A biosensor and an active material layer thereof and a method of manufacturing the same
By using a batch coating method, the problems of wetting, spreading, and boundary overflow caused by ink droplet overlap in inkjet printing are avoided. This enables high-precision patterned deposition of the active material layer of the biosensor, solves the problems of large fluctuations in sensor detection performance and difficulty in standardized mass production, and improves the stability and measurement accuracy of the sensor.
Patent Information
- Application Number
- CN202610877212.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-16
- Publication Date
- 2026-08-25
AI Technical Summary
In the continuous coating process of inkjet printing, the wet overlap of ink droplets is easily affected by interfacial tension, resulting in wetting, spreading and boundary overflow. This leads to poor consistency in the pattern accuracy, film thickness and site density of the active material layer, causing large fluctuations in sensor detection performance and making it difficult to standardize mass production.
By employing a batch coating method, multiple sensing units formed in a single batch are spaced apart, and sensing units formed in multiple batches are connected to avoid random droplet diffusion, accurately control the forming shape and position of the sensing units, and solve the problem of uncontrolled forming in sequential continuous inkjet coating by splicing multiple batches with staggered filling points.
It achieves high-precision control of the shape and position of the sensing unit, improves the stability and consistency of the sensor, reduces the scrap cost of mass production, supports standardized mass production, and expands the detection range and measurement accuracy.
Smart Images
Figure CN122631730A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of electrochemical analysis and detection technology, specifically to a biosensor, its active material layer, and its preparation method. Background Technology
[0002] Biosensors are devices that utilize the specific recognition capabilities of biomolecules to convert the concentration of a analyte into a detectable signal, and are widely used in the medical and health fields. Compared with in vitro monitoring technologies, implantable flexible sensors can achieve continuous, real-time, and accurate monitoring of physiological information in vivo, showing broad application prospects in medical diagnosis, health management, and drug development. For example, continuous glucose monitoring (CGM) has been commercially applied in diabetes management, revolutionizing chronic disease management. However, existing CGM sensors still suffer from problems such as frequent replacement and easy loss or detachment of bioactive materials, seriously affecting the long-term stability and monitoring accuracy of the sensors. Therefore, improving the immobilization technology of active materials in sensors is key to the preparation of high-efficiency biosensors.
[0003] The active material layer (containing enzymes, antibodies, nucleic acids, cell receptors, etc.) is the core structure of a biosensor. It detects target molecules through specific recognition and signal conversion. Therefore, the amount and area of active material coating in the working area of the sensor are crucial, directly determining the sensor's key performance characteristics such as sensitivity, response speed, and long-term stability.
[0004] To meet the miniaturization demands of sensors, device sizes need to be controlled at the millimeter level or even smaller. The fabrication process must simultaneously possess characteristics such as high precision (micrometer-level resolution and accurate positioning), digitalization (flexibility and programmability), non-contact (avoiding substrate damage or cross-contamination caused by physical contact), low loss, and gentle processing (bio-friendly). Inkjet printing technology, due to its advantages of high precision, process control, and wide applicability to a wide range of materials, is widely used for coating bioactive substances onto the working areas of sensors.
[0005] In the coating process of microscale bioactive material layers, inkjet printing technology is typically used to deposit the active material as multiple discrete points on the sensor's working area. This coating process is mature and easy to control. Compared to isolated coating points, connecting the coating points facilitates the dispersion and transmission of external forces, while also constraining and supporting the active material layer, thereby extending the sensor's lifespan. For the fabrication requirement of interconnected coating points, sequential coating is a common method to achieve the connection of active materials. However, in the continuous coating process of inkjet printing, the liquid active functional material in the overlapping area of adjacent coating droplets is prone to random wetting, lateral spreading, and boundary overflow under the drive of gas-liquid-solid three-phase interfacial tension. This leads to blurred coating area boundaries, uncontrolled distribution of effective coating area and film thickness, and difficulty in achieving high-precision patterned deposition. This phenomenon directly results in significant dispersion in the effective active site density and signal intensity of mass-produced sensor sensing units, manifesting as large fluctuations in sensor sensitivity, detection limit, and response consistency, severely restricting the large-scale, standardized, and highly consistent commercial mass production of sensors. Summary of the Invention
[0006] The main technical problem solved by this invention is that the continuous coating process of inkjet printing is susceptible to wetting, spreading, and boundary overflow due to the wet overlap of ink droplets and the influence of interfacial tension. This results in poor consistency of the pattern accuracy, film thickness, and site density of the active material layer, causing large fluctuations in sensor detection performance and making it difficult to standardize mass production.
[0007] Based on one of the above objectives, some embodiments of this application provide a method for preparing an active material layer of a biosensor. The biosensor includes a substrate, and the active material layer is formed by coating an active functional reagent onto the surface of the substrate. The steps include:
[0008] Multiple coating point groups are preset on the surface of the substrate. Each group of coating point groups corresponds to a single coating batch, and the coating points in each group of coating point groups are set at intervals.
[0009] Select any one of the coating point groups as the first coating point group, and sequentially coat all the coating points in the first coating point group with the active functional reagent to form the first group of sensing units;
[0010] The remaining coating point groups are selected sequentially and coated in staggered batches to form multiple sets of sensing units. Multiple sensing units in the multiple sets of sensing units are connected, any two adjacent sensing units are in contact, and different coating batches are formed.
[0011] In some optional embodiments, the sensing units formed in each subsequent coating batch are distributed at the gap positions of the sensing units formed in the preceding coating batch.
[0012] In some alternative embodiments, the sensing unit formed in a subsequent coating batch comes into contact with the sensing unit formed in an adjacent coating batch.
[0013] In some alternative embodiments, the plurality of coating point groups are arranged in a single row, a single column, or an array;
[0014] The array can be a rectangular array or a cellular array.
[0015] In some optional embodiments, the active functional reagent includes active substance reagents and auxiliary reagents for identifying and detecting target analytes.
[0016] In some optional embodiments, the active functional reagents corresponding to different coating batches are different; and / or,
[0017] The sensing units formed in different coating batches have at least two morphologies and / or two sizes, while the sensing units within the same coating batch have the same morphology and size.
[0018] In some alternative embodiments, the sensing unit satisfies at least one of the following conditions (1)-(3):
[0019] (1) The diameter of each sensing unit ranges from 0.1 μm to 2000 μm;
[0020] (2) The thickness of each sensing unit ranges from 0.1 μm to 200 μm;
[0021] (3) The total coating volume of the multiple sensing units ranges from 10 pL to 1 μL.
[0022] Based on one of the above objectives, some embodiments of this application provide an active material layer for a biosensor, which is formed by coating an active functional reagent onto a substrate surface for identifying and detecting target analytes. The biosensor includes a plurality of interconnected sensing units, with any two adjacent sensing units in contact with each other, and the plurality of sensing units having at least two sizes.
[0023] In some optional embodiments, at least one sensing unit of a different size is provided between any two adjacent sensing units of the same size.
[0024] In some optional embodiments, the sensing unit includes a main sensing unit and a secondary sensing unit of different sizes, wherein the size of the main sensing unit is larger than the size of the secondary sensing unit, and a secondary sensing unit is provided between any two adjacent main sensing units.
[0025] In some alternative embodiments, the plurality of the sensing units are arranged in a single row, a single column, or an array;
[0026] The array can be a rectangular array or a cellular array.
[0027] In some optional embodiments, the plurality of sensing units are sequentially coated in multiple coating batches, the sensing units formed in the same coating batch are arranged at intervals along the surface of the substrate, and any adjacent sensing units come from different coating batches, and the sensing units within the same coating batch are of the same size.
[0028] To achieve one of the above objectives, some embodiments of this application provide a method for preparing a biosensor, the steps of which include the preparation method of the active material layer as described in any of the preceding claims.
[0029] In some alternative embodiments, a flow-limiting agent is further coated onto the surface of the substrate to form an outer membrane layer that covers the active material layer.
[0030] Based on one of the above objectives, some embodiments of this application provide a biosensor, including a substrate and an active material layer prepared by the method described in any of the preceding claims, or an active material layer as described above, wherein the active material layer is formed by coating the surface of the substrate with an active functional reagent, or the biosensor is made using the biosensor preparation method described above.
[0031] The preparation method of the active material layer of the biosensor according to the above embodiments employs a single-batch, spaced-point coating process. This avoids mutual wetting and overflow deformation of adjacent liquid reagents, solving the molding runaway problem of sequential continuous inkjet coating. This allows for precise control of the molding shape and position of each sensing unit. Through multi-batch staggered-point splicing molding, the process is simple, controllable, and highly stable. Furthermore, each sensing unit is molded independently with controllable shape, significantly reducing defects such as coating blurring, uneven thickness, and site failure. This effectively improves the product yield, reduces scrap costs and quality control difficulties in mass production, and ultimately enables the stable production of highly consistent biosensors, facilitating standardized mass production. In addition, different coating batches can flexibly select the same or different active functional reagents according to detection requirements, enabling the preparation of sensors with single detection functions or multifunctional composite sensors in a one-step process, thereby improving process adaptability.
[0032] The active material layer of the biosensor according to the above embodiment includes a plurality of interconnected sensing units, adjacent sensing units are in contact, and the plurality of sensing units have at least two sizes, so that the active material layer can be matched to detect different concentration ranges of analytes, thereby broadening the detection range of the biosensor; at the same time, the multi-size sensing units can form a differential detection structure, thereby canceling environmental common-mode interference and improving measurement accuracy. Attached Figure Description
[0033] Figure 1 This is a structural flowchart of a method for preparing the active material layer of a biosensor in one embodiment of this application;
[0034] Figure 2 This is a schematic diagram of a structure in which multiple sensing units are arranged in a single row in one embodiment of this application, wherein: a) is a schematic diagram of a structure in which multiple sensing units are tangent in one example; b) is a schematic diagram of a structure in which multiple sensing units intersect in one example; c) is a schematic diagram of a structure in which multiple sensing units are tangent in another example; d) is a schematic diagram of a structure in which multiple sensing units of the same size are tangent in one example; e) is a schematic diagram of a structure in which multiple sensing units of the same size intersect in one example; and f) is a schematic diagram of a structure in which multiple sensing units of the same size both intersect and are tangent in yet another example.
[0035] Figure 3 This is a schematic diagram of the structure in the first embodiment of this application, showing multiple sensing units arranged in a rectangular array.
[0036] Figure 4 This is a schematic diagram of the structure in the second embodiment of this application, showing multiple sensing units arranged in a rectangular array.
[0037] Figure 5 This is a schematic diagram of the structure in the third embodiment of this application, showing multiple sensing units arranged in a rectangular array.
[0038] Figure 6 This is a schematic diagram of the structure in the fourth embodiment of this application, showing multiple sensing units arranged in a rectangular array.
[0039] Figure 7 This is a schematic diagram of the structure in the fifth embodiment of this application, showing multiple sensing units arranged in a rectangular array.
[0040] Figure 8 This is a schematic diagram of a structure in which multiple sensing units are distributed in a honeycomb array in one embodiment of this application;
[0041] Figure 9 This is a schematic diagram of a structure in which multiple sensing units are distributed in a honeycomb array in another embodiment of this application;
[0042] Figure 10 This is a structural flowchart of a method for preparing a biosensor in one embodiment of this application;
[0043] Figure 11 This is a schematic diagram of the coating of the sensing unit corresponding to Embodiment 1 of this application;
[0044] Figure 12The images shown are physical diagrams of the electrodes corresponding to Embodiment 1 and the comparative example of this application, wherein a is a physical diagram of the first batch of electrodes in Embodiment 1; b is a physical diagram of the second batch of electrodes in Embodiment 1; c is a physical diagram of the third batch of electrodes in Embodiment 1; and d is a physical diagram of the electrodes of the comparative example.
[0045] Figure 13 The graph shows the in vitro electrochemical test results of the monitoring probes for the electrodes corresponding to the embodiments and comparative examples of this application. Detailed Implementation
[0046] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. Similar elements in different embodiments are referred to by associated similar element reference numerals. In the following embodiments, many details are described to facilitate a better understanding of the invention. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other elements, materials, or methods. In some cases, certain operations related to the present invention are not shown or described in the specification. This is to avoid obscuring the core parts of the invention with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.
[0047] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.
[0048] The serial numbers assigned to components in this document, such as "first" and "second," are used only to distinguish the described objects and have no sequential or technical meaning. The terms "connection" and "linkage" used in this invention, unless otherwise specified, include both direct and indirect connections (linkages).
[0049] Some embodiments of this application provide a method for preparing an active material layer for a biosensor. This method employs a batch coating approach, with multiple sensing units formed in a single batch spaced apart. Ultimately, multiple sensing units formed in multiple batches are connected, meaning adjacent sensing units are in contact, but not formed in the same batch. This allows sufficient drying time at the points where the active functional reagent coating points are connected, avoiding uncontrolled coating area due to random droplet diffusion. Throughout the process, it prevents adjacent liquid reagents from wetting each other, overflowing, or deforming, solving the problem of uncontrolled forming in sequential continuous inkjet coating. This allows for precise control of the forming shape and position of each sensing unit. The size, position, and coating sequence of the sensing units are flexibly adjustable, facilitating multi-path pattern design. Furthermore, it allows for flexible selection of active functional reagents with the same or different components, ensuring improved activity, stability, and accuracy of microsensors and supporting large-scale production, while also enhancing process adaptability. In addition, by adjusting the size, number, and arrangement of the sensing units, the overall shape, porosity, and effective reaction area of the active area of the sensing units on the substrate can be easily programmed, thereby fine-tuning the sensor's sensitivity, response time, and other performance parameters. This provides a guarantee for improving the activity, stability, and accuracy of micro sensors and for their mass production.
[0050] Biosensors include, but are not limited to, electrochemical or optical sensors. These biosensors include a substrate, which can be a conductive or insulating substrate. The conductive substrate can serve as the working electrode; for example, the substrate can be a carbon substrate (including at least one of activated carbon, carbon nanofibers, graphene, and carbon nanotubes) or a metal substrate (including at least one of nickel foam, Au, and Ag) used to fabricate the working electrode. The insulating substrate can be a polymer film or a paper-based substrate used to support or carry the working electrode, etc. The active material layer is formed by coating an active functional reagent onto the surface of the substrate.
[0051] Furthermore, the substrate has a working area, and the active material layer covers (including full or partial coverage) the working area.
[0052] Please see Figure 1 The method for preparing the active material layer of a biosensor includes the following steps:
[0053] S101: Multiple coating point groups are preset on the surface of the substrate. Each coating point group corresponds to a single coating batch, and the coating points in each coating point group are set at intervals.
[0054] S102: Select any one of the coating point groups as the first coating point group, and sequentially coat all the coating points in the first coating point group with active functional reagents to form the first group of sensing units.
[0055] S103: Select the remaining coating point groups in sequence and coat them in batches in a staggered manner to form multiple sets of sensing units. Multiple sensing units in the multiple sets of sensing units are connected, any two adjacent sensing units are in contact, and different coating batches are formed.
[0056] In this application, "contact" can be understood as intersecting or tangent, where intersecting means that at least a portion of the area between the two sensing units overlaps. For example... Figure 2 As shown in a), c), and d), two adjacent sensing units in a plurality of sensing units are arranged tangentially, as follows: Figure 2 As shown in b) and e), two adjacent sensing units in a plurality of sensing units are arranged intersectingly, as follows: Figure 2 As shown in f), two adjacent sensing units in the multiple sensing units are arranged to both intersect and be tangent.
[0057] In this application, "misaligned coating" refers to a coating pattern in which the positions of coating areas formed in different batches are misaligned, partially overlapping, or partially missing. For example, such as... Figure 2 As shown in a), when coating in two batches, the coating points of the second batch and the first batch are staggered and tangent. Figure 2 As shown in b), when coating in two batches, the coating points of the second batch and the first batch are staggered and partially overlap (i.e. intersect).
[0058] In some embodiments, the coating process for the active functional reagent includes, but is not limited to, inkjet printing and aerosol jet printing.
[0059] In some embodiments, the number of multiple coating point groups can be two or more, and the corresponding coating batch can also be two or more times. For example, the coating batch can be two, three, or four times, which can be selected according to the coating requirements, and adjacent points in the coating point group corresponding to each batch can be arranged at intervals.
[0060] In some examples, please refer to Figure 2(b) As shown in the diagram, multiple sensing units are numbered sequentially from left to right as 1 to 11. The coating points corresponding to these 11 sensing units are divided into two groups: the first group includes 6 points (1, 3, 5, 7, 9, 11), and the second group includes 5 points (2, 4, 6, 8, 10). Coating is performed in two batches: first, the 6 points (1, 3, 5, 7, 9, 11) are coated, then the 5 points (2, 4, 6, 8, 10) are coated. Points 1, 3, 5, 7, 9, and 11 are spaced apart, while points 2, 4, 6, 8, and 10 are spaced apart and located in the gaps between points 1, 3, 5, 7, 9, and 11. Finally, multiple interconnected sensing units are coated, with any two adjacent sensing units intersecting. Alternatively, the coating order can be reversed, i.e., the second group of coating points is coated first, followed by the first group. Alternatively, the 11 points can be divided into three groups of points to be coated. For example, the first group of points to be coated includes 1, 4, 6, and 9; the second group includes 3, 5, 7, and 10; and the third group includes 2, 8, and 11. Any one of these groups can be selected as the first group of points to be coated, and then the remaining two groups of points can be coated, such as in the order of the first group of points, the second group of points, and the third group of points.
[0061] In some embodiments, a biosensor may include a single biosensor unit or multiple (e.g., 2-20,000) biosensor units, which may be arranged in a single row, single column, or array. When coating a biosensor composed of multiple biosensor units, multiple coating point groups may be pre-set on one biosensor unit. After coating that unit, other biosensor units are coated sequentially, thus completing the coating of one sensor unit in one pass. Alternatively, multiple coating point groups may be pre-set on all biosensor units, and coating may be performed in batches according to different groups, thereby achieving coating of all biosensor units in one pass. Alternatively, a row or column of an array of multiple biosensor units may be coated in one pass.
[0062] It should be noted that the term "one round" refers to a complete coating process, after which several interconnected sensing units are formed. A single coating process consists of multiple coating batches. For example, if a single coating round corresponds to the fabrication of a single sensing unit, it means that all coating points of the sensing unit are grouped and coated sequentially in multiple batches to form all sensing units of that unit; after completing a single round of processing, multiple rounds of processes are carried out sequentially to complete the coating fabrication of the remaining sensing units.
[0063] In some embodiments, the sensing units formed in each subsequent coating batch are distributed at the gap positions between the sensing units formed in the previous coating batch. For example... Figure 2As shown in b), the 11 points are divided into two groups. The first group includes 6 points: 1, 3, 5, 7, 9, and 11. The second group includes 5 points: 2, 4, 6, 8, and 10. In the second group, point 2 is located between points 1 and 3; point 4 between points 3 and 5; point 6 between points 5 and 7; point 8 between points 7 and 9; and point 10 between points 9 and 11. This means that adjacent sensing units formed belong to different batches. Alternatively, when dividing the coating into three batches, the first batch can be coated with a large gap, the second batch can be staggered within the gaps of the first batch, and the third batch can continue to be coated with staggered layers to form a continuous structure. This process can be repeated to coat points divided into four or more batches.
[0064] It should be noted that the subsequently formed sensing units are distributed in the gaps between the preceding sensing units, and the subsequent sensing units and the two preceding sensing units that form the gaps can be arranged at intervals, tangent to each other, or intersecting.
[0065] Of course, in other embodiments, the sensing units formed in each subsequent coating batch may not correspond to the gap positions of the sensing units formed in the previous coating batch. For example... Figure 2 As shown in b), the 11 points are divided into three groups. The first group of coating points includes 1, 3, and 5; the second group of coating points includes 7, 9, and 11; and the third group of coating points includes 2, 4, 6, 8, and 10. The second group of coating points and the first group of coating points are distributed alternately from left to right. The third group of coating points are located in the gap between the first group of coating points and the second group of coating points.
[0066] It should be noted that "preceding" is understood as all coating batches preceding the current coating batch, and does not specifically refer to the adjacent preceding coating batch.
[0067] In some embodiments, the sensing unit formed in a subsequent coating batch comes into contact with the sensing unit formed in an adjacent coating batch. For example, as... Figure 2 As shown in c), the sensing unit formed in the second coating batch is in contact with the sensing unit formed in the first coating batch. The sensing unit of the first coating batch is formed at four points: 1, 4, 7, and 10. The sensing unit of the second coating batch is formed at three points: 2, 5, and 8. The sensing unit of the third coating batch is formed at three points: 3, 6, and 9.
[0068] In some embodiments, multiple coating point groups are arranged in a single row, single column, or array, wherein the array is a rectangular array or a cellular array. For example... Figure 2 As shown, multiple coating sites are arranged in a single row. Figures 3 to 7 As shown, multiple coating points are distributed in a rectangular array. Figure 8 and Figure 9As shown, multiple coating points are distributed in a honeycomb array. This array distribution maximizes the number of sensing units within a limited area, improving space utilization and thus maximizing the density and signal uniformity of the reaction points. This effect is particularly optimal with a honeycomb array distribution.
[0069] In some embodiments, the active functional reagent includes an active substance reagent for identifying and detecting the target analyte and auxiliary reagents. The inclusion of auxiliary reagents can prevent diffusion interference between different enzyme systems.
[0070] In some embodiments, the active substance reagent includes bioactive substances and / or electron transporters. Bioactive substances include enzymes, antibodies and antigens, nucleic acids, nucleic acid aptamers, cells, fluorescent dyes, and fluorescent proteins; electron transporters include metal complexes / chelates, carbon nanotubes, graphene, and conductive polymers, and the active substance reagent may include one or more of the above.
[0071] In some embodiments, different coating batches correspond to different active functional reagents, thereby enabling the identification of multiple target analytes in the same working area. This allows for the fabrication of sensors with single detection functions as well as multifunctional composite sensors in a one-step process, thus improving process adaptability. For examples, please continue reading. Figures 3 to 7 The sensing unit includes three active functional reagents: M for type A analyte, S1 for type B analyte, and S2 for type C analyte. Please continue reading. Figure 8 and Figure 9 The sensing unit includes three active functional reagents and auxiliary reagents, such as M for type A analyte, S1 for type B analyte, S2 for type C analyte, and S3 for type C analyte. 1 As an auxiliary reagent.
[0072] Of course, in other embodiments, the active functional reagents corresponding to different coating batches can also be the same.
[0073] In some embodiments, at least two morphologies and / or two sizes are provided, and the morphology and size of the sensing units within the same coating batch are consistent. That is, the morphology and size of the sensing units formed in different coating batches are different, or one of the morphology and size of the sensing units formed in different coating batches is different. This helps to increase the overall effective area of the sensing unit and improve the sensitivity of the response. For an example, please refer to [link to example]. Figure 2 In diagrams a) and b), the multiple sensing units can be formed from two coating batches. They all have the same shape (circular) but come in two different sizes: the sensing units formed in the first coating batch are larger, and the sensing units formed in the second coating batch are smaller. Please refer to... Figure 2(c) The multiple sensing units in the figure can be formed by three coating batches. They are all the same in shape, which is circular, but there are two different sizes. The larger sensing units formed in the first coating batch are 1, 4, 7, and 10. The second coating batch is formed in 2, 5, and 8. The third coating batch is formed in 3, 6, and 9. The sensing units formed in the second and third coating batches are the same size.
[0074] In this application, morphology specifically refers to geometric features such as surface shape, outline, and structural appearance. For example, the outline of the sensing unit can be circular, elliptical, square, polygonal, triangular, pentagonal, petal-shaped, or other irregularly shaped structures. Dimensions refer to parameters such as thickness and diameter.
[0075] In some embodiments, the diameter of each sensing unit ranges from 0.1 μm to 2000 μm; and / or, the thickness of each sensing unit ranges from 0.1 μm to 200 μm; and / or, the total coating volume of multiple (i.e., all) sensing units ranges from 10 pL to 1 μL. This size limitation facilitates applications in micro-sized biosensors, and controlling the diameter and thickness of individual sensing units helps ensure that the volume coated each time is small, avoiding uncontrolled coating area due to random diffusion.
[0076] Furthermore, by controlling the volume V or diameter D of a single sensing unit and the spacing d between two adjacent sensing units, the minimum overlap or coverage required to form a continuous boundary can be satisfied, thereby enabling the interconnection of multiple sensing units without significant spreading. For example, the dot-coating volume of a single sensing unit is 10 pL-5000 pL, and the spacing d between two adjacent sensing units is ≤ diameter D.
[0077] Please see Figures 2 to 9 Embodiments of this application also provide an active material layer for a biosensor, formed by coating an active functional reagent onto a substrate surface, for identifying and detecting target analytes, comprising multiple interconnected sensing units, wherein any two adjacent sensing units are in contact (i.e., tangential or intersecting), and the multiple sensing units may be the same or different in size. Figure 2 As shown in d), e), and f), the sensing units in the figures are the same size, as... Figure 2 As shown in a), b), and c), the sensing unit in the figure has two sizes. Of course, in other embodiments, it may also have three or more sizes.
[0078] In some embodiments, at least one sensing unit of a different size is provided between any two adjacent sensing units of the same size. For example, such as... Figure 2As shown in a) and b), a sensing unit of a different size is provided between two sensing units of the same size. That is, the sensing units at points 1 and 3 are of the same size, while the sensing unit at point 2, which is located between them, is of a different size than the sensing units corresponding to points 1 and 3. Figure 2 As shown in c), between two sensing units of the same size, there are two sensing units of different sizes. Specifically, the sensing units at points 1 and 4 are the same size, and the sensing units at points 2 and 3 in between are the same size. However, the sensing units corresponding to points 1 and 4 are different in size; that is, between points 1 and 4, there are two sensing units of different sizes, and these two sensing units are the same size. Of course, in other examples, the sensing units at points 2 and 3 between points 1 and 4 can also be different in size; that is, between points 1 and 4, there are two sensing units of different sizes, and these two sensing units are also different in size.
[0079] In some embodiments, the sensing unit includes a main sensing unit and a secondary sensing unit of different sizes, wherein the size of the main sensing unit is larger than the size of the secondary sensing unit, and a secondary sensing unit is provided between any two adjacent main sensing units. See also Figure 2 In section a), the sensing units corresponding to 1, 3, 5, 7, and 9 are primary sensing units, while the sensing units corresponding to 2, 4, 6, and 8 are secondary sensing units. Please continue reading. Figure 2 In section b), the sensing units corresponding to 1, 3, 5, 7, 9, and 11 are the main sensing units, and the sensing units corresponding to 2, 4, 6, 8, and 10 are the auxiliary sensing units.
[0080] Please continue reading. Figures 2 to 9 Multiple sensing units are arranged in a single row, a single column, or an array; among them, the array is a rectangular array or a cellular array.
[0081] In some embodiments, multiple sensing units are sequentially coated and formed by multiple coating batches. Sensing units formed in the same coating batch are arranged at intervals along the surface of the substrate, and any adjacent sensing units come from different coating batches. The sensing units in the same coating batch have the same size. That is, multiple sensing units are prepared by the preparation method described above, which involves coating sensing units formed in different batches at intervals. The specific method has been described in detail above and will not be repeated here.
[0082] The multiple sensing units in this application are coated and molded in different batches, avoiding the problems of interfacial tension wetting, lateral spreading, and boundary overflow caused by wet ink droplet overlap. This results in regular boundaries of the active material layer, uniform film thickness, and stable effective sensing area, thereby solving the problem of poor pattern forming accuracy. The multiple sensing units are connected and adjacent sensing units are in contact, which can also effectively reinforce the active material layer, reduce the risk of peeling during long-term use, improve the sensor lifespan, and improve the utilization rate of the active material layer coating area within the limited working area of the sensor.
[0083] Of course, in other embodiments, multiple sensing units can also be continuously coated and molded, and the multiple sensing units have at least two sizes.
[0084] Please see Figure 10 The embodiments of this application also provide a method for preparing a biosensor, the steps of which include the preparation method of the active material layer as described in any of the above embodiments. The specific preparation method of the active material layer has been described in detail above and will not be repeated here.
[0085] In some embodiments, S104: further includes coating the surface of the substrate with a flow-limiting reagent to form an outer membrane layer, the outer membrane layer covering the active material layer. The outer membrane layer may be a flow-limiting membrane made of one or more polymer materials, which can limit the diffusion rate of analytes (such as glucose) to the active region, reduce non-specific protein adsorption, reduce rejection reactions, or protect the active material layer from detachment.
[0086] In some embodiments, after coating the active material layer, the active material is further cross-linked. Specifically, the substrate coated with the sensing unit is placed in a constant temperature and humidity chamber for cross-linking and curing, thereby forming a stable active material layer and preventing it from peeling off. The cross-linking temperature of the constant temperature and humidity chamber can be 10℃-50℃, the cross-linking time can be 24h-96h, and the relative humidity can be 45%Rh-80%Rh. For example, the cross-linking time is 48h, the cross-linking temperature is 25℃, and the relative humidity is 65%Rh.
[0087] In some embodiments, after the outer film layer is coated, the substrate coated with the active material layer and the outer film layer is placed again in a constant temperature and humidity chamber for crosslinking and curing to form a stable outer film layer. The crosslinking temperature of the constant temperature and humidity chamber can be 10℃-50℃, the crosslinking time can be 24h-96h, and the relative humidity can be 45%Rh-80%Rh. For example, the crosslinking time is 48h, the crosslinking temperature is 25℃, and the relative humidity is 65%Rh.
[0088] Embodiments of this application also provide a biosensor, comprising a substrate and an active substance layer prepared by the method described in any of the above embodiments. The active substance layer is formed by coating an active functional reagent onto the surface of the substrate. The method for preparing the active substance layer has been described in detail above and will not be repeated here. Alternatively, the biosensor comprises a substrate and an active substance layer as described in any of the above embodiments. The active substance layer is formed by coating an active functional reagent onto the surface of the substrate. This active substance layer has been described in detail above and will not be repeated here. Alternatively, the biosensor is manufactured using the method for preparing the sensor in any of the above embodiments. This method for preparing the biosensor has been described in detail above and will not be repeated here.
[0089] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0090] It should be noted that experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer; all reagents and raw materials involved are commercially available products.
[0091] Example 1
[0092] Prepare a carbon electrode as a substrate, with a length of 5000 μm and a width of 350 μm; clean the carbon electrode surface before coating to ensure it is clean; uniformly mix glucose oxidase (GOD), electron mediator, and crosslinking agent (PEGDGE), and then load the mixture onto an inkjet printer; pre-determine 13 sensing unit points along the length of the working electrode in the working area of the substrate, such as... Figure 11 As shown, the electrodes are divided into two groups, named main points and auxiliary points, respectively. The main points consist of 7 main sensing units (M1, M2, M3…) and 6 auxiliary sensing units (S1, S2, S3…). The main and auxiliary points are spatially separated, but connected in an intersecting manner. The 13 sensing units are arranged in a single row along the length of the working electrode. Coating is performed in an alternating sequence, first coating the main points (M1→M2→M3→…→M7), then coating the auxiliary points (S1→S2→…→S6). After coating, the electrodes are placed in a constant temperature and humidity chamber for cross-linking and curing to form a stable active material layer. A flow-limiting membrane is then coated on the working area of the substrate. After the flow-limiting membrane is coated, the electrodes are placed in a constant temperature and humidity chamber for cross-linking and curing to form a stable outer membrane layer.
[0093] Repeat the above steps three times to complete the coating of the sensing unit on the electrode in three batches, which are respectively referred to as the first batch, the second batch and the third batch.
[0094] The mean and coefficient of variation of the diameter of the sensing unit prepared in the above embodiments are shown in Table 1 below.
[0095] Table 1
[0096]
[0097] As shown in Table 1, the coefficients of variation for the coating diameter and coating area of the three batches of electrodes are 0.15% and 0.28%, respectively, both less than 5%. This indicates that the spaced coating method of this application achieves precise coating of the connected active materials, and the average coefficient of variation for sensitivity is 1.90%, less than 5%. This demonstrates that electrodes coated with active material layers using this method have high sensitivity and good consistency.
[0098] Comparative Example
[0099] Using the components described in the embodiments, 13 sensing unit points are pre-defined along the length of the working electrode in the working area of the substrate. Multiple interconnected sensing units are formed on the working electrode using a continuous coating method to complete the coating of the active material layer. Then, the active material layer is cross-linked and cured, and the outer film layer is formed according to the method described in the embodiments.
[0100] The electrodes prepared in the above embodiments and comparative examples are compared. A physical image of the electrodes prepared in the embodiments is shown below. Figure 12 As shown in images a, b, and c, the circular outlines of the multiple connected sensing units are clear and regular, with no overflow or random flow, indicating that the spaced coating method described in this application can achieve precise coating of connected active materials. A comparative example of the prepared physical sample is shown below. Figure 12 As shown in Figure d, it can be seen that after coating, the droplets diffuse to form a diffusion region. The diffusion regions at adjacent points have a large overlap, and the thickness of this region increases while the edges become thinner. The coating consistency is poor, and it is not suitable for coating samples for array detection. Figure 13 The figure shows a comparison of the in vitro electrochemical test results of the biosensors prepared in the examples and comparative examples. The biosensor with the spaced coating method in the examples has a sensitivity that is about 7% higher, indicating that this application can improve the utilization rate of the active material coating area within the limited working area of the sensor, which is beneficial to further improving the sensitivity. Furthermore, after continuous monitoring of sensitivity changes for 23 days, it was found that the continuous coating attenuation in the comparative example was twice that of the spaced coating in the examples. This shows that the method of this application, by connecting the spaced coating points to each other in the spatial design, is beneficial to strengthening the active material layer, reducing the risk of shedding during long-term use, and improving the lifespan of the sensor.
[0101] This document describes various exemplary embodiments with reference to them. However, those skilled in the art will recognize that changes and modifications can be made to the exemplary embodiments without departing from the scope of this document. For example, various operational steps and components for performing operational steps can be implemented in different ways depending on the specific application or considering any number of cost functions associated with the operation of the system (e.g., one or more steps can be deleted, modified, or combined with other steps).
[0102] While the principles herein have been illustrated in various embodiments, numerous modifications to the structures, arrangements, proportions, elements, materials, and components, particularly suited to specific environments and operational requirements, may be used without departing from the principles and scope of this disclosure. These modifications and other alterations or alterations will be included within the scope of this document. Those skilled in the art will recognize that many changes can be made to the details of the above embodiments without departing from the fundamental principles of the invention.
Claims
1. A method for preparing an active material layer of a biosensor, the biosensor comprising a substrate, the active material layer being formed by coating an active functional reagent onto the surface of the substrate, characterized in that, The steps include: Multiple coating point groups are preset on the surface of the substrate. Each group of coating point groups corresponds to a single coating batch, and the coating points in each group of coating point groups are set at intervals. Select any one of the coating point groups as the first coating point group, and sequentially coat all the coating points in the first coating point group with the active functional reagent to form the first group of sensing units; The remaining coating point groups are selected sequentially and coated in staggered batches to form multiple sets of sensing units. Multiple sensing units in the multiple sets of sensing units are connected, any two adjacent sensing units are in contact, and different coating batches are formed.
2. The method for preparing the active material layer of the biosensor according to claim 1, characterized in that, The sensing units formed in each subsequent coating batch are distributed at the gap positions of the sensing units formed in the preceding coating batch.
3. The method for preparing the active material layer of the biosensor according to claim 1 or 2, characterized in that, The sensing unit formed in the subsequent coating batch comes into contact with the sensing unit formed in the adjacent coating batch.
4. The method for preparing the active material layer of the biosensor according to claim 1, characterized in that, Multiple groups of coating points are distributed in a single row, a single column, or an array; The array can be a rectangular array or a cellular array.
5. The method for preparing the active material layer of the biosensor according to claim 1, characterized in that, The active functional reagents include active substance reagents and auxiliary reagents for identifying and detecting target analytes.
6. The method for preparing the active material layer of the biosensor according to claim 1, characterized in that, The active functional reagents corresponding to different coating batches are different; and / or, The sensing units formed in different coating batches have at least two morphologies and / or two sizes, while the sensing units within the same coating batch have the same morphology and size.
7. The method for preparing the active material layer of the biosensor according to claim 1, characterized in that, The sensing unit satisfies at least one of the following conditions (1)-(3): (1) The diameter of each sensing unit ranges from 0.1 μm to 2000 μm; (2) The thickness of each sensing unit ranges from 0.1 μm to 200 μm; (3) The total coating volume of the multiple sensing units ranges from 10 pL to 1 μL.
8. An active material layer of a biosensor, formed by coating an active functional reagent onto a substrate surface, for identifying and detecting target analytes, characterized in that, It includes multiple interconnected sensing units, with any two adjacent sensing units in contact with each other, and the multiple sensing units have at least two sizes.
9. The active material layer according to claim 8, characterized in that, At least one sensing unit of a different size is provided between any two adjacent sensing units of the same size.
10. The active material layer according to claim 9, characterized in that, The sensing unit includes a main sensing unit and a secondary sensing unit of different sizes. The size of the main sensing unit is larger than that of the secondary sensing unit, and a secondary sensing unit is provided between any two adjacent main sensing units.
11. The active material layer according to claim 8, characterized in that, The multiple sensing units are arranged in a single row, a single column, or an array; The array can be a rectangular array or a cellular array.
12. The active material layer according to any one of claims 8-11, characterized in that, Multiple sensing units are formed by sequentially coating in multiple coating batches. The sensing units formed in the same coating batch are arranged at intervals along the surface of the substrate, and any adjacent sensing units come from different coating batches. The sensing units within the same coating batch have the same size.
13. A method for preparing a biosensor, characterized in that, The steps include the method for preparing the active substance layer as described in any one of claims 1-7.
14. The method for preparing a biosensor according to claim 13, characterized in that, It also includes coating the surface of the substrate with a flow-limiting agent to form an outer membrane layer, the outer membrane layer covering the active substance layer.
15. A biosensor, characterized in that, The biosensor may include a substrate and an active material layer prepared by the method described in any one of claims 1-7 or an active material layer prepared by any one of claims 8-12; or, the biosensor may be prepared by the method described in claim 13 or 14.