aqueous solution, was deposited on the working electrode as an effective catalyst for glucose oxidation reaction. To achieve better deposition results, a stable and reliable electrochemical deposition method was explored, namely, cyclic voltammetry scanning once, with a voltage scan range of 0-0.5 V and a scan rate of 20 mV / s. Drying was carried out at room temperature. 3 μl of glucose oxidase-chitosan mixed solution was added dropwise to the working electrode of the paper-based graphene glucose detection unit and allowed to stand at 4°C for 2 hours. The mixed solution was prepared by mixing 10 mg of glucose oxidase in 1 ml of 1% (w / w) chitosan solution, with 2% glacial acetic acid as the solvent. The chitosan solution can effectively serve as the immobilization matrix in the paper-based graphene enzymatic molecular detection unit.

[0040] 2) Modification method of working electrode of paper-based graphene lactic acid detection unit: 3 μl of lactase solution prepared by 20 mg lactase in 1 ml of chitosan solution with a mass fraction of 1% was added dropwise to the working electrode of paper-based graphene lactic acid detection unit. The solvent of the 1% chitosan solution was 2% glacial acetic acid. The solution was left to stand in a refrigerator at 4°C for 2 hours.

[0041] 3) Modification method of working electrode of paper-based graphene uric acid detection unit: 3 μl of urase solution prepared by 5 mg of urase in 1 ml of chitosan solution with a mass fraction of 1% was added dropwise to the working electrode of paper-based graphene uric acid detection unit. The solvent of the 1% chitosan solution was 2% glacial acetic acid. The solution was left to stand in a refrigerator at 4°C for 2 hours.

[0042] Sensing performance calibration of each paper-based graphene enzymatic molecular detection unit in this embodiment: 1) The paper-based graphene enzyme-catalyzed molecular detection units for detecting glucose, lactic acid, and uric acid signals were connected to the electrochemical workstation using specially designed fixtures, and each unit was calibrated individually using the chronoamperometry method.

[0043] 2) Performance calibration of the paper-based graphene glucose detection unit: Calibration was performed in PBS. The glucose concentration in the electrochemical reaction cell was increased in a gradient of 7.5 mM. The voltage set for the chronoamperometry was -0.05 V. The time-current curve was recorded. The glucose detection and calibration results are shown in Figure 5(a). The paper-based graphene glucose detection unit can detect glucose from 0 to 30 mM.

[0044] 3) Performance calibration of paper-based graphene lactic acid detection unit: Calibration was performed in PBS. The lactic acid concentration in the electrochemical reaction cell was increased in a gradient of 5 mM. The voltage set for the chronoamperometry was -0.2 V. The time-current curve was recorded. The lactic acid detection and calibration results are shown in Figure 5(b). This detection unit can detect lactic acid from 0 to 20 mM.

[0045] 4) Performance calibration of the paper-based graphene uric acid detection unit: Calibration was performed in PBS. The uric acid concentration in the electrochemical reaction cell was increased in a gradient of 0.5 mM. The voltage set for the chronoamperometry was 0.6 V. The time-current curve was recorded. The uric acid detection and calibration results are shown in Figure 5(c). This detection unit can detect uric acid from 0 to 2 mM.

[0046] As can be seen from the above scheme, this invention uses readily available and manufactured paper as the main raw material for molecular chip manufacturing. It is environmentally friendly and biodegradable, significantly reducing detection and processing costs. The capillary action of paper-based fibers drives liquid flow, eliminating the need for external pumps, thus broadening its applicability and facilitating integrated sample transfer and detection. Laser-induced modulation and high-precision cutting enable integrated laser manufacturing of the paper-based graphene working electrode / microchannel, greatly improving manufacturing controllability and efficiency. The customized integrated paper-based graphene enzyme biosensor supports simultaneous detection of multiple target signals, well-suited to the flexible application scenarios of molecular detection chips, and efficiently provides richer data references for medical applications.

[0047] The above embodiments are merely illustrative of the detection capability and reliability of the paper-based multi-index molecules, and are intended only to aid in understanding the technical solution and feasibility of the present invention. They are not limited to the three molecules exemplified above. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A paper-based graphene enzyme biosensor, characterized in that, The paper substrate is flame-retardant treated and is made of cellulose paper. The paper substrate includes a sensor area, a liquid droplet application area (4), and interconnected microchannels (5). The liquid droplet application area (4) is connected to each sensor area through the interconnected microchannels (5). The sensor area includes a liquid absorption area (11), an auxiliary electrode (1), a working electrode (2) and a reference electrode (3). One end of the auxiliary electrode (1), one end of the working electrode (2) and one end of the reference electrode (3) are connected to the liquid absorption area (11). The liquid absorption area (11) is connected to the interconnected microchannel (5). The auxiliary electrode (1) and the working electrode (2) are both graphene nanoelectrodes formed on the surface of a paper substrate by laser processing. The reference electrode (3) is an electrode formed after coating an electrode layer on the surface of a paper substrate. The working electrode (2) is provided with a specific enzyme for identifying the target index. The specific enzymes for identifying the target index set in the working electrode area of ​​all graphene nanoelectrodes are not all the same. When the graphene nanoelectrodes formed on the surface of the paper substrate by laser processing are performed in the laser grating mode, a defocused laser scanning method is adopted, the optimized defocus distance is set to 1.5~1.7mm, the laser power is set to 2.4~3W, the laser scanning rate is set to 112.5~137.5mm / s, the number of scans is 3~5, and the pixel density is 480~520PPI. The auxiliary electrode (1), working electrode (2) and reference electrode (3) are provided with a liquid blocking zone on the side near the liquid absorption area (11); The flame retardant treatment process of the paper substrate includes: uniformly spraying the flame retardant onto the surface of the paper substrate and drying it to complete the flame retardant treatment; the flame retardant is an ammonium polyphosphate solution with a concentration of 0.1~0.2g / ml. After uniformly spraying the flame retardant onto the surface of the paper substrate, it is dried at room temperature while keeping the surface of the paper substrate flat.

2. The paper-based graphene enzyme biosensor according to claim 1, characterized in that, The auxiliary electrode (1), working electrode (2) and reference electrode (3) are all strip-shaped. The working electrode (2) is located between the auxiliary electrode (1) and the reference electrode (3). One end of the working electrode (2) on the liquid absorption area (11) is set as a circular area (2-1). The diameter of the circular area (2-1) is larger than the width of the working electrode (2). One end of the auxiliary electrode (1) on the liquid absorption area (11) is set as an arc segment (1-1). The arc segment (1-1) is concentric with the circular area (2-1) of the working electrode (2). The end of the arc segment (1-1) extends to the end of the reference electrode (3) on the liquid absorption area (11) and leaves a gap between it and the end of the reference electrode (3). The end of the interconnecting microchannel (5) connected to the liquid absorption area (11) is located between the end of the arc segment (1-1) of the auxiliary electrode (1) and the end of the reference electrode (3).

3. The paper-based graphene enzyme biosensor according to claim 1, characterized in that, The electrode layer uses an Ag / AgCl electrode, and the liquid blocking region is located at the edge of the liquid absorption region (11).

4. The method for preparing the paper-based graphene enzyme biosensor according to any one of claims 1-3, characterized in that, The process includes the following: The flame retardant is evenly sprayed onto the paper substrate surface and dried to complete the flame retardant treatment; the flame retardant is an ammonium polyphosphate solution with a concentration of 0.1~0.2g / ml. After the flame retardant is evenly sprayed onto the paper substrate surface, it is dried at room temperature while keeping the paper substrate surface flat. An auxiliary electrode (1) and a working electrode (2) are fabricated on a flame-retardant paper substrate using laser processing. The process is carried out in laser grating mode, using a defocused laser scanning method. The optimized defocus distance is set to 1.5~1.7mm, the laser power is set to 2.4~3W, the laser scanning rate is set to 112.5~137.5mm / s, the number of scans is 3~5, and the pixel density is 480~520PPI. The working electrode (2) is modified to deposit and fix a specific enzyme for identifying the target indicator; the electrode layer is coated to form a reference electrode (3). After the auxiliary electrode (1), working electrode (2) and reference electrode (3) are processed, a hydrophobic agent is dropped on the side of the auxiliary electrode (1), working electrode (2) and reference electrode (3) near the liquid absorption area (11) to form a liquid blocking area on the side of the auxiliary electrode (1), working electrode (2) and reference electrode (3) near the liquid absorption area (11). Finally, the paper-based graphene enzyme biosensor was obtained by cutting interconnected microchannels (5) and liquid droplet addition area (4) on the flame-retardant paper substrate using laser processing.

5. The method for preparing the paper-based graphene enzyme biosensor according to claim 4, characterized in that, The flame retardant is an ammonium polyphosphate solution.

6. The method for preparing the paper-based graphene enzyme biosensor according to claim 4, characterized in that, When cutting interconnected microchannels (5) and liquid droplet addition area (4) on a flame-retardant paper substrate using laser processing, the laser process parameters are laser power of 17.7~18.3W and scanning speed of 362.5~387.5mm / s.

7. The method for preparing the paper-based graphene enzyme biosensor according to claim 4, characterized in that, The hydrophobic agent is polydimethylsiloxane.

8. The method for preparing the paper-based graphene enzyme biosensor according to claim 4, characterized in that, When modifying the working electrode (2) to deposit and immobilize specific enzymes for identifying the target indicator, the modifications include those for glucose detection sensors, lactate detection sensors, and uric acid detection sensors, wherein: When modifying the glucose detection sensor: deposit Prussian blue solution in the working electrode area and fix 2~4 μl of glucose oxidase-chitosan solution, then let it stand at 2~6℃ for 2~5 hours; When modifying the lactate detection sensor: fix 2~4μl of lactase-chitosan solution in the working electrode area, and then let it stand for 2~5 hours at 2~62℃; When modifying the uric acid detection sensor: fix 2~4μl of uricase-chitosan solution in the working electrode area, and then let it stand for 2~5 hours at 2~6℃.

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