Paper-based graphene enzyme biosensor and preparation method thereof

Abstract

The invention discloses a paper-based graphene enzyme biosensor and a preparation method thereof, a paper substrate of the sensor comprises sensor areas, a test liquid dropwise adding area and interconnection micro-channels, and the test liquid dropwise adding area is connected with each sensor area through the interconnection micro-channels; one ends of the auxiliary electrode, the working electrode and the reference electrode in the sensor area are connected with the liquid absorption area, and the liquid absorption area is connected with the interconnected micro-channel; the auxiliary electrode and the working electrode are graphene nano material electrodes formed by processing the surface of the paper substrate, the reference electrode is an electrode formed by coating the surface of the paper substrate with an electrode layer, the working electrode is provided with specific enzymes for identifying indexes to be detected, and all the specific enzymes can not be completely the same; a liquid blocking area is arranged on one side, close to the liquid absorption area, of each of the auxiliary electrode, the working electrode and the reference electrode. According to the invention, the problems of complex introduction of nano materials and structures, manufacturing separation of micro-channel electrodes, low efficiency and single detection target of a current paper-based biomolecule detection chip are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of sensor manufacturing and biomolecule detection, and particularly relates to a paper-based graphene enzyme biosensor and a preparation method thereof. Background Art

[0002] Molecular detection technology is an important means of health monitoring and disease diagnosis, but the commonly used detection methods often rely on complex equipment and devices, require professional operation, are costly and have limited application scenarios. Electrochemical biosensors are usually in the form of chips, consisting of sensing electrodes and microfluidics as basic units. They do not require large equipment and professionals, significantly simplify operation, and are suitable for a variety of scenarios. They can achieve sensitive and convenient detection of disease marker molecules 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 the advantages of no external pump drive, flexibility, and low cost, and are expected to promote the widespread application of molecular detection technology.

[0003] In paper-based biosensors, the sensing electrodes play a detection function, and the microfluidic structure can support convenient multi-detection operations. However, the manufacturing process of the sensing electrodes and microfluidic channels in existing paper-based biosensors is separate and inefficient, and the method of introducing nanomaterials or structures to improve the performance of the sensing electrodes is complicated, which limits the detection diversity and application flexibility of paper-based sensors. Therefore, it is necessary to provide a manufacturing method that can efficiently produce the sensing electrodes and microfluidic channel structures in paper-based biosensors. Summary of the invention

[0004] In order to solve the problems of separation and low efficiency of the sensing electrode / microchannel manufacturing process in current paper-based biosensors, as well as complicated steps for introducing nanomaterials and structures, the purpose of the present invention is to provide a paper-based graphene enzyme biosensor and a preparation method thereof.

[0005] The technical solution adopted by the present invention is as follows: A paper-based graphene enzyme biosensor comprises a paper substrate, wherein the paper substrate comprises a sensor region, a measuring liquid drop region and interconnected microfluidic channels, wherein the measuring liquid drop region is connected to each sensor region via the interconnected microfluidic channels; The sensor region includes a liquid imbibition region, 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 imbibition region, and the liquid imbibition region is connected to the interconnected microfluidic channel; The auxiliary electrode and the working electrode are both graphene nanomaterial electrodes (also referred to as graphene nanoelectrodes) formed on the surface of the paper substrate, the reference electrode is an electrode formed after the electrode layer is coated on the surface of the paper substrate, the working electrode is provided with a specific enzyme for identifying the index to be measured, and the specific enzymes for identifying the index to be measured set in the working electrode regions of all graphene nanomaterial electrodes are not all the same; A liquid blocking area is arranged on the auxiliary electrode, the working electrode and the reference electrode at a side close to the liquid absorption area.

[0006] Preferably, the auxiliary electrode, the working electrode and the reference electrode all adopt a strip structure, the working electrode is located between the auxiliary electrode and the reference electrode, the end of the working electrode located on the liquid absorption area is set as a circular area, the diameter of the circular area is greater than the width of the working electrode, the end of the auxiliary electrode located on the liquid absorption area is set as an arc segment, the arc segment 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 the end of the reference electrode, and the end of the interconnected microfluidic channel 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 made of Ag / AgCl electrode, the paper substrate is made of cellulose paper, and the liquid blocking area is located at the edge of the liquid absorption area.

[0008] The method for preparing the paper-based graphene enzyme biosensor of the present invention comprises the following steps: By laser processing, auxiliary electrodes and working electrodes are processed on a flame-retardant paper substrate; The working electrode is modified to deposit and fix a specific enzyme for identifying the index to be measured; an electrode layer is coated to form a reference electrode; After the auxiliary electrode, the working electrode and the reference electrode are processed, a hydrophobic agent is dropped on the side of the auxiliary electrode, the working electrode and the reference electrode close to the liquid absorption area, so that a liquid blocking area is formed on the auxiliary electrode, the working electrode and the reference electrode on the side close to the liquid absorption area; Finally, through laser processing, interconnected microchannels and liquid droplet addition areas were cut on the 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 of the paper substrate comprises: The flame retardant is evenly sprayed on the surface of the paper substrate and dried to complete the flame retardant treatment, wherein the flame retardant is an ammonium polyphosphate solution.

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

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

[0012] Preferably, when the interconnected microchannels and the liquid droplet adding area are cut out on the flame-retardant treated paper substrate by laser processing, the laser process parameters are laser power of 17.7-18.3 W and scanning speed of 362.5-387.5 mm / s.

[0013] Preferably, the hydrophobic agent is polydimethylsiloxane.

[0014] Preferably, the working electrode is modified to deposit and fix a specific enzyme for identifying the index to be measured, including modification of a glucose detection sensor, modification of a lactic acid detection sensor, and modification of a uric acid detection sensor, wherein: When modifying the glucose detection sensor: deposit a Prussian blue solution on the working electrode area, fix 2-4 μl of glucose oxidase-chitosan solution, and then let it stand at 2-6°C for 2-5 hours; When modifying the lactate detection sensor: fix 2~4μl of lactase-chitosan solution on 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 on the working electrode area, and then let it stand at 2~6℃ for 2~5 hours.

[0015] The present invention has the following beneficial effects: The paper-based graphene enzyme biosensor of the present invention uses a paper substrate that is easy to obtain and manufacture as a raw material, is environmentally friendly and biodegradable, and greatly reduces the detection and processing costs. In addition, the capillary action of the paper-based fiber drives the liquid to flow, and no external pump is required, so the applicability is wider, which is conducive to the integration of sample transfer and detection. There are several sensor areas on the paper substrate, and the sensor area adopts a three-electrode system, including a liquid absorption area, an auxiliary electrode, a working electrode and a reference electrode. The working electrode area is provided with a specific enzyme for identifying the index to be measured, so the structure of each graphene nanomaterial electrode in the present invention can be used as a separate biomolecule detection unit, and the specific enzymes for identifying the index to be measured provided in all working electrode areas may not be completely the same. At this time, the paper-based graphene enzyme biosensor of the present invention may have more than two different biomolecule detection units, thereby supporting the convenient detection of multiple target signals, being able to adapt well to the flexible application scenarios of molecular detection, and efficiently providing more abundant data references for medical treatment; or the paper-based graphene enzyme biosensor of the present invention may have multiple identical biomolecule detection units, thereby supporting multiple detections of the same target signal. In summary, the present invention solves the problems of the current paper-based biosensor in which the sensing electrode / microchannel manufacturing process is separated and inefficient, and the steps for introducing nanomaterials and structures are complicated. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of the manufacturing principle and process of processing graphene nanomaterial electrodes and microchannels in the present invention; Figure 2 This is a physical picture of the paper-based sensor designed and manufactured by the present invention using three indicators detection as an example; Figure 3 The Raman spectrum characterization results of the laser-induced paper-based graphene of the present invention; Figure 4 cyclic voltammetry curves of the paper-based graphene enzymatic molecular detection unit under different laser powers in an embodiment of the present invention; FIG5(a) is a graph showing the response of the paper-based detection unit to glucose solutions of different concentrations in an embodiment of the present invention; FIG5(b) is a graph showing the response of the paper-based detection unit to lactic acid solutions of different concentrations in an embodiment of the present invention; FIG5(c) is a graph showing the response of the paper-based detection unit to uric acid solutions of different concentrations in an embodiment of the present invention; In the figure, 1-auxiliary electrode, 1-1-arc segment, 2-working electrode, 2-1-circular area, 3-reference electrode, 4-liquid drop adding area, 5-interconnected microfluidic channel, 6-first graphene sensor, 7-second graphene sensor, 8-third graphene sensor, 9-paper substrate, 10-graphene nanomaterial electrode, 11-liquid absorption area. DETAILED DESCRIPTION

[0017] The exemplary embodiments of the present application are described below with reference to the accompanying drawings. It should be understood that these specific descriptions are only used to teach those skilled in the art how to implement the present application, and are not intended to exhaust all possible methods of the present application, nor to limit the scope of the present application.

[0018] The basic method of the present invention is to induce and cut a sensor integrated with multiple structure-controllable graphene nanomaterial sensing electrodes and microchannels through laser control on a paper base, and quickly manufacture a paper-based biomolecule detection chip that can support the simultaneous detection of multiple signals. According to the detection requirements, the type of enzymatic molecules of the signals to be tested can be controlled by selecting specific enzymes. Taking the detection of three different enzymatic molecular indicators as an example, the specific implementation of the present invention is as follows: like Figure 2 As shown, the paper-based graphene enzyme biosensor designed and manufactured in this embodiment and used for the detection of three indicators includes a paper substrate, wherein the paper substrate includes a sensor area (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 measuring drop area 4 and an interconnected microfluidic channel 5, wherein the liquid measuring drop area 4 is connected to each sensor area through the interconnected microfluidic channel 5; the sensor area includes a liquid imbibition area 11, an auxiliary electrode 1, a working electrode 2 and a reference electrode 3, wherein 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 imbibition area 11, and the liquid imbibition area 11 is connected to the interconnected microfluidic channel 5; the auxiliary electrode 1 and the working electrode 2 are both graphene nanomaterial electrodes formed on the surface of the paper substrate by 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 index to be measured. The specific enzymes for identifying the index to be measured provided in the working electrode regions of all graphene nanomaterial electrodes may be different or the same. In this case, the present invention can realize the detection of multiple indexes at one time (when the specific enzymes are different), or the present invention can realize multiple detections of the same index 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 area on one side close to the liquid absorption area 11. The liquid blocking area is not drawn in the figure and is as close to the liquid absorption area 11 as possible. Its function is to block the liquid in the liquid absorption area 11 from penetrating into the auxiliary electrode 1, the working electrode 2 and the reference electrode 3. The graphene nanomaterial electrode is a three-dimensional structure containing graphene nanomaterials in situ carbonized by a moderate pyrolysis effect of laser, presenting a micro-nanoscale porous morphology.

[0019] When the paper-based multi-index molecular detection chip for three index detection is used, the liquid to be tested is dropped into the liquid drop adding area 4, and the liquid to be tested infiltrates into the liquid absorption area 11 of each sensor area along the interconnected microchannel 5. Figure 2Taking the orientation shown in the third graphene sensor 6 below as an example, the test liquid absorbed by the liquid absorption area 11 is infiltrated into the left ends of the auxiliary electrode 1, the working electrode 2 and the reference electrode 3 to meet the measurement requirements. The test liquid droplet addition area 4, the interconnected microchannel 5 and the liquid absorption area 11 in the sensor area in the paper-based graphene enzyme biosensor of the present invention are all made of unprocessed paper substrate materials. The auxiliary electrode 1 and the working electrode 2 are both in-situ processed 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 the present invention is paper. Therefore, the paper-based graphene enzyme biosensor of the present invention can also be called a paper-based multi-index molecular detection chip. As a preferred embodiment of the present invention, see Figure 1 and Figure 2 The auxiliary electrode 1, the working electrode 2 and the 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 greater than the width of the working electrode 2. The circular area 2-1 facilitates the diffusion of the liquid to be tested. One end of the auxiliary electrode 1 located 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 to maximize the ratio of the auxiliary electrode area to the working electrode area and reduce the measurement error caused by the polarization of the auxiliary electrode. The end of the arc segment 1-1 extends to one end of the reference electrode 3 located on the liquid absorption area 11 and there is a gap between the end of the reference electrode 3. The gap enables the liquid to be tested guided by the interconnected microchannel 5 to enter between the arc segment of the auxiliary electrode 1 and the circular part of the right end of the working electrode 2 on the liquid absorption area 11, so that the right ends of the auxiliary electrode 1, the working electrode 2 and the reference electrode 3 can all be in full contact with the liquid to be tested. The end of the interconnected 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.

[0020] As a preferred embodiment of the present invention, in the above embodiment of the present invention, the electrode layer may be Ag / AgCl electrode, the paper substrate may be cellulose paper (such as 3MM cellulose paper), and the liquid blocking area is located at the edge of the liquid absorption area 11.

[0021] See also Figure 1 The preparation method of the paper-based graphene enzyme biosensor of the present invention comprises the following steps: Step 1: Flame retardant treatment of paper The flammable paper is pretreated with an effective flame retardant to prevent the paper from burning through under laser irradiation, and then the paper treated with the flame retardant is placed at room temperature to dry. Specifically, in the experiment of treating flammable paper with different flame retardant methods, ammonium polyphosphate whose graphene nanomaterial electrode does not fall off and has good induction effect is selected for subsequent research. It can usually achieve a flame retardant effect by reacting with organic matter, dehydrating to form a protective layer, releasing non-flammable gas, etc. at high temperature.

[0022] The invention adopts 0.1-0.2 g / ml ammonium polyphosphate solution, sprays it evenly on cellulose paper, and performs drying treatment at room temperature to avoid paper wrinkles caused by rapid drying.

[0023] Step 2: Graphene nanomaterial electrode manufacturing Draw the required electrode group and size of the paper-based graphene enzyme biosensor in AutoCAD drawing software, import it into the laser processing equipment, and then use tape to firmly fix the flame-retardant treated paper to a specific position on the laser processing platform to reduce the error caused by fluctuations in the laser spot height, thereby preventing the deformation and displacement that occur during the printing process from affecting the induction of nanomaterials, and prepare for laser printing.

[0024] In order to optimize the electrical properties 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 is carried out in the grating mode of the laser, and the induction degree of graphene is adjusted by multi-parameter control of the laser. When the laser induction of graphene nanomaterial electrodes is carried out in the grating mode, the power is 2.4~3.0W, the scanning rate is 112.5~137.5mm / s, the number of scans is 3~5 times, and the pixel density is 480~520PPI.

[0026] Step 3: Laser cutting microchannels The microchannel and chip model cutting was performed in the 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 the vector mode.

[0027] Step 4: Cut the microfluidic chip body By laser processing, interconnected microchannels 5 and liquid droplet addition areas 4 are cut out on paper, 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 is modified, and a specific enzyme for identifying the index to be measured is deposited and fixed; in order to enhance the temperature resistance and reliability of electrochemical sensing, Ag / AgCl is coated on the reference electrode RE position reserved in the sensor and dried at 60°C.

[0029] A hydrophobic agent (such as polydimethylsiloxane (PDMS)) is dropped on the side of the auxiliary electrode 1 , the working electrode 2 and the reference electrode 3 close to the liquid absorption area 11 , so that a liquid blocking area is formed on the auxiliary electrode 1 , the working electrode 2 and the reference electrode 3 close to the liquid absorption area 11 .

[0030] When modifying the working electrodes of each sensor in the obtained multi-index molecular detection chip, the specific enzyme that identifies the index to be measured is deposited and fixed at the corresponding position, and then left to stand at 2-6°C. The specific process of modifying the paper-based graphene enzymatic biosensor with a specific enzyme can be: 1) Modification of glucose detection sensor: Prussian blue solution was deposited on the working electrode, and 2-5 μl glucose oxidase-chitosan solution was fixed, and then allowed to stand at 2-6°C for 2-5 hours.

[0031] 2) Modification of lactate detection sensor: 2-5 μl lactase-chitosan solution was fixed on the working electrode and allowed to stand at 2-6°C for 2-5 hours.

[0032] 3) Modification of uric acid detection sensor: 2-5 μl uricase-chitosan solution was fixed on the working electrode and allowed to stand at 2-6°C for 2-5 hours.

[0033] In the above scheme, the mixture is allowed to stand at 2 to 6°C for 2 to 5 hours, usually in a refrigerator, for no less than 2 hours.

[0034] Example like Figure 2 As shown, this embodiment proposes that 3MM paper can be used to prepare a paper-based multi-index molecular detection chip that can simultaneously support the detection of three indicators. The chip is mainly composed of three graphene sensors (i.e., a first graphene sensor 6, a second graphene sensor 7, and a third graphene sensor 8) for respectively detecting enzymatic molecular indicators, a liquid droplet addition area 4, and interconnected microfluidic channels 5.

[0035] The mechanism of multi-target detection achieved by using the paper-based multi-indicator molecular detection chip is as follows: the sample liquid to be tested is added to the test liquid drop area 4, and the sample liquid enters the modified working electrodes in the three sensor areas corresponding to the three indicators along the flow channel under the capillary action of the paper base. The electrical changes produced by the redox principle of the enzymatic reaction can be captured in real time by the electrochemical workstation, thereby realizing multi-concentration detection of glucose, lactic acid and uric acid in the test liquid.

[0036] To demonstrate the feasibility of the paper-based multi-index molecular detection chip, specifically, in this embodiment, independent sensor areas for glucose, lactate and uric acid detection were manufactured and modified respectively. Each independent sensor area can be called a paper-based graphene enzymatic molecular detection unit, which is used to demonstrate the detection response of the independent sensor area to different concentrations of the test solution.

[0037] like Figure 3 As shown, relatively clear D peak, G peak and 2D peak appeared in the Raman spectrum characterization results, indicating the induced formation of graphene on the paper-based material.

[0038] like Figure 4 As shown in the figure, the electrochemical performance of the paper-based graphene enzymatic molecular detection unit manufactured under the power range of 2.4~3W was characterized by cyclic voltammetry. The paper-based graphene enzymatic molecular detection unit is an independent sensor area with a power of 100×10 –3 5×10 –3 MK3[Fe(CN)6] / K4[Fe(CN)6] solution served as electrolyte. Although the paper-based graphene sensing electrode has better electrochemical properties under 3.0W laser power, its mechanical properties are poor and it is easy to be damaged during use. Therefore, the induction parameters of the graphene nanomaterial electrode of the paper-based graphene enzymatic molecular detection unit in the embodiment are selected as: power 2.7W, speed 125mm / s, and scanning times 4.

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

[0040] 2) Method for modifying the working electrode of the paper-based graphene lactate detection unit: 3 μl of a lactate enzyme solution prepared by dissolving 20 mg of lactate enzyme in 1 ml of a 1% chitosan solution was added dropwise to the working electrode of the paper-based graphene lactate detection unit, wherein the solvent of the 1% chitosan solution was 2% glacial acetic acid, and the mixture was placed in a refrigerator at 4°C for 2 hours.

[0041] 3) Method for modifying the working electrode of the paper-based graphene uric acid detection unit: 3 μl of a uricase solution prepared by dissolving 5 mg of uricase in 1 ml of a 1% chitosan solution was added dropwise to the working electrode of the paper-based graphene uric acid detection unit, wherein the solvent of the 1% chitosan solution was 2% glacial acetic acid, and the mixture was allowed to stand in a refrigerator at 4°C for 2 hours.

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

[0043] 2) Performance calibration of the paper-based graphene glucose detection unit: The calibration was carried out in PBS. The glucose concentration in the electrochemical reaction cell increased in a gradient of 7.5 mM. The voltage set by 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 achieve 0~30 mM glucose detection.

[0044] 3) Performance calibration of paper-based graphene lactic acid detection unit: The calibration was carried out in PBS. The lactic acid concentration in the electrochemical reaction cell increased in a gradient of 5 mM. The voltage set by 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). The detection unit can achieve 0~20 mM lactic acid detection.

[0045] 4) Performance calibration of the paper-based graphene uric acid detection unit: The calibration was carried out in PBS. The uric acid concentration in the electrochemical reaction cell increased in a gradient of 0.5 mM. The voltage set by 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). The detection unit can achieve 0~2 mM uric acid detection.

[0046] It can be seen from the above scheme that the present invention uses paper that is easy to obtain and manufacture as the main raw material for the manufacture of molecular chips. It is environmentally friendly and biodegradable, which greatly reduces the detection and processing costs. The capillary action of paper-based fibers drives the flow of liquid, and no external pump is required. It has a wider applicability and is conducive to the integration of sample transfer and detection. The integrated laser manufacturing of paper-based graphene working electrode / microfluidic channel is achieved by laser induced regulation and high-precision cutting, which greatly improves the controllability and efficiency of manufacturing. The customized integrated paper-based graphene enzyme biosensor can support the simultaneous detection of multiple target signals, can adapt well to the flexible application scenarios of molecular detection chips, and efficiently provide more abundant data references for medical treatment.

[0047] The above examples are only for clarifying the detection capability and reliability of the paper-based multi-indicator molecule, and are only used to help understand the technical solution and feasibility of the present invention, and are not limited to the three molecules mentioned above. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, 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 comprises a sensor region, a liquid measurement drop region (4) and interconnected microfluidic channels (5), wherein the liquid measurement drop region (4) is connected to each sensor region via the interconnected microfluidic channels (5); The sensor region comprises a liquid imbibition 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 imbibition region (11), and the liquid imbibition region (11) is connected to the interconnected microfluidic channel (5); The auxiliary electrode (1) and the working electrode (2) are both graphene nanoelectrodes formed by processing on the surface of a paper substrate; the reference electrode (3) is an electrode formed by coating an electrode layer on the surface of the paper substrate; the working electrode (2) is provided with a specific enzyme for identifying the index to be measured; and the specific enzymes for identifying the index to be measured provided in the working electrode regions of all graphene nanoelectrodes are not all the same; A liquid blocking area is provided on the auxiliary electrode (1), the working electrode (2) and the reference electrode (3) on a side close to the liquid absorption area (11).

2. The paper-based graphene enzyme biosensor according to claim 1, characterized in that: The auxiliary electrode (1), the working electrode (2) and the 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 greater than the width of the working electrode (2). One end of the auxiliary electrode (1) located 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) located on the liquid absorption area (11) and a gap is left between the end of the arc segment (1-1) of the auxiliary electrode (1) and the end of the reference electrode (3). The end of the interconnected 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 adopts Ag / AgCl electrode, the paper substrate adopts cellulose paper, and the liquid blocking area is located at the edge of the liquid absorption area (11).

4. The method for preparing the paper-based graphene enzyme biosensor according to any one of claims 1 to 3, characterized in that: The process includes the following: An auxiliary electrode (1) and a working electrode (2) are processed on a flame-retardant paper substrate by laser processing; The working electrode (2) is modified to deposit and fix a specific enzyme for identifying the index to be measured; an electrode layer is coated to form a reference electrode (3); After the auxiliary electrode (1), the working electrode (2) and the reference electrode (3) are processed, a hydrophobic agent is dropped on the side of the auxiliary electrode (1), the working electrode (2) and the reference electrode (3) close to the liquid absorption area (11), so that a liquid blocking area is formed on the side of the auxiliary electrode (1), the working electrode (2) and the reference electrode (3) close to the liquid absorption area (11); Finally, by laser processing, interconnected microchannels (5) and a liquid measurement droplet addition area (4) are cut out on the flame-retardant treated paper substrate to obtain the paper-based graphene enzyme biosensor.

5. The method for preparing a paper-based graphene enzyme biosensor according to claim 4, characterized in that: The flame retardant treatment process of paper substrate includes: The flame retardant is evenly sprayed on the surface of the paper substrate and dried to complete the flame retardant treatment, wherein the flame retardant is an ammonium polyphosphate solution.

6. The method for preparing a paper-based graphene enzyme biosensor according to claim 5, characterized in that: The flame retardant is an ammonium polyphosphate solution with a concentration of 0.1-0.2 g / ml. After the flame retardant is evenly sprayed on the surface of the paper substrate, it is dried at room temperature and the surface of the paper substrate is kept flat.

7. The method for preparing a paper-based graphene enzyme biosensor according to claim 4, characterized in that: When an auxiliary electrode (1) and a working electrode (2) are processed on a flame-retardant paper substrate by laser processing, the process is performed in a laser raster mode, using a defocused laser scanning method, setting an optimized defocus distance of 1.5 to 1.7 mm, setting a laser power of 2.4 to 3 W, a laser scanning rate of 112.5 to 137.5 mm / s, a scanning number of 3 to 5, and a pixel density of 480 to 520 PPI.

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

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

10. The method for preparing a paper-based graphene enzyme biosensor according to claim 4, characterized in that: The working electrode (2) is modified to deposit and fix a specific enzyme for identifying the index to be measured, including the modification of a glucose detection sensor, the modification of a lactic acid detection sensor and the modification of a uric acid detection sensor, wherein: When modifying the glucose detection sensor: deposit a Prussian blue solution on the working electrode area, fix 2-4 μl of glucose oxidase-chitosan solution, and then let it stand at 2-6°C for 2-5 hours; When modifying the lactate detection sensor: fix 2~4μl of lactase-chitosan solution on the working electrode area, and then let it stand at 2~62℃ for 2~5 hours; When modifying the uric acid detection sensor: fix 2~4μl of uricase-chitosan solution on the working electrode area, and then let it stand at 2~6℃ for 2~5 hours.

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