Preparation method of magnetic paper-based fluorescent biosensor

Abstract

The invention belongs to the technical field of biological detection sensors, and particularly relates to a preparation method of a magnetic paper-based fluorescent biosensor. The preparation method comprises the following steps: preparing a paper-based sensing chip, modifying an antibody, and coating a silver nanowire. The ferromagnetic material FeSiB and the fluorescent carbon dots are doped on a paper-based platform, biological functional modification on the surface of the test paper is realized by utilizing the characteristics that the carbon dot surface amino is coupled with an antibody and has a fluorescence characteristic, and the fluorescent carbon dots can be verified to be uniformly distributed on the surface of the sensor through luminescence development of fluorescence. The efficient combination of carboxyl on the antibody and amino on the fluorescent carbon dots is facilitated; the biosensor prepared by adopting the composite structure has the advantages of low cost, simplicity in operation, high sensitivity and the like; the method can be applied to the fields of health detection equipment and biological and medical detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection sensors, and specifically relates to a preparation method of a magnetic paper-based fluorescent biosensor. Background Art

[0002] Currently, the common methods for detecting CRP clinically mainly include immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), electrochemistry, colloidal gold immunochromatography technology, etc. However, these detection methods have some disadvantages: Immunoturbidimetry: Although this method is relatively simple to operate, it may be interfered by various factors during the detection process, such as the turbidity of the sample, non-specific reactions, etc., thus affecting the accuracy of the detection results.

[0003] ELISA: Although ELISA has high sensitivity and strong specificity, its operation is relatively cumbersome, requiring multiple washing and incubation steps, which takes a long time. In addition, this method is also easily affected by environmental factors such as temperature and humidity, resulting in unstable results.

[0004] Electrochemistry: Although electrochemistry has advantages such as high sensitivity and fast response speed, its detection results are easily affected by factors such as the surface state of the electrode and the composition of the electrolyte solution, resulting in fluctuations and instability of the results. At the same time, the equipment cost and maintenance cost of electrochemistry are relatively high, restricting its application in some scenarios.

[0005] Colloidal gold immunochromatography technology: Although colloidal gold immunochromatography technology is simple and fast to operate, its sensitivity is relatively low, and it may not be able to detect low-concentration target molecules.

[0006] Moreover, the flexible substrates used in most biosensors have great limitations, resulting in a relatively complex sensor manufacturing process and very time-consuming operation, thus restricting their application. In recent years, due to characteristics such as light weight, good adsorption, easy storage, and good biocompatibility, paper has been widely used as the substrate of biosensors. Compared with traditional PDMS and glass, it has advantages such as low cost and good portability, integrating various materials on a thin and light paper. Currently, paper-based sensing platforms have been applied in different fields such as flexible sensors, food safety monitoring, and clinical diagnosis. However, they also have some significant disadvantages. Among them, the most important disadvantage is that ordinary luminescent materials are difficult to be fixed on the paper substrate, which restricts the stability and durability of the sensor. At the same time, the optical activity of these luminescent materials is also easily lost, which will lead to a decrease in the sensitivity and accuracy of the sensor. These disadvantages limit the reliability and practicality of paper-based sensors in some application scenarios. Summary of the Invention

[0007] The present invention overcomes the deficiencies of the prior art and provides a method for preparing a magnetic paper-based fluorescent biosensor; the present invention is achieved through the following technical solutions: A method for preparing a magnetic paper-based fluorescent biosensor, comprising the following steps: S1. Preparation of a paper-based sensing chip S1.1. Soak photo paper in FeSiB nanofluid so that FeSiB nanoparticles uniformly cover the photo paper; S1.2. Then dry the photo paper; S1.3. Next, soak the photo paper doped with FeSiB in a fluorescent carbon dot solution so that the fluorescent carbon dots uniformly adhere to the photo paper to obtain a paper-based sensing chip; S2. Antibody modification S2.1. Mix the CRP antibody solution with an activating reagent and shake it to activate the carboxyl group on the CRP antibody into NHS ester, so that the CRP antibody combines with the amino group on the fluorescent carbon dots; S2.2. Then soak the paper-based sensing chip in the activated CRP antibody solution and allow the CRP antibody to fully combine with the fluorescent carbon dots; S2.3. Block the paper-based sensing chip in a BSA solution to reduce the influence of electrostatic adsorption and steric hindrance effects, and then wash the surface of the paper-based sensing chip; S3. Coating with silver nanowires: Coat silver nanowires on the smooth side of the paper-based sensing chip to make the paper-based sensing chip conductive.

[0008] Further, the preparation method of the FeSiB nanofluid is: disperse FeSiB powder into deionized water and disperse FeSiB nanoparticles through ultrasonic waves.

[0009] Furthermore, use an ultrasonic cell disruptor to disperse the FeSiB nanoparticles for 15 - 30 min to obtain a uniform FeSiB nanofluid.

[0010] Further, soak the photo paper in the FeSiB nanofluid for 15 - 30 min.

[0011] Further, in step S1.3, soak the photo paper doped with FeSiB in the fluorescent carbon dot solution for 10 - 15 h, and obtain the paper-based sensing chip after natural drying.

[0012] Further, the components of the activating reagent include NHS and EDC.

[0013] Further, in step S2.2, after soaking the paper-based sensing chip in the activated CRP antibody solution, place it in an environment of 36 - 38 °C for 1 h - 1.5 h to allow the CRP antibody to fully combine with the fluorescent carbon dots.

[0014] Furthermore, after the CRP antibody is fully combined with the fluorescent carbon dots, the surface-attached excess antibody is removed by rinsing with PBS.

[0015] Further, in step S2.3, the surface of the paper-based sensing chip is cleaned with PBS and deionized water in sequence.

[0016] Further, after coating with silver nanowires, the paper-based sensing chip is allowed to dry naturally.

[0017] The beneficial effects of the present invention compared with the prior art are as follows: 1. Based on the advantages of the high magnetic permeability and saturation magnetic induction intensity of FeSiB, the fluorescence characteristics of CDs, the excellent electrical conductivity of AgNWs, and the good biocompatibility of photo paper, the present invention prepares a magnetic paper-based fluorescent biosensor. Using photo paper as the substrate of the sensor, FeSiB and CDs are uniformly covered on the paper-based platform, and AgNWs are spin-coated on the smooth side of the paper to prepare the biosensor. It has good sensing performance, as well as the advantages of simple preparation, easy storage, and good biocompatibility.

[0018] 2. The specific binding of the antibody and the antigen will generate surface stress on the paper-based sensor. Based on the magnetic properties of FeSiB, under the condition of an external magnetic field, the CRP concentration can be quantified by detecting the change in magnetic permeability. After introducing AgNWs, the CRP concentration can be quantified by measuring the change in resistance, thereby realizing two measurement methods.

[0019] 3. FeSiB has excellent magnetic properties, including a high saturation magnetic induction intensity, and is a soft magnetic material, and its magnetic properties will change with the change of stress or strain. The functional group - amino group on the surface of the fluorescent carbon dots can be well coupled with the carboxyl group of the antibody to realize the immobilization of the antibody. Based on the specific recognition of antigen-antibody, the detection of various substances can be realized. The present invention has the advantages of high sensitivity, simple preparation, and good biocompatibility, and is of great significance for the rapid and accurate clinical detection of CRP.

[0020] 4. The present invention dopes ferromagnetic material FeSiB and fluorescent carbon dots on the paper-based platform, and utilizes the characteristics that the carbon dots have amino groups on the surface to couple antibodies and have fluorescence characteristics to realize the biofunctionalization modification of the test paper surface. And through the fluorescence imaging, it can be verified that the fluorescent carbon dots are uniformly distributed on the sensor surface, which is beneficial to the efficient binding of the carboxyl group on the antibody and the amino group on the fluorescent carbon dots. It is expected to be applied to health detection equipment and the fields of biological and medical detection. The biosensor prepared by adopting this composite structure has the advantages of low cost, simple operation, and high sensitivity. Description of the Drawings

[0021] Figure 1This is the flow chart of the preparation method of the magnetic paper-based fluorescence biosensor of the present invention. Detailed implementation manners

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail in combination with embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The technical solutions of the present invention will be described in detail below in combination with embodiments and drawings, but the protection scope is not limited by this.

[0023] See Figure 1 , this embodiment proposes a preparation method of a magnetic paper-based fluorescence biosensor; specifically includes the following steps: Select basic materials: FeSiB, silver nanowires, fluorescent carbon dots, photo paper, absolute ethanol, PBS (polybutylene succinate), EDS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide); S1. Preparation of paper-based sensing chip S1.1. First, take 3 g of FeSiB powder and disperse it into a beaker containing deionized water, and use an ultrasonic cell disruptor to disperse the FeSiB nanoparticles for 20 min to obtain a uniform FeSiB nanofluid.

[0024] S1.2. Then soak the prepared 4 mm × 4 mm photo paper in the FeSiB nanofluid for 20 minutes, so that the FeSiB nanoparticles are evenly covered on the photo paper-based platform.

[0025] S1.3. Then place the paper-based platform on a heating table and dry it at 60 °C for 10 min.

[0026] S1.4. Then soak the paper-based platform doped with FeSiB in the fluorescent carbon dot solution for 12 h, and obtain the paper-based sensing chip after natural drying for storage and standby.

[0027] S2. Antibody modification Dilute the CRP antibody solution with 0.01 M PBS solution to prepare an antibody solution of 25 μg / mL. At room temperature, add 0.0064 g of NHS and 0.0064 g of EDC to 1 ml of deionized water and mix to make an activation reagent. Mix the antibody solution and the NHS / EDC solution in a volume ratio of 1:1 and shake for 30 min to activate the carboxyl group on the antibody into NHS ester, which is beneficial to the combination of the antibody and the amino group on the fluorescent carbon dots.

[0028] S2.2. Then, immerse the paper-based sensing chip into the activated antibody solution, and place it in an incubator at 37 °C for 1 h to allow the antibodies to fully bind to the fluorescent carbon dots. Next, take out the paper-based sensing chip modified with antibodies, and rinse it with PBS to remove the excess antibodies attached to the surface.

[0029] S2.3. Finally, seal the paper-based sensing chip in a BSA solution with a mass percentage concentration of 0.1% for 30 min to reduce the influence of electrostatic adsorption and steric hindrance effects, which is beneficial to preventing non-specific binding. Then, wash the surface of the paper-based sensing chip with PBS and deionized water successively to complete the preparation of the magnetic paper-based fluorescence biosensor for CRP detection.

[0030] S3. Coating silver nanowires: Use a pipette to coat a uniform layer of silver nanowires on the smooth side of the paper-based sensing chip, and then dry it naturally to make the sensor conductive, which is convenient for subsequent piezoresistive testing and antigen concentration testing.

[0031] The proposed AgNWs / FeSiB / CDs photo paper composite film in the present invention uses photo paper as the substrate of the sensor, has good biocompatibility, and can be used for the detection of CRP.

[0032] After adding the antigen solution, when the antibody specifically binds to the antigen, stress will be generated, causing the sensor to deform, and the magnetic domain arrangement of the internal magnetic material will change, resulting in a change in magnetic permeability. On the other hand, the deformation of the sensor will cause a change in the distance between AgNWs on the sensor surface, thus changing the resistance. Therefore, the stress generated by the binding of antibody and antigen on the sensor surface can be converted into electrical and magnetic signals, and CRP can be detected by these two methods of magnetics and electronics, realizing dual-mode detection and effectively improving the functionality of the sensor.

[0033] By changing the types of modified antibodies, different antigens can be detected, increasing the utilization rate of the test strip and expanding the application range.

[0034] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific embodiments of the present invention are limited to this. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the premise of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the patent protection scope determined by the claims submitted by the present invention.

Claims

1. A method for preparing a magnetic paper-based fluorescent biosensor, characterized in that: The following steps are involved: S1. Preparation of paper-based sensor chip S1.1, soaking the photo paper in FeSiB nanofluid, so that the FeSiB nanoparticles are evenly covered on the photo paper; S1.2, then drying the photo paper; S1.3, then immersing the photo paper doped with FeSiB into the fluorescent carbon dot solution so that the fluorescent carbon dots are evenly attached to the photo paper to obtain a paper-based sensor chip; S2. Antibody modification S2.1, mixing the CRP antibody solution and the activation reagent and shaking them to activate the carboxyl group on the CRP antibody into NHS ester, so that the CRP antibody and the amino group on the fluorescent carbon dots are combined; S2.2, then immersing the paper-based sensor chip into the activated CRP antibody solution, and allowing the CRP antibody to fully combine with the fluorescent carbon dots; S2.3, sealing the paper-based sensor chip in the BSA solution to reduce the influence of electrostatic adsorption and steric hindrance effect, and then cleaning the surface of the paper-based sensor chip; S3. Silver nanowire coating: Silver nanowires are coated on the smooth side of the paper-based sensor chip to make the paper-based sensor chip conductive.

2. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: The preparation method of FeSiB nanofluid is as follows: dispersing FeSiB powder into deionized water, and dispersing FeSiB nanoparticles by ultrasonic wave.

3. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 2, characterized in that: The FeSiB nanoparticles were dispersed for 15-30 min using an ultrasonic cell crusher to obtain a uniform FeSiB nanofluid.

4. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: Soak the photo paper in the FeSiB nanofluid for 15-30 minutes.

5. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: Step S1.3 is to immerse the photo paper doped with FeSiB into the fluorescent carbon dot solution for 10-15 hours, and obtain the paper-based sensor chip after natural drying.

6. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: The components of the activation reagent include NHS and EDC.

7. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: In step S2.2, the paper-based sensor chip is immersed in the activated CRP antibody solution and then placed in an environment of 36-38° C. for 1 h-1.5 h to allow the CRP antibody to fully bind to the fluorescent carbon dots.

8. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 7, characterized in that: After the CRP antibody is fully combined with the fluorescent carbon dots, the excess antibody attached to the surface is removed by washing with PBS.

9. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: In step S2.3, the cleaning of the paper-based sensor chip surface is to clean the paper-based sensor chip surface with PBS and deionized water in sequence.

10. The method for preparing a magnetic paper-based fluorescent biosensor according to claim 1, characterized in that: After coating the silver nanowires, the paper-based sensor chip was allowed to dry naturally.

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