Portable electrochemical biosensing platform based on paper-based microfluidics

Abstract

The invention discloses a portable electrochemical biosensing platform based on paper-based microfluidics, which relates to the technical field of biological detection equipment and comprises a biosensing device for detecting biomarkers and an electrochemical workstation connected with the biosensing device, and the electrochemical workstation realizes instant detection of the biosensing device and the biomarker through the POCT system. According to the platform disclosed by the invention, the platform is combined with the biosensing device, the electrochemical workstation connected with the biosensing device and the smart phone, a patient can directly collect a sample by himself / herself without the help of a professional through a point-of-care testing (POCT) technology, and after the sample is added into the detection device, a detection result can be obtained in a short time; the platform has the advantages of being low in cost, convenient to carry, easy and convenient to operate and the like, meanwhile, the platform can detect various biomarkers, the universality of a biosensor is improved, the requirement for on-site rapid detection is met, and meanwhile virus propagation can be effectively prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection equipment, in particular to a portable electrochemical biosensing platform based on paper-based microfluidics. Background Art

[0002] Point-of-care testing (POCT) is also called bedside testing. Simply put, POCT refers to a detection mode that can detect and report the test results in a timely manner only at the subject end. Compared with traditional laboratory testing, this rapid detection method does not require a fixed, dedicated testing site, occupies fewer resources, and does not require complex professional technical training to stably and accurately detect specific analytes; a biosensor is an analytical device that uses biological molecules as recognition elements and converts the detected biological signals into measurable physical or chemical signals through signal converters to detect specific targets. Generally speaking, after the biological molecules specifically recognize the target, the corresponding signal sensor will convert or amplify the recognition element into an easy-to-read detection signal, thereby realizing the detection of specific targets. Biosensors have the advantages of high sensitivity, good selectivity, and fast response. Therefore, they have been widely used in environmental monitoring, food analysis, medical testing, etc.

[0003] In recent years, highly sensitive and highly specific biosensors have gradually emerged in the development and application of POCT, especially precise and miniature portable biosensors, which have become the general trend of its development. As POCT detection technology penetrates into various places and gradually expands in emergency rooms, the wild, communities, and users' homes, "miniaturization", "portability", "integration", and "intelligence" have become the new pursuit of POCT detection technology innovation and its integration with other fields. The development of fast, simple, efficient, integrated, and user-friendly instant detection equipment is of great significance both in application scenarios and application value.

[0004] For example, the Chinese patent CN117969626A, "A new strategy for directional immobilization of antibodies based on aptamers", is to obtain a polymethylene blue modified electrode by modifying the screen-printed electrode with polymethylene blue, label the KIM-1 antibody with MB-DNA, and use cyclic voltammetry to directionally immobilize the antibody to achieve the detection of KIM-1. PMB not only increases the electron transfer rate, but also provides a platform for the directional immobilization of antibodies.

[0005] Although traditional biosensors have the advantages of high sensitivity, good selectivity, and fast response speed, their signal detection devices are mostly expensive, bulky, cannot be carried around, and are complicated to operate. They require specific professional technicians to use. Therefore, traditional biosensors are not universal and cannot meet the needs of rapid on-site detection. To address the above problems, a portable electrochemical biosensor platform based on paper-based microfluidics is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a portable electrochemical biosensing platform based on paper-based microfluidics to solve the problems raised by the above background technology.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a portable electrochemical biosensor platform based on paper-based microfluidics, comprising a biosensor device for detecting biomarkers and an electrochemical workstation connected thereto, wherein the electrochemical workstation realizes instant detection of the biosensor device and the biomarker through a POCT system, wherein the POCT system is composed of a smart phone and an electrochemical detection module EDM, and is controlled by a microcontroller MCU, wherein the biosensor device comprises an upper cover, a lower cover, a biosensor and a paper-based microfluidic, wherein the biosensor is obtained by modifying a carbon mesh electrode with polymethylene blue to obtain a polymethylene blue working electrode, and then connecting an antibody / aptamer of a biomarker labeled with methylene blue to the working electrode through cyclic voltammetry.

[0008] Preferably, the carbon mesh electrode is modified with polymethylene blue by electropolymerizing methylene blue in a methylene blue solution using cyclic voltammetry.

[0009] Preferably, the antibody / aptamer of the biomarker labeled with methylene blue is connected to the working electrode by cyclic voltammetry, and then 1% bovine serum albumin plus the biomarker to be tested are incubated and then detected by differential pulse voltammetry to prevent nonspecific adsorption.

[0010] Preferably, the biosensor is prepared by a method comprising the following steps: A1. Electropolymerization of methylene blue: A carbon mesh electrode was used as the working electrode, and a buffer solution of 0.07-0.22 M PBS was used as the base solution. The carbon mesh electrode was connected to an electrochemical workstation. In a 0.3-1.4 mM methylene blue solution, an organic polymer polymethylene blue was polymerized on the surface of the working electrode using cyclic voltammetry to obtain a polymethylene blue working electrode. The voltage was -0.5 to 1.2 V, the scan rate was 50 mV / s, and the number of cycles was 30. A2. Immobilization of antibodies / aptamers of biomarkers labeled with methylene blue: The polymethylene blue working electrode was cleaned with deionized water and dried at room temperature. Then, a solution of antibodies / aptamers of biomarkers labeled with methylene blue was added to the surface of the polymethylene blue working electrode. The cyclic voltammetry voltage was -0.5 to 1.2 V, the scan rate was 50-55 mV / s, and the number of cycles was 2-5. The antibodies / aptamers of biomarkers labeled with methylene blue were connected to the working electrode by cyclic voltammetry to achieve directional immobilization of the antibodies / aptamers. A3. Casein blocking: 1% bovine serum albumin plus the biomarker to be tested is incubated and then detected by differential pulse voltammetry to prevent nonspecific adsorption, and the electrode is rinsed again to obtain the biosensor.

[0011] Preferably, the paper-based microfluid is prepared by a method comprising the following steps: B1. According to the paper-based microfluidic shape, filter paper with a size of 10.0 mm × 35.0 mm is used, and solid paraffin is repeatedly brushed on the filter paper. The solid paraffin is printed on the surface of the pure cellulose paper of the filter paper to obtain wax-printed paper; B2. Heat the wax printing paper in an oven at 90°C for 2 minutes to allow the solid paraffin to diffuse completely. Due to the porous structure of the filter paper, the wax on the surface of the filter paper is re-melted and penetrates into the paper during heating, forming a hydrophobic area within the fiber of the paper, and finally forming a hydrophobic fence. B3. Cool the heated filter paper at room temperature to obtain a microfluidic paper chip, i.e., paper-based microfluidics.

[0012] Preferably, a drip hole is formed through one end of the top of the upper cover. When the biosensor device is assembled, the biosensor and the paper-based microfluid are bonded and fixed by double-sided tape, and the biosensor and the paper-based microfluid are placed between the upper cover and the lower cover. The bottom of the upper cover is fixedly connected to the top of the lower cover. When the biomarker is detected by the electrochemical workstation and the biosensor device, the electrochemical workstation is connected to the biosensor, and the smart phone can be connected to the electrochemical workstation via a Bluetooth or WIFI interface. Finally, the test results are viewed using the smart phone.

[0013] Preferably, the chip used in the electrochemical workstation is mainly composed of a Bluetooth ceramic antenna, a battery management module, a Bluetooth low power system and an MCU module.

[0014] Preferably, the MCU module consists of four parts: an analog front end, a digital-to-analog converter, an analog-to-digital converter and a programmable read-only memory.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, the platform combines a biosensor device, an electrochemical workstation connected to the biosensor device, and a smart phone. Through the point-of-care detection technology POCT, patients can directly collect samples by themselves without the help of professionals. After adding the samples to the detection device, the test results can be obtained in a short time. The platform has the advantages of low cost, easy to carry, and simple operation. At the same time, the platform can detect a variety of biomarkers, which solves the defects of traditional biosensor signal detection devices that are expensive, bulky, cannot be carried around, and are complicated to operate. Patients can use the device independently to complete the test without the need for guidance from specific professional technicians. The universality of biosensors is improved, and the needs of rapid on-site detection are met. By detecting biomarkers of different diseases in blood / urine, the platform can be used as an early warning device for preventing certain diseases in advance, which can effectively prevent the spread of viruses.

[0016] 2. In the present invention, the platform is composed of a biosensor device, an electrochemical workstation connected to the biosensor device, and a smart phone. The biosensor device is composed of an upper cover, a lower cover, a biosensor and a paper-based microfluid. When preparing the biosensor, the carbon mesh electrode is modified with polymethylene blue to obtain a polymethylene blue working electrode, and then the antibody / aptamer of the biomarker labeled with methylene blue is connected to the working electrode by cyclic voltammetry to achieve directional immobilization of the antibody / aptamer. Finally, 1% bovine serum albumin is added to the biomarker to be tested and incubated for differential pulse voltammetry detection to prevent nonspecific adsorption. When preparing the paper-based microfluid, solid paraffin is repeatedly brushed on the filter paper according to the shape of the paper-based microfluidic control, and then the filter paper is heated to melt the wax film and immerse it into the paper to form a hydrophobic fence to obtain the paper-based microfluid. When the polymethylene blue / carbon mesh electrode sensor of the present invention is used for biomarker detection, it has a wide linear range and good anti-interference performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic structural diagram of a biosensor device based on a paper-based microfluidic portable electrochemical biosensor platform of the present invention; Figure 2 An exploded view of a biosensing device based on a paper-based microfluidic portable electrochemical biosensing platform of the present invention; Figure 3 The overall design block diagram of a microfluidic electrochemical biosensor device based on a paper-based microfluidic portable electrochemical biosensor platform of the present invention; Figure 4 The figure is a schematic diagram of the shape and size of the paper-based microfluidics in the portable electrochemical biosensing platform based on paper-based microfluidics of the present invention.

[0018] In the figure: 1. upper cover; 2. lower cover; 3. biosensor; 4. paper-based microfluidics; 5. drip hole. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Reference Figure 1 - Figure 4 As shown: a portable electrochemical biosensor platform based on paper-based microfluidics, including a biosensor device for detecting biomarkers and an electrochemical workstation connected thereto, the electrochemical workstation realizes instant detection of the biosensor device and the biomarker through a POCT system, the POCT system is composed of a smart phone and an electrochemical detection module EDM, and is controlled by a microcontroller MCU, the biosensor device includes an upper cover 1, a lower cover 2, a biosensor 3 and a paper-based microfluid 4, the biosensor 3 is obtained by modifying a carbon mesh electrode with polymethylene blue to obtain a polymethylene blue working electrode, and then connecting an antibody / aptamer of a biomarker labeled with methylene blue to the working electrode by cyclic voltammetry; using a method of electropolymerizing methylene blue, the carbon mesh electrode is modified with polymethylene blue using cyclic voltammetry in a methylene blue solution; connecting an antibody / aptamer of a biomarker labeled with methylene blue to the working electrode by cyclic voltammetry, and then incubating with 1% bovine serum albumin and the biomarker to be tested, and then performing differential pulse voltammetry detection to prevent nonspecific adsorption.

[0021] The biosensor 3 is prepared by a method comprising the following steps: A1. Electropolymerization of methylene blue: A carbon mesh electrode was used as the working electrode, and a buffer solution of 0.07-0.22 M PBS was used as the base solution. The carbon mesh electrode was connected to an electrochemical workstation. In a 0.3-1.4 mM methylene blue solution, an organic polymer polymethylene blue was polymerized on the surface of the working electrode using cyclic voltammetry to obtain a polymethylene blue working electrode. The voltage was -0.5 to 1.2 V, the scan rate was 50 mV / s, and the number of cycles was 30. A2. Immobilization of antibodies / aptamers of biomarkers labeled with methylene blue: The polymethylene blue working electrode was cleaned with deionized water and dried at room temperature. Then, a solution of antibodies / aptamers of biomarkers labeled with methylene blue was added to the surface of the polymethylene blue working electrode. The cyclic voltammetry voltage was -0.5 to 1.2 V, the scan rate was 50-55 mV / s, and the number of cycles was 2-5. The antibodies / aptamers of biomarkers labeled with methylene blue were connected to the working electrode by cyclic voltammetry to achieve directional immobilization of the antibodies / aptamers. A3. Casein blocking: 1% bovine serum albumin plus the biomarker to be tested was incubated and then detected by differential pulse voltammetry to prevent nonspecific adsorption, and the electrode was rinsed again to obtain biosensor 3.

[0022] The paper-based microfluid 4 is prepared by a method comprising the following steps: B1. According to the paper-based microfluidic shape (such as Figure 4 As shown in the figure), use filter paper with a size of 10.0 mm × 35.0 mm (select Whatman No. 1 filter paper), brush the filter paper repeatedly with solid paraffin wax, and the solid paraffin wax is printed on the surface of the pure cellulose paper of the filter paper to obtain wax-printed paper; B2. Heat the wax printing paper in an oven at 90°C for 2 minutes to allow the solid paraffin to diffuse completely. Due to the porous structure of the filter paper, the wax on the surface of the filter paper is re-melted and penetrates into the paper during heating, forming a hydrophobic area within the fiber of the paper, and finally forming a hydrophobic fence. B3. Cool the heated filter paper at room temperature to obtain a microfluidic paper chip, i.e., paper-based microfluidic 4.

[0023] like Figure 3 As shown, in this embodiment, Figure 3 In (A), the chip used in the electrochemical workstation is mainly composed of a Bluetooth ceramic antenna, a battery management module, a Bluetooth low-power system and an MCU module. Figure 3 In (B), the MCU module consists of four parts: an analog front end (AFE), a digital-to-analog converter (DAC), an analog-to-digital converter (ADC), and an EEPROM. A drip hole 5 is provided at one end of the top cover 1. Figure 3 (E), b and c represent the carbon mesh electrodes in paper-based microfluidics 4 and biosensor 3, respectively. Figure 3In (Eb), light gray represents the hydrophobic area used for wax printing, and the remaining white areas are hydrophilic areas. When assembling the biosensor device, the biosensor 3 and the paper-based microfluidic 4 are fixed by double-sided tape, and the biosensor 3 and the paper-based microfluidic 4 are placed between the upper cover 1 and the lower cover 2. The bottom of the upper cover 1 is fixedly connected to the top of the lower cover 2. When the biomarker is detected by the electrochemical workstation and the biosensor device, the electrochemical workstation is connected to the biosensor 3. The smartphone can be connected to the electrochemical workstation via a Bluetooth or WIFI interface, and finally the smartphone is used to view the test results.

[0024] The use method and working principle of the device: In the present invention, the portable electrochemical biosensor platform based on paper-based microfluidics is composed of a biosensor device, an electrochemical workstation connected to the biosensor device, and a smart phone. The biosensor device is composed of an upper cover 1, a lower cover 2, a biosensor 3 and a paper-based microfluidic 4. A drip hole 5 for dripping liquid is provided on the top of the upper cover 1. When preparing the biosensor 3, a carbon wire mesh electrode is used as a working electrode, and an organic polymer polymethylene blue is polymerized on the surface of the working electrode by cyclic voltammetry to obtain a polymethylene blue working electrode. The polymethylene blue working electrode is then washed with deionized water and dried at room temperature. Then, an antibody / aptamer of a biomarker labeled with methylene blue is connected to the working electrode by cyclic voltammetry to achieve Directed immobilization of antibodies / aptamers, and finally incubation with 1% bovine serum albumin and the biomarker to be tested for differential pulse voltammetry detection to prevent nonspecific adsorption, and then the electrodes are rinsed again to obtain biosensor 3; when preparing paper-based microfluidics 4, solid paraffin is repeatedly brushed on the filter paper according to the paper-based microfluidic shape, and the solid paraffin is printed on the surface of the pure cellulose paper of the filter paper to obtain wax-printed paper, and then the wax-printed paper is placed in an oven at a temperature of 90°C and heated for 2 minutes to allow the solid paraffin to diffuse completely. Due to the porous structure of the filter paper, when heated, the wax on the surface of the filter paper is re-melted and penetrates into the paper, forming a hydrophobic area in the fiber of the paper, and finally forming a hydrophobic fence. Finally, the heated filter paper is cooled at room temperature to obtain a microfluidic paper chip, namely, paper-based microfluidics 4; After the biosensor 3 and the paper-based microfluid 4 are prepared, the biosensor 3 and the paper-based microfluid 4 are bonded and fixed by double-sided tape, and the biosensor 3 and the paper-based microfluid 4 are placed between the upper cover 1 and the lower cover 2, and the bottom of the upper cover 1 is fixed to the top of the lower cover 2. After the biosensor device is assembled, when the biomarker is detected by the electrochemical workstation (the chip used in the electrochemical workstation is mainly composed of a Bluetooth ceramic antenna, a battery management module, a Bluetooth low-power system and an MCU module, and the MCU module is composed of four parts: an analog front end AFE, a digital-to-analog converter DAC, an analog-to-digital converter ADC and a programmable read-only memory EEPREOM) and the biosensor device, the electrochemical workstation is connected to the biosensor 3, and the smart phone can be connected to the electrochemical workstation through a Bluetooth or WIFI interface. The patient directly collects the sample by himself, and then adds the sample to the device through the drip hole 5, and finally uses the smart phone to view the test results.

[0025] The platform combines a biosensor device, an electrochemical workstation connected to the biosensor device, and a smart phone. Through the point-of-care testing technology POCT, patients can collect samples directly by themselves without the help of professionals. After adding the samples to the testing device, the test results can be obtained in a short time. It has the advantages of low cost, easy to carry, and simple operation. At the same time, the platform can detect a variety of biomarkers, which solves the defects of traditional biosensor signal detection devices, such as high price, large size, cannot be carried around, and complicated operation. Patients can use the device to complete the test independently without the guidance of specific professional technicians, thereby improving the universality of biosensors and meeting the needs of rapid on-site testing. By detecting biomarkers of different diseases in blood / urine, it can be used as an early warning device for preventing certain diseases in advance, which can effectively prevent the spread of viruses.

[0026] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A portable electrochemical biosensing platform based on paper-based microfluidics, characterized by: The invention comprises a biosensor device for detecting biomarkers and an electrochemical workstation connected thereto. The electrochemical workstation realizes instant detection of the biosensor device and the biomarker through a POCT system. The POCT system is composed of a smart phone and an electrochemical detection module EDM and is controlled by a microcontroller MCU. The biosensor device comprises an upper cover (1), a lower cover (2), a biosensor (3) and a paper-based microfluid (4). The biosensor (3) is obtained by modifying a carbon mesh electrode with polymethylene blue to obtain a polymethylene blue working electrode, and then connecting an antibody / aptamer of a biomarker labeled with methylene blue to the working electrode through cyclic voltammetry.

2. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 1, characterized in that: The carbon mesh electrode was modified with polymethylene blue by electropolymerization of methylene blue in methylene blue solution using cyclic voltammetry.

3. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 2, characterized in that: The antibody / aptamer of the biomarker labeled with methylene blue is connected to the working electrode by cyclic voltammetry, and then 1% bovine serum albumin plus the biomarker to be tested are incubated and then detected by differential pulse voltammetry to prevent nonspecific adsorption.

4. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 3, characterized in that: The biosensor (3) is prepared by a method comprising the following steps: A1. Electropolymerization of methylene blue: A carbon mesh electrode was used as the working electrode, and a buffer solution of 0.07-0.22 M PBS was used as the base solution. The carbon mesh electrode was connected to an electrochemical workstation. In a 0.3-1.4 mM methylene blue solution, an organic polymer polymethylene blue was polymerized on the surface of the working electrode using cyclic voltammetry to obtain a polymethylene blue working electrode. The voltage was -0.5 to 1.2 V, the scan rate was 50 mV / s, and the number of cycles was 30. A2. Immobilization of antibodies / aptamers of biomarkers labeled with methylene blue: The polymethylene blue working electrode was cleaned with deionized water and dried at room temperature. Then, a solution of antibodies / aptamers of biomarkers labeled with methylene blue was added to the surface of the polymethylene blue working electrode. The cyclic voltammetry voltage was -0.5 to 1.2 V, the scan rate was 50-55 mV / s, and the number of cycles was 2-5. The antibodies / aptamers of biomarkers labeled with methylene blue were connected to the working electrode by cyclic voltammetry to achieve directional immobilization of the antibodies / aptamers. A3. Casein blocking: 1% bovine serum albumin plus the biomarker to be tested is incubated and then detected by differential pulse voltammetry to prevent nonspecific adsorption. The electrode is rinsed again to obtain the biosensor (3).

5. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 4, characterized in that: The paper-based microfluid (4) is prepared by a method comprising the following steps: B1. According to the paper-based microfluidic shape, filter paper with a size of 10.0 mm × 35.0 mm is used, and solid paraffin is repeatedly brushed on the filter paper. The solid paraffin is printed on the surface of the pure cellulose paper of the filter paper to obtain wax-printed paper; B2. Heat the wax printing paper in an oven at 90°C for 2 minutes to allow the solid paraffin to diffuse completely. Due to the porous structure of the filter paper, the wax on the surface of the filter paper is re-melted and penetrates into the paper during heating, forming a hydrophobic area within the fiber of the paper, and finally forming a hydrophobic fence. B3. Cool the heated filter paper at room temperature to obtain a microfluidic paper chip, i.e., paper-based microfluidics (4).

6. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 1, characterized in that: A drip hole (5) is formed through one end of the top of the upper cover (1). When assembling the biosensor device, the biosensor (3) and the paper-based microfluid (4) are bonded and fixed by double-sided adhesive, and the biosensor (3) and the paper-based microfluid (4) are placed between the upper cover (1) and the lower cover (2). The bottom of the upper cover (1) is fixedly connected to the top of the lower cover (2). When the biomarker is detected by the electrochemical workstation and the biosensor device, the electrochemical workstation is connected to the biosensor (3). The smart phone can be connected to the electrochemical workstation via a Bluetooth or WIFI interface. Finally, the detection result is viewed using the smart phone.

7. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 1, characterized in that: The chip used in the electrochemical workstation is mainly composed of a Bluetooth ceramic antenna, a battery management module, a Bluetooth low-power system and an MCU module.

8. The portable electrochemical biosensing platform based on paper-based microfluidics according to claim 7, characterized in that: The MCU module consists of four parts: an analog front end, a digital-to-analog converter, an analog-to-digital converter and a programmable read-only memory.

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