Method for preparing a sensitive layer of a biosensor

Abstract

The present disclosure provides a preparation method of a sensitive layer of a biosensor, the biosensor comprising a working electrode, the sensitive layer being formed by drop coating a sensitive layer reagent on a surface of the working electrode, the preparation method comprising: presetting a plurality of point positions on the surface of the working electrode along a length direction of the working electrode; and drop coating the sensitive layer reagent on the plurality of point positions in sequence, the sensitive layer reagent comprising a sensitive substance; wherein a spacing between the plurality of point positions is less than a preset distance, so that the sensitive layer reagents on two adjacent point positions are in contact after being drop coated on the working electrode. According to the present disclosure, a preparation method of a sensitive layer of a biosensor is provided, which can increase the amount of the sensitive substance and reduce the overflow of the sensitive substance from the electrode as much as possible.

Description

Technical Field

[0001] This disclosure relates to the cutting-edge field of new materials, specifically to a method for preparing the sensitive layer of a biosensor. Background Technology

[0002] A biosensor is an analytical device that tightly integrates biological materials, bio-derived materials, or biomimetic materials with physicochemical sensors or sensing microsystems based on optics, electrochemistry, temperature, piezoelectricity, magnetism, or micromechanics. Biosensors typically consist of a biosensitive element and a corresponding transducer, enabling the measurement of specific chemical or biological substances. The biosensitive element in a biosensor is a substance that can bind to the analyte and generate a corresponding signal, such as proteins, enzymes, antigens / antibodies, DNA, and nucleic acids. The biosensitive element usually needs to be coated onto the surface of the sensor's electrode. Electrodes, especially implantable electrodes (such as those in glucose monitoring sensors), are generally small, typically on the micrometer scale. Common coating methods include dip-coating, spraying, and inkjet printing. Dip-coating typically coats the entire surface of the sample immersed in the solution, making targeted coating at specific locations impossible. Spraying can coat a single surface of the sample, often covering a large area, but its coating capability is insufficient for micrometer-sized samples. Inkjet printing has a wide range of applications and generally meets the spotting requirements of the electrode.

[0003] When coating sensitive substances onto micrometer-scale electrodes, insufficient coating can lead to low sensitivity in biosensors, while excessive coating may cause the sensitive substance to overflow the electrode, resulting in poor biosensor consistency. Currently, the coating method for the sensitive layer typically involves drop-coating the sensitive substance onto the working electrode in the form of multiple discrete dots. This drop-coating method is easier to control; however, for sensors with high sensitivity requirements, this method has low utilization of the electrode area, resulting in a smaller area of ​​sensitive substance on the working electrode, thus making it difficult to meet the required sensitivity.

[0004] Therefore, there is a need to provide a method that can improve the sensitivity and consistency of biosensors. Summary of the Invention

[0005] This disclosure was made in view of the above-mentioned state of the prior art, and its purpose is to provide a method for preparing the sensitive layer of a biosensor that can increase the amount of sensitive substance while minimizing the leakage of sensitive substance from the electrode.

[0006] To this end, the first aspect of this disclosure provides a method for preparing a sensitive layer of a biosensor, the biosensor including a working electrode, the sensitive layer being formed by drop-coating a sensitive layer reagent onto the surface of the working electrode, characterized in that the preparation method includes: pre-setting a plurality of points along the length direction of the working electrode on the surface of the working electrode; sequentially drop-coating a sensitive layer reagent, the sensitive layer reagent including a sensitive substance, onto the plurality of points; wherein the spacing between the plurality of points is less than a preset distance such that the sensitive layer reagent on two adjacent points comes into contact after being drop-coated onto the working electrode. In the preparation method disclosed herein, a sensitive layer is formed on the surface of the working electrode by drop-coating a sensitive layer reagent, which is suitable for the processing of micro-sized electrodes. Pre-setting points along the length of the working electrode minimizes the overflow of the sensitive layer reagent compared to methods such as dip-coating, spraying, or dotting more than one point along the width of the working electrode. This reduces the likelihood of the sensitive layer reagent overflowing from the working electrode, thus reducing poor electrode uniformity caused by the overflow of sensitive substances. By drop-coating the sensitive layer reagent at multiple points on the working electrode with a spacing less than a preset distance, adjacent points of the sensitive layer reagent come into contact. Compared to drop-coating the sensitive layer reagent onto the electrode surface in a discrete manner, this method makes better use of the working electrode area, thereby increasing the content of the sensitive layer reagent on the working electrode, increasing the content of sensitive substances in the biosensor, and ultimately improving the sensitivity of the biosensor.

[0007] In the preparation method disclosed herein, optionally, the preset distance is related to the fluidity of the sensitive layer reagent and the volume of the droplets applied each time. In this case, a more suitable preset distance can be obtained based on the properties and volume of the sensitive layer reagent.

[0008] In the preparation method disclosed herein, optionally, at least two drops of the sensitive layer reagent are continuously applied to each point. This reduces the uneven distribution of sensitive substances caused by the coffee ring effect. Specifically, the coffee ring effect is a phenomenon observed when a drop of coffee falls onto a table and, after a period of time, its particles leave a stain. The stain is uneven in color, with the edges being darker than the center, forming a ring-shaped spot. The main causes of the coffee ring effect are the shape of the liquid particles and the direction of their flow. In this case, after applying the first drop of sensitive layer reagent to a point, and before the first drop has completely dried and formed a coffee ring, immediately applying the second drop of sensitive layer reagent can weaken the coffee ring effect of the first drop, thereby making the distribution of sensitive substances at each point more uniform.

[0009] In the preparation method disclosed herein, optionally, the sensitive layer reagent includes a volatile solvent. Thus, after the sensitive layer reagent is drop-coated onto the surface of the working electrode, the solvent can easily evaporate, leaving the sensitive substance on the surface of the working electrode.

[0010] In the preparation method disclosed herein, optionally, multiple rounds of drop coating are performed on the plurality of sites, each round of drop coating comprising sequentially drop coating each site, with a predetermined interval between each round of drop coating to allow the solvent to evaporate or partially evaporate. In this case, performing multiple rounds of drop coating on each site can increase the amount of sensitive material at each site, thereby increasing the content of sensitive material on the surface of the working electrode, thereby improving the sensitivity of the biosensor.

[0011] In the preparation method disclosed herein, optionally, the working electrode is elongated and projected along a direction orthogonal to the surface of the working electrode, such that the maximum width of the sensitive layer reagent before it has diffused at the designated point is no greater than two-thirds of the width of the working electrode. In this case, the possibility of sensitive substances overflowing from the working electrode can be minimized.

[0012] In the preparation method disclosed herein, optionally, the plurality of sites are uniformly arranged along the length direction of the working electrode, the width of the working electrode is 200 μm to 400 μm, the distance between two adjacent sites is 50 μm to 150 μm, and 20 pL to 150 pL of the sensitive layer reagent is drop-coated onto each site. This facilitates the drop-coating of the sensitive layer reagent onto the surface of the working electrode.

[0013] In the preparation method disclosed herein, optionally, after the sensitive layer reagent is drop-coated, it diffuses to form a diffusion region, and the diffusion regions at adjacent sites have overlapping areas, the area of ​​which accounts for 10% to 30% of the area of ​​the diffusion region. This allows for more efficient utilization of the surface area of ​​the working electrode.

[0014] In the preparation method disclosed herein, optionally, the sensitive layer reagent is stirred or shaken before drop-coating the working electrode. This facilitates the dissolution of the sensitive substance in the sensitive layer reagent and makes the distribution of the sensitive substance in the sensitive layer reagent more uniform, thereby improving the uniformity of the distribution of the sensitive substance on the electrode surface.

[0015] In the preparation method disclosed herein, optionally, after drop coating, a semi-permeable membrane reagent is coated on the surface of the working electrode to form a semi-permeable membrane, which completely covers the sensitive layer. Thus, the sensitive substance can be located between the working electrode and the semi-permeable membrane, thereby providing some protection to the sensitive substance and reducing the likelihood of it detaching from the working electrode.

[0016] The second aspect of this disclosure also provides a biosensor comprising a sensitive layer prepared by the preparation method described in the first aspect of this disclosure. The biosensor provided by this disclosure forms a sensitive layer on the surface of the working electrode by drop-coating a sensitive layer reagent, making it suitable for the fabrication of micro-sized electrodes. By pre-setting points along the length of the working electrode, compared to methods such as dip-coating, spraying, or dotting more than one point along the width of the working electrode, the overflow of the sensitive layer reagent from the working electrode can be minimized, thereby reducing poor electrode uniformity caused by the overflow of sensitive substances. By drop-coating the sensitive layer reagent at multiple points on the working electrode with a spacing less than a preset distance, ensuring contact between adjacent points, the area of ​​the working electrode is utilized more effectively than by drop-coating the sensitive layer reagent onto the electrode surface in a discrete manner. This increases the content of the sensitive layer reagent on the working electrode, thereby increasing the content of sensitive substances in the biosensor and ultimately improving the sensitivity of the biosensor.

[0017] According to this disclosure, a method for preparing the sensitive layer of a biosensor that can increase the amount of sensitive substance while minimizing the leakage of sensitive substance from the electrode can be provided. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the usage of the biosensor involved in this disclosure.

[0019] Figure 2 This is a schematic diagram illustrating the structure of the biosensor involved in this disclosure.

[0020] Figure 3 This is a flowchart illustrating a method for preparing the sensitive layer of a biosensor involved in the examples of this disclosure.

[0021] Figure 4 This is a schematic diagram illustrating the spacing between two adjacent points involved in the examples of this disclosure.

[0022] Figure 5 This is a schematic diagram illustrating the droplet size and electrode width involved in the examples of this disclosure.

[0023] Figure 6 This is a schematic diagram illustrating the overlapping areas involved in the examples of this disclosure.

[0024] Figure 7 This is a planar schematic diagram illustrating the sensitive layer involved in the example of this disclosure.

[0025] Figure 8 This is a diagram showing the electrode test results related to an example of this disclosure. Detailed Implementation

[0026] All references cited in this disclosure are incorporated herein by reference in their entirety, as fully illustrated. Unless otherwise defined, the technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0027] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals will be used for the same components, and repeated descriptions will be omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the components or the shapes of the components may differ from actual figures.

[0028] The biosensors disclosed herein can be implantable biosensors. In some examples, the biosensor may include an implantable electrode with a sensitive substance on its surface. In this case, the implantable electrode is often small, on the micrometer scale, and the amount of sensitive substance added to the electrode surface during processing is limited by the electrode size. Therefore, a processing method is needed that can increase the amount of sensitive substance while minimizing leakage of the sensitive substance from the electrode.

[0029] This disclosure relates to a method for preparing a sensitive layer of a biosensor. The preparation method disclosed herein can increase the content of the sensitive substance on the working electrode, thereby improving the sensitivity of the biosensor; and can minimize the leakage of the sensitive substance from the electrode, thereby improving the consistency of the biosensor. The method for preparing the sensitive layer of the biosensor disclosed herein can be referred to as a "processing method," a "spotting method," or simply a "preparation method."

[0030] The following description, in conjunction with the accompanying drawings, illustrates the method for preparing the sensitive layer of the biosensor disclosed herein.

[0031] Figure 1 This is a schematic diagram showing the usage state of the biosensor 1 involved in this disclosure.

[0032] In this embodiment, the biosensor 1 may include a monitoring probe 10 and an electronic system 20 (see...). Figure 1 In some examples, the monitoring probe 10 can be implanted on, for example, the surface of a human body and come into contact with tissue fluid, thereby sensing the signal of the analyte in the tissue fluid to obtain information about the analyte in the human body (e.g., concentration information). In some examples, the monitoring probe 10 can be connected to an electronic system 20. In this case, by contacting the monitoring probe 10 with the tissue fluid or blood of the human body, the biosensor 1 can sense a signal related to the analyte in the tissue fluid, and by transmitting this analyte signal to the electronic system 20, the corresponding analyte information can be obtained. Although Figure 1The location of the biosensor 1 is shown, but this embodiment is not limited to this. For example, the biosensor 1 can also be configured in the abdomen, waist, leg, etc.

[0033] In some examples, the monitoring probe 10 can acquire information about the components (i.e., the analyte) in human body fluids. These components can be at least one of the following: glucose, lactic acid, uric acid, acetylcholine, amylase, bilirubin, cholesterol, human chorionic gonadotropin (hCG), creatine kinase, creatine, creatine anhydride, DNA, fructosamine, glucose, glutamine, growth hormone, hormones, ketone bodies, lactate, oxygen, peroxides, prothrombin, RNA, thyroid-stimulating hormone (TSH), troponin, antigens, and antibodies. Understandably, the types of body fluid components that the monitoring probe 10 can acquire are related to the types of sensitive substances in the biosensor. Sensitive substances are those that can cause the analyte to produce a corresponding signal. Examples include glucose oxidase and antigens.

[0034] Figure 2 This is a schematic diagram illustrating the structure of the biosensor involved in this disclosure.

[0035] In this embodiment, the monitoring probe 10 of sensor 1 may include a working electrode 12 and a sensitive layer 13 (see...). Figure 2 In some examples, the sensitive layer 13 may be disposed on the surface of the working electrode 12. In some examples, the sensitive layer 13 may contain a sensitive substance. In some examples, the sensitive layer 13 may be formed by drop-applying a sensitive layer reagent onto the surface of the working electrode 12. In this case, by implanting the monitoring probe 10 subcutaneously, the sensor 1 can easily sense signals in the tissue fluid related to the analyte.

[0036] In some examples, the monitoring probe 10 may also include a substrate 11. In some examples, the working electrode 12 may be disposed on the substrate 11. In some examples, the substrate 11 may be insulating. In some examples, the substrate 11 may be a flexible substrate 11. In some examples, the substrate 11 may be a flexible insulating substrate 11. In some examples, the flexible insulating substrate 11 may be made of at least one of polyimide (PI), polyethylene terephthalate (PET), parylene, silicone resin, polydimethylsiloxane (PDMS), polyethylene glycol (PEG), or polytetrafluoroethylene resin (Teflon), polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyethylene terephthalate (PEN). Thus, the substrate 11 can possess both flexibility and insulation, thereby helping to reduce discomfort after implantation.

[0037] In some examples, the monitoring probe 10 may also include a counter electrode 15. In some examples, the counter electrode 15 may be made of platinum, silver, silver chloride, palladium, titanium, or iridium. In some examples, the counter electrode 15 may be disposed on both sides of the substrate 11, respectively, along with the working electrode 12.

[0038] In some examples, the monitoring probe 10 may also include a reference electrode 14. In some examples, the working electrode 12 and the reference electrode 14 may be stacked on the same side of the substrate 11. In some examples, an insulating layer may be provided between the working electrode 12 and the reference electrode 14. This facilitates the formation of a stable current path. In some examples, the reference electrode 14 may be made of silver or silver chloride (Ag or Ag / Cl). In some examples, the reference electrode 14 may form a known and fixed potential difference with the tissue fluid or blood. In this case, the potential difference between the working electrode 12 and the tissue fluid or blood can be measured by the potential difference formed between the reference electrode 14 and the working electrode 12, thereby more accurately determining the voltage generated by the working electrode 12. Thus, the electronic system 20 can automatically adjust and maintain the stability of the voltage at the working electrode 12 according to a preset voltage value, further ensuring that the measured current signal accurately reflects the signal value of the analyte.

[0039] In some examples, the monitoring probe 10 may also include a semi-permeable membrane 16. In some examples, the semi-permeable membrane 16 may cover the sensitive layer 13. Thus, the sensitive substance can be located between the working electrode and the semi-permeable membrane, thereby the semi-permeable membrane can provide a certain degree of protection for the sensitive substance and reduce the possibility of the sensitive substance detaching from the working electrode.

[0040] In some examples, the semi-permeable membrane 16 may have selective permeability. In some examples, the semi-permeable membrane 16 can reduce the amount of analyte entering. Specifically, the semi-permeable membrane 16 can effectively reduce the amount of analyte diffusing to the sensitive layer 13 by a certain proportion. In this case, by having the semi-permeable membrane 16 at least cover the sensitive layer 13, it is beneficial to reduce the amount of analyte diffusing to the sensitive layer 13 from the external environment, and it is beneficial to improve the linear range of the biosensor 1 in monitoring the analyte signal. Thus, the magnitude of the current monitored by the sensor can more accurately reflect the concentration of the analyte through the semi-permeable membrane 16. In some examples, the semi-permeable membrane 16 can reduce the amount of analyte entering by a factor of 10 to 200. Thus, the amount of analyte diffusing to the sensitive layer can be reduced by a certain proportion, limiting the amount of analyte in contact with the sensitive layer 13 and controlling it within the linear range of the sensor. In this disclosure, the ratio by which the semipermeable membrane 16 reduces the amount of analyte entering the membrane can refer to the permeability of the analyte. For example, if the semipermeable membrane 16 reduces the amount of analyte entering the membrane by a factor of 10, then the permeability of the analyte in the semipermeable membrane 16 is 10%.

[0041] In some examples, the monitoring probe 10 may include a biocompatible membrane. In some examples, the biocompatible membrane may be located on the semipermeable membrane 16. In some examples, the biocompatible membrane may serve as the outermost layer of the monitoring probe 10 and cover the implanted portion of the monitoring probe 10. Thus, by providing a biocompatible membrane, it is possible to reduce the immune rejection response of the human body to the monitoring probe.

[0042] In some examples, the semi-permeable membrane 16 may also be biocompatible. Therefore, after the biosensor's monitoring probe is implanted in the human body, the immune response to the probe can be reduced, thereby extending the lifespan of the biosensor. In this case, the monitoring probe 10 of the biosensor 1 may not require a separate biocompatible membrane.

[0043] The "Preparation Method" will be described in further detail below. Figure 3 This is a flowchart illustrating a method for preparing the sensitive layer of a biosensor involved in the examples of this disclosure.

[0044] In this embodiment, the preparation method may include: pre-setting multiple points on the surface of the working electrode (step S10) and sequentially applying a sensitive layer reagent to the multiple points so that the sensitive layer reagents on two adjacent points come into contact (step S20).

[0045] In some examples, the preparation of the working electrode 12 may be included. In some examples, the working electrode 12 may be formed from a nanoslurry. In some examples, the nanoslurry is a conductive slurry containing nanoparticles. This improves the conductivity of the working electrode 12, thereby enhancing the sensitivity of the lactic acid sensor 1. In some examples, the nanoparticles may be uniformly distributed within the working electrode 12. This further improves the conductivity of the working electrode 12. In some examples, the nanoparticles may be at least one of gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

[0046] In some examples, the working electrode 12 can be cleaned before drop coating. In some examples, the surface of the working electrode 12 can be cleaned sequentially with anhydrous ethanol and deionized water. In some examples, the cleaned working electrode 12 can also be dried.

[0047] In some examples, the working electrode 12 can be elongated. In some examples, the length of the working electrode 12 can be from 100 μm to 5000 μm. Preferably, the length of the working electrode 12 can be from 200 μm to 400 μm. For example, the length of the working electrode 12 can be 200 μm, 250 μm, 300 μm, 350 μm, or 400 μm. In some examples, the width of the working electrode 12 can be from 50 μm to 400 μm. Preferably, the width of the working electrode 12 can be from 50 μm to 200 μm. For example, the width of the working electrode 12 can be 50 μm, 80 μm, 100 μm, 150 μm, or 200 μm. Thus, the electrode size can be more suitable for use as an implantable electrode.

[0048] Figure 4 This is a schematic diagram illustrating the spacing between two adjacent points involved in the examples of this disclosure. Figure 5 This is a schematic diagram illustrating the droplet size and electrode width involved in the examples of this disclosure. Figure 6 This is a schematic diagram illustrating the overlapping areas involved in the examples of this disclosure. Figure 7 This is a planar schematic diagram illustrating the sensitive layer 13 involved in the example of this disclosure.

[0049] In some examples, multiple points can be pre-set sequentially along the length of the working electrode 12. In some examples, the multiple points can be evenly arranged. In other words, the distance between any two adjacent points can be the same.

[0050] In some examples, the spacing between multiple dots can be less than a preset distance, so that the sensitive layer reagent on two adjacent dots comes into contact after being dropped onto the working electrode 12. In some examples, the sensitive layer reagent diffuses to form a diffusion ring after being dropped onto the working electrode 12, and the diffusion rings of the sensitive layer reagent on two adjacent dots can come into contact. In this case, compared to dropping the sensitive layer reagent onto the electrode surface in a discrete dot pattern, the area of ​​the working electrode can be utilized more effectively, thereby increasing the content of the sensitive layer reagent on the working electrode, thus increasing the content of the sensitive substance in the biosensor, and consequently improving the sensitivity of the biosensor.

[0051] In some examples, the distance between two adjacent points can range from 50 μm to 150 μm. For example, the distance between two adjacent points can be 50 μm, 75 μm, 100 μm, 125 μm, or 150 μm. See also Figure 4 The distance between two adjacent points can be schematically described by the distance between points A and B.

[0052] In some examples, the preset distance can be related to the fluidity of the sensitive layer reagent and the volume of each droplet. In this case, a suitable preset distance can be obtained based on the fluid properties of the sensitive layer reagent and the droplet volume. In some examples, the preset distance can be determined in advance by performing a droplet application experiment using the sensitive layer reagent and electrodes.

[0053] In some examples, multiple points can be evenly arranged along the length of the working electrode 12. This facilitates the setup and execution of the drop coating operation.

[0054] In some examples, the sensitive layer reagent may include a sensitive substance. A sensitive substance is a substance that can cause the analyte to produce a corresponding signal. Examples include glucose oxidase and antigens. Thus, the analyte can be detected by the biosensor 1.

[0055] In some examples, the sensing layer reagent may also include a solvent. In some examples, the solvent may be volatile. Therefore, after the sensing layer reagent is drop-coated onto the working electrode surface, the solvent can easily evaporate, leaving the sensitive substance on the working electrode surface. In some examples, the solvent may also be less volatile. In this case, after drop-coating, methods such as natural drying, vacuuming, or baking can be used to promote the drying of the sensing layer reagent, ensuring that the sensitive substance remains on the working electrode surface.

[0056] In some examples, the sensing layer reagent may also include a cross-linking agent. In some examples, the cross-linking agent facilitates the cross-linking of the sensing material to the surface of the working electrode. In some examples, the sensing layer 13 may be formed by drop-coating the sensing layer reagent onto the working electrode 12 and then cross-linking it. This facilitates better fixation of the sensing material to the surface of the working electrode 12.

[0057] In some examples, graphene, porous titanium dioxide, or conductive organic salts may also be added to the sensitive layer reagent. In this case, when the sensitive substance is a redox enzyme, it can better promote the enzymatic reaction.

[0058] In some examples, the sensitive layer reagent can be stirred or shaken before drop-coating the working electrode 12. This facilitates the dissolution of the sensitive substance in the sensitive layer reagent and makes the distribution of the sensitive substance in the sensitive layer reagent more uniform, thereby improving the uniformity of the distribution of the sensitive substance on the electrode surface.

[0059] In some examples, the sensitive layer reagent can be stirred or shaken, and the supernatant can be centrifuged and used as the sensitive layer reagent for drop coating. In this case, undissolved substances in the sensitive layer reagent can be reduced, thereby reducing the likelihood of clogging at the outlet of the drop coating instrument.

[0060] In some examples, at least two drops of the sensitive layer reagent can be continuously applied to each point. For example, two, three, four, or five drops of the sensitive layer reagent can be continuously applied to each point. This can reduce the effect of uneven distribution of sensitive material on the electrode surface caused by the coffee ring effect. Specifically, the concept of the coffee ring effect comes from an everyday phenomenon: when a drop of coffee falls on a table, its particulate matter leaves a stain on the table after a period of time. The stain is uneven in color, with the edges being darker than the center, forming a ring-shaped spot. The main reason for the coffee ring effect is the influence of the shape of the liquid droplets and the direction of flow. In this case, after applying the first drop of sensitive layer reagent to a point, before the first drop has completely dried and formed a coffee ring, immediately applying the second drop of sensitive layer reagent can reduce the coffee ring effect of the first drop, thereby making the distribution of sensitive material at each point more uniform.

[0061] In some examples, multiple rounds of drop application can be performed on multiple sites. In some examples, a single round of drop application can refer to applying the sensitive layer reagent to each site sequentially. In other words, multiple rounds of drop application means repeating the drop application operation (applying the sensitive layer reagent to all sites sequentially) after applying the sensitive layer reagent to all sites sequentially. In some examples, each round of drop application can be performed on pre-defined sites. In other words, the drop application location does not need to be changed in each round; the droplets are always dropped onto the pre-defined sites.

[0062] In some examples, a predetermined time interval can be allowed between each round of drop-coating to allow the solvent to evaporate or partially evaporate. Preferably, a predetermined time interval can be allowed between each round of drop-coating to allow the solvent to evaporate completely. In this case, allowing the solvent of the sensitive layer reagent from the previous round of drop-coating to evaporate before performing the next round of drop-coating can reduce the possibility of sensitive material overflowing from the working electrode due to excessive droplets at a single point.

[0063] In some examples, projecting along a direction orthogonal to the surface of the working electrode 12, the maximum width of the sensitive layer reagent droplet before it diffuses at the application point is no greater than two-thirds of the width of the working electrode. In other examples, this means that the width of the droplet before it diffuses at the working electrode 12 is no greater than two-thirds of the width of the working electrode. See also Figure 5 The width of a droplet of sensitive reagent added to the working electrode 12 before it diffuses can be represented by L1, and the width of the working electrode can be represented by L2, i.e., L1≤(2 / 3)*L2. This minimizes the occurrence of sensitive substances overflowing from the electrode.

[0064] In some examples, 20 pL to 150 pL of the sensitive layer reagent can be applied to one site at a time. For example, 20 pL, 50 pL, 80 pL, 100 pL, 130 pL, or 150 pL of the sensitive layer reagent can be applied to one site at a time.

[0065] In some examples, an automated dispensing applicator can be used to perform pre-defined spot application and dispensing operations on the working electrode 12. In some examples, the automated dispensing applicator may have a CCD positioning system that positions the working electrode 12 and pre-defined spots on its surface. This allows for the dispensing of the sensitive layer reagent onto the surface of the working electrode 12 according to a pre-set program. In some examples, the dispensing program may include the volume of each dispensing, the number of drops per spot, the distance moved after each spot is applied, and the number of dispensing cycles. In some examples, a picoliter dispensing applicator can be used for the dispensing operation.

[0066] In some examples, the reagent in the sensitive layer can diffuse after being dropped onto the substrate, forming a diffusion region M. The diffusion regions M at adjacent sites can overlap with each other (see [reference needed]). Figure 6 The area of ​​the overlapping region N can account for 10% to 30% of the area of ​​the diffusion region M. Preferably, the area of ​​the overlapping region N can account for 20% of the area of ​​the diffusion region M. This allows for more efficient utilization of the surface area of ​​the working electrode.

[0067] In some examples, the diffusion region M can refer to the area formed by diffusion after one round of drop application to a single point. For instance, if each round of drop application involves continuously applying 3 drops of the sensitive layer reagent to a single point, then the diffusion region M refers to the area formed by diffusion after continuously applying 3 drops of the sensitive layer reagent to a single point.

[0068] In some examples, multiple sites are arranged in a row along the length of the working electrode 12. In some examples, the sensitive layer reagents on adjacent sites can be in contact with each other.

[0069] In some examples, after the drop-coating is completed, the sensing layer reagent forms a sensing layer 13 on the surface of the working electrode 12. In some examples, after the sensing layer reagent is dropped onto all points of the working electrode 12, a linear sensing layer 13 is formed on the surface of the working electrode 12 (see [reference]). Figure 7 In this case, the length of the working electrode can be utilized more fully, thereby increasing the amount of sensitive substance on the working electrode and improving the sensitivity of the working electrode; and the width of the sensitive layer can be compressed, reducing the leakage of reagent from the sensitive layer onto the working electrode, thereby improving the consistency of the electrode.

[0070] In some examples, after drop coating, a semi-permeable membrane reagent can be coated onto the surface of the working electrode 12 to form a semi-permeable membrane 16, which completely covers the sensitive layer 13. In some examples, a semi-permeable membrane reagent can also be coated onto the surface of the sensitive layer 13 to form a semi-permeable membrane 16. This allows the sensitive layer to be encapsulated by the working electrode and the semi-permeable membrane, thereby protecting the sensitive layer and reducing the likelihood of sensitive substances escaping from the biosensor.

[0071] The embodiment also provides a biosensor 1 including a sensitive layer prepared by the above-described preparation method. The description of the working electrode 12 and the sensitive layer 13 can be found in the foregoing content and will not be repeated here. The biosensor 1 provided in this embodiment forms a sensitive layer on the surface of the working electrode by drop-coating a sensitive layer reagent, making it suitable for processing micro-sized electrodes. By pre-setting points along the length of the working electrode, compared to methods such as dip-coating, spraying, or dotting more than one point along the width of the working electrode, the overflow of the sensitive layer reagent from the working electrode can be minimized, thereby reducing poor electrode uniformity caused by the overflow of sensitive substances. By drop-coating the sensitive layer reagent at multiple points on the working electrode with a spacing less than a preset distance, allowing the sensitive layer reagent at adjacent points to contact each other, the area of ​​the working electrode can be utilized more effectively than by drop-coating the sensitive layer reagent onto the electrode surface in a discrete manner. This increases the content of the sensitive layer reagent on the working electrode, thereby increasing the content of sensitive substances in the biosensor and ultimately improving the sensitivity of the biosensor.

[0072] The following describes in detail the preparation method of the sensitive layer of the biosensor provided in this disclosure with reference to the embodiments, but it should not be construed as limiting the scope of protection of this disclosure.

[0073] Example:

[0074] 1. Prepare a plate of electrodes (an electrode array consisting of 8*24 electrodes). Clean all electrodes with anhydrous ethanol for 3-5 minutes, then clean with deionized water for 3-5 minutes, and bake at 40-60℃ for 2 hours.

[0075] 2. Prepare the sensitive layer reagent (glucose oxidase, cross-linking agent, metal polymer, buffer solution) and store it in a refrigerator at about 4°C until use.

[0076] 3. Take out an appropriate amount of the sensitive layer reagent, shake for 1-3 minutes, centrifuge at 15000 rpm for 5 minutes, and take about 200 μL of the supernatant for later use.

[0077] 4. The CCD positioning system of the drop coater is used to position the electrodes. Each electrode has 18 preset points along its length, and the distance between two points is designed to be 100μm.

[0078] 5. Using a picoliter dropper, take 5 μL of the sensitive substance.

[0079] 6. Apply the sensitive substance to each electrode in sequence, with 3 drops of the substance applied to each point, each droplet being 75pl.

[0080] 7. After the first coat of coating is completed for each electrode, wait 5 minutes before repeating step 6.

[0081] 8. Move to the next electrode for drop coating, as in steps 6 and 7.

[0082] 9. After the electrodes and sensitive substances have been cross-linked, the electrode array on one plate is laser-cut into individual electrodes.

[0083] 10. A semi-permeable membrane reagent (a mixed alcohol solution of polyvinylpyridine and Nafion) is coated onto the electrode surface by dip-coating to form a biocompatible semi-permeable membrane.

[0084] 11. This process yields 8*24 monitoring probes.

[0085] 12. Three monitoring probes were selected from each of the middle, upper, lower, left, and right sections of the electrode array for testing. Glucose solutions with concentrations of 0.00, 2.20, 5.00, 10.00, 15.00, 20.00, and 25.00 mmol / L were used for the tests, and the current values ​​of the monitoring probes at different concentrations were recorded. The test results are shown in Table 1 below.

[0086] Table 1 Test Results

[0087]

[0088]

[0089] Figure 8 This is a graph showing the electrode test results involved in the examples of this disclosure. (From Table 1 above and...) Figure 8 It can be seen that the monitoring probe obtained by the preparation method in this embodiment has high linearity of current within a certain range in the test liquid, and R 2 The efficiency reached 0.99, with a sensitivity of approximately 1.3 nA / (mmol / L), and the coefficient of variation (CV) for different monitoring probes was 1.77%. This indicates that the electrode with a sensitive layer prepared in this embodiment exhibits high consistency and sensitivity.

[0090] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the foregoing description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations shall fall within the scope of the present disclosure.

Claims

1. A method for preparing a sensitive layer of a biosensor, the biosensor comprising a working electrode, wherein the sensitive layer is formed by drop-coating a sensitive layer reagent onto the surface of the working electrode, characterized in that, The preparation method includes: Along the length of the working electrode, multiple points are pre-set on the surface of the working electrode; A sensitive layer reagent is sequentially applied to the plurality of points, the sensitive layer reagent comprising a sensitive substance; The spacing between the multiple points is less than a preset distance, ensuring that the sensitive layer reagent at adjacent points comes into contact after being applied to the working electrode. At least two drops of the sensitive layer reagent are continuously applied to each point, with the next drop applied before the previous drop has completely dried. The working electrode is elongated, and when projected along a direction orthogonal to its surface, the maximum width of the sensitive layer reagent applied to the point before diffusion is no greater than two-thirds of the width of the working electrode. After application, the sensitive layer reagent diffuses to form a diffusion area, with overlapping areas between adjacent points. The area of ​​the overlapping area accounts for 10% to 30% of the area of ​​the diffusion area.

2. The preparation method according to claim 1, characterized in that, The preset distance is related to the fluidity of the sensitive layer reagent and the volume of the droplet applied each time.

3. The preparation method according to claim 2, characterized in that, The sensitive layer reagent includes a volatile solvent.

4. The preparation method according to claim 3, characterized in that, Multiple rounds of drop coating are performed on the multiple points, each round of drop coating includes sequentially drop coating each point, and a predetermined time interval is given between each round of drop coating to allow the solvent to evaporate or partially evaporate.

5. The preparation method according to claim 1, characterized in that, The plurality of points are evenly arranged along the length of the working electrode, the width of the working electrode is 200μm to 400μm, the distance between two adjacent points is 50μm to 150μm, and 20pL to 150pL of the sensitive layer reagent is dropped onto the points each time.

6. The preparation method according to claim 1, characterized in that, Before drop-coating the working electrode, the reagent in the sensitive layer is stirred or shaken.

7. The preparation method according to claim 1, characterized in that, After the drop coating is completed, a semi-permeable membrane reagent is coated on the surface of the working electrode to form a semi-permeable membrane that completely covers the sensitive layer.

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