A microsensor enzyme method and microsensor

Abstract

The present invention discloses a micro-sensor enzyme spotting method and a micro-sensor. The method comprises the following steps: forming a working electrode base on a substrate, adopting a micro-dot coating process of enzyme spot streaking on a working electrode of the working electrode base to form an enzyme spot sensing layer, adopting a laser cutting method to limit an effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer; uniformly coating an enzyme line or multiple enzyme lines close to each other on the working electrode to form an enzyme spot sensing layer of a whole area, adopting a laser cutting method to remove irregular parts of the edge of the enzyme spot sensing layer, thereby ensuring the consistency of the area size of the target enzyme sensing layer, having low requirements for micro-dot coating process equipment and convenient operation, and being able to accurately control the total area of the enzyme spot, thereby ensuring the final consistency of the product, improving the product yield, greatly reducing the requirements for precise ultra-micro dot sampling solution, and expanding the applicability of the enzyme spot streaking process.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical biosensors, and in particular relates to a spot enzyme method of a microsensor and a microsensor. Background Art

[0002] Currently, the trend for commercially available microanalyte sensors, such as those for continuous glucose monitoring systems (CGMS), is toward factory calibration. This requires excellent process consistency. Traditionally, the enzyme spotting process for CGMS sensors utilizes an ultramicro-dosing system (a picoliter-scale piezoelectric jet spotting device) to spot the enzyme. This involves uniformly spotting one or more circular enzyme spots onto the center of the sensor's working electrode. This method places high demands on the spotting equipment. Firstly, the equipment must be able to stably control picoliter-scale droplets, with single droplets below 1nL, preferably below 300pL, or even below 100pL or even smaller, requiring extremely high droplet volume control accuracy. Secondly, the enzyme preparation liquid used for spotting has stringent requirements, requiring extremely small or even particle-free particles and extremely low viscosity (e.g., below 20cps). Furthermore, positioning accuracy must be below 20µm. Overall, achieving consistent enzyme spotting requires high equipment and process requirements for existing ultramicro-dosing technology, significantly increasing R&D and production costs. Summary of the Invention

[0003] In view of this, the present invention provides a microsensor enzyme spotting method and a microsensor, which adopts the enzyme spotting and streaking method to produce the enzyme sensing layer. Without the need for high-precision spotting equipment and extremely low-viscosity enzyme solution, the total area of the enzyme spot can be accurately controlled while ensuring the final consistency of the product, thereby reducing production costs. The following technical solutions are specifically adopted to achieve this.

[0004] In a first aspect, the present invention provides a method for producing an enzyme-doped microsensor, comprising the following steps:

[0005] providing a substrate on which at least one working electrode base is formed;

[0006] A working electrode is arranged on the working electrode substrate, and an enzyme spot sensing layer is formed on the working electrode by a micro-dot coating process of enzyme streaking;

[0007] Laser cutting is used to define an effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer.

[0008] As a preferred embodiment of the above technical solution, a working electrode is provided on the working electrode substrate, and an enzyme spot sensing layer is formed on the working electrode by a micro-dot coating process of enzyme streaking, comprising:

[0009] An enzyme layer liquid is coated on the working electrode by a continuous or spaced enzyme streaking method to form an enzyme spot sensing layer;

[0010] When the enzyme dot streaking method is continuous streaking, the enzyme layer liquid is uniformly coated on the working electrode in sequence to form multiple enzyme lines that are closely arranged or partially overlapped, wherein the time interval between the coating of each two enzyme lines is the first time, and the multiple enzyme lines together form an enzyme spot sensing layer of an entire area.

[0011] As a preferred embodiment of the above technical solution, when the dot enzyme streaking method is spaced streaking, the enzyme layer liquid is uniformly coated on the working electrode in sequence to form a first enzyme line, a second enzyme line and a third enzyme line arranged at intervals;

[0012] After the enzyme layer liquid of each enzyme line evaporates and dries for a second time, the enzyme layer liquid is filled into the gaps between the first enzyme line and the second enzyme line, and between the second enzyme line and the third enzyme line, respectively, to form an enzyme spot sensing layer of a whole area.

[0013] As a preferred embodiment of the above technical solution, an enzyme plaque sensing layer is formed by coating an enzyme layer liquid on the working electrode by using an enzyme streaking method with spaced lines, comprising:

[0014] uniformly coating a line of enzyme coating liquid on the working electrode along the first line to form a fourth enzyme line;

[0015] At the end point of the fourth enzyme line, the starting point of the next enzyme line of the fourth enzyme line is adjusted according to the second stroke line;

[0016] uniformly coating an enzyme coating liquid along the starting point and parallel to the fourth enzyme line to form a fifth enzyme line, and uniformly coating an enzyme coating liquid in accordance with the second scoring line and the first scoring line in sequence to form a sixth enzyme line;

[0017] According to the first marking line, the enzyme layer liquid is evenly coated between the fourth enzyme line and the fifth enzyme line, and between the fifth enzyme line and the sixth enzyme line to form the enzyme spot sensing layer in a whole area.

[0018] As a preferred embodiment of the above technical solution, the first marking movement is the enzyme dot movement, the second marking movement is the needle lifting and running movement, the first marking movement and the second marking movement are at a preset angle, wherein a micro-dispensing system is used to perform a micro-dispensing process on the working electrode by using the enzyme dot marking method, and the micro-dispensing process uses a screw propulsion or a piston injection valve to perform ultra-micro enzyme dot.

[0019] As a preferred embodiment of the above technical solution, when the preset angle is 90 degrees and the first scribing line is parallel to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second scribing line are arranged in a "J" shape or a reverse "J" shape;

[0020] When the preset angle is 90 degrees and the first marking line is perpendicular to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second marking line are moved in the reverse direction of the Chinese character "J".

[0021] As a preferred embodiment of the above technical solution, when the preset angle is less than 90 degrees and the first scribing line is parallel to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second scribing line form a "Z" shape or a reverse "Z" shape;

[0022] When the preset angle is less than 90 degrees and the first marking line is perpendicular to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second marking line form an inverted "Z" shape or an inverted "Z" shape in the opposite direction.

[0023] In a second aspect, the present invention also provides a microsensor comprising a working electrode substrate, a working electrode arranged on the working electrode substrate, and a sensing element arranged on the working electrode, wherein the sensing element comprises a target enzyme sensing layer prepared by the point enzyme method of the above-mentioned microsensor and a membrane layer on the target enzyme sensing layer.

[0024] As a preferred embodiment of the above technical solution, the area of the target enzyme sensing layer is less than or equal to the area of the working electrode, the working electrode is prepared by a printing process, the thickness of the working electrode is less than 300 μm, and the electrode tip width of the working electrode is less than 400 μm.

[0025] As a preferred embodiment of the above technical solution, the microsensor is a sensor for detecting analytes including glucose, blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides or inorganic salts.

[0026] The present invention provides a microsensor enzyme spot method and microsensor, which forms a working electrode base on a substrate, forms an enzyme spot sensing layer on the working electrode of the working electrode base by a micro-dot coating process of enzyme spot streaking, and uses laser cutting to limit the effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer. Uniformly coating an enzyme line or multiple enzyme lines close to each other on the working electrode can form an enzyme spot sensing layer of a whole area, and laser cutting is used to remove the irregular parts of the edge of the enzyme spot sensing layer, thereby ensuring the consistency of the area size of the enzyme spot sensing layer. The enzyme spot streaking method has low requirements for micro-dot coating process equipment and the enzyme layer liquid itself, is easy to operate, and can accurately control the total area of the enzyme spot, thereby ensuring the final consistency of the product, improving the product yield, greatly reducing the requirements for precise ultra-micro spotting solution, and expanding the applicability of the enzyme spot streaking process. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 A flow chart of the enzyme spot method of the microsensor provided by the present invention;

[0029] Figure 2 Schematic diagram of the dot enzyme effect of three horizontal sensing layers and three vertical sensing layers provided by the present invention;

[0030] Figure 3 Schematic diagram of the dot enzyme effect of five horizontal sensing layers and five vertical sensing layers provided by the present invention;

[0031] Figure 4 Schematic diagram of the enzyme effect of the "self" movement and the reverse "self" movement provided by the present invention;

[0032] Figure 5 Schematic diagram of the effect of the "Z"-shaped and reverse "Z"-shaped enzymes provided by the present invention;

[0033] Figure 6 Schematic diagram of the effect of the enzyme provided by the present invention in the reverse direction of the "self" movement and the reverse direction of the "self" movement;

[0034] Figure 7 Schematic diagram of the effect of the enzyme provided by the present invention in the inverted "Z" shape and the reverse inverted "Z" shape;

[0035] Figure 8 This is a schematic structural diagram of the microsensor provided by the present invention.

[0036] The main component symbols are described as follows:

[0037] 1-working electrode substrate; 2-enzyme spot sensing layer; 3-first enzyme line; 4-second enzyme line; 5-third enzyme line; 6-fourth enzyme line; 7-fifth enzyme line; 8-sixth enzyme line; 9-working electrode; 10-target enzyme sensing layer; 11-membrane layer. DETAILED DESCRIPTION

[0038] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0039] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be an intermediate element. Conversely, when an element is referred to as being "directly on" another element, there is no intermediate element. The terms "vertical," "horizontal," "left," "right," and similar expressions used herein are for illustrative purposes only.

[0040] See Figure 1 The present invention provides a microsensor spot enzyme method, comprising the following steps:

[0041] S1: providing a substrate, and forming at least one working electrode base on the substrate;

[0042] In this embodiment, the substrate is a plastic film substrate, and one or more working electrode substrates are prepared on the substrate by screen printing. When there are multiple working electrode substrates, the multiple working electrode substrates are arranged in multiple rows.

[0043] S2: arranging a working electrode on the working electrode substrate, and forming an enzyme spot sensing layer on the working electrode by a micro-dot coating process of enzyme streaking;

[0044] In this embodiment, a working electrode and a conductive circuit are provided on each working electrode substrate. The working electrode and the conductive circuit are connected. A micro-dispensing system is used to apply the enzyme to the working electrode (i.e., the upper surface of the working electrode). The printed working electrode area can be larger than its actual usable area to facilitate subsequent enzyme application. After enzyme application is complete, a laser is used to remove excess working electrode and irregularly shaped portions of the enzyme application. A micro-dispensing process (also known as micro-dispensing) is performed on the working electrode using a micro-dispensing system by streaking enzyme lines. This micro-dispensing system utilizes a relatively low-cost screw propulsion or piston-type dispensing valve for ultra-micro enzyme application. Because the droplets sprayed by the screw propulsion or piston-type dispensing valve are large and continuous, the enzyme layer liquid or enzyme preparation solution in this embodiment does not require extremely low viscosity or extremely small particles. Enzyme streaking refers to uniformly coating one or more closely adjacent enzyme lines on the working electrode (the upper surface of the working electrode) to form a single, integrated enzyme plaque sensing layer.

[0045] It should be noted that the enzyme spot sensing layer is formed on the working electrode by a micro-dot coating process of enzyme streaking, including: coating the enzyme layer liquid on the working electrode by continuous streaking or spaced streaking to form the enzyme spot sensing layer; when the enzyme streaking method is continuous streaking, the enzyme layer liquid is uniformly coated on the working electrode in sequence to form a plurality of enzyme lines that are closely arranged or partially overlapped, wherein the time interval between the coating of each two enzyme lines is the first time, and the plurality of enzyme lines together form an enzyme spot sensing layer of an entire area. In other words, the enzyme streaking method mainly includes continuous streaking and spaced streaking. Continuous streaking refers to the positions of the enzyme lines streaked in sequence being close to each other or partially overlapping, and a certain waiting time is required between streaking each enzyme line, that is, after the enzyme layer liquid of a single enzyme line evaporates and dries for a certain period of time, the next enzyme line is continuously streaked. Interval marking means first marking enzyme lines at intervals, and then after the enzyme layer liquid evaporates and dries for a certain period of time, the gaps (the gaps between two enzyme lines) are partially filled with enzyme layer liquid lines, thereby accurately controlling the total area of the enzyme spot and improving the efficiency of enzyme spotting.

[0046] It is understood that by means of a screw propulsion or piston-type dispensing valve, the enzyme layer liquid can be output in strips (forming enzyme lines). By continuously drawing lines with each enzyme layer liquid arranged closely together or partially overlapping (a first waiting time is required when drawing each enzyme layer liquid enzyme line), or by first drawing out intervals between each enzyme layer liquid and then filling the gaps between each enzyme layer liquid with intervals (a second waiting time is required when filling the gaps with enzyme layer liquid), a continuous, single enzyme plaque sensing layer can be formed on the working electrode. The cost of a screw propulsion or piston-type dispensing valve is low, and the liquid particle size of the enzyme layer liquid (enzyme line) output in strips is large, so the requirements for the enzyme layer liquid, also known as the enzyme preparation solution, are low, and the cost is also reduced. However, the detection accuracy of the enzyme plaque sensing layer finally formed is not reduced. The first time and the second time can be the same or different, and are not limited here.

[0047] It is worth further explaining that the substrate is a plastic film, which includes one or more working electrode substrates, each of which includes a conductive circuit and a working electrode. The multiple working electrode substrates can be arranged in multiple columns. Within the same row of working electrode substrates, a continuous dot enzyme streaking method can first streak the first enzyme line on the multiple working electrodes in the same row, then, after a first waiting time (time starts from the completion of the first enzyme line on the first working electrode), streak the second enzyme line on the multiple working electrodes in the same row, and so on, until a complete enzyme plaque sensing layer is formed on each working electrode. Alternatively, an intermittent dot enzyme streaking method can first streak the first enzyme line on the multiple working electrodes in the same row, then sequentially streak the second and third enzyme lines on the multiple working electrodes in the same row, then, after a second waiting time (time starts from the completion of the first enzyme line on the first working electrode), fill the gap between the first and second enzyme lines, and so on, until a complete enzyme plaque sensing layer is formed on each working electrode. This further improves the efficiency of the dot enzyme method of the microsensor.

[0048] S3: using laser cutting to define an effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer.

[0049] In this embodiment, laser cutting involves irradiating the material being cut with a high-power density laser beam, rapidly heating the material to its vaporization temperature and evaporating it to form holes. As the beam moves across the material, the holes continuously form narrow slits (e.g., approximately 0.01 mm), completing the cutting of the material. During the enzyme spot marking process, the edges of the enzyme spot sensing layer may abut or partially overlap, resulting in irregularities along the enzyme spot edge. Laser cutting removes these irregularities and ensures consistent enzyme spot size. The target enzyme sensing layer is the enzyme spot sensing layer cut from a defined area, i.e., the entire area.

[0050] It should be noted that the active area refers to the area on the working electrode that senses the chemical. The parameters or characteristics of the active area are reproducible, such that the coefficient of variation (CV) of the active area between microsensors is less than about 5%, for example, less than about 3% or 1%, etc. This can be achieved through manufacturing process control and definition procedures that define the active area during microsensor manufacturing. Furthermore, by maintaining the active area, i.e., the dimensions (width, length, diameter, and thickness) of the working electrode, substantially constant across the microsensor, the reproducibility of the active area of the microsensor minimizes sensitivity to sensor-to-sensor variations. For example, the active area of the working electrode may not be defined until the values of parameters related to sensor batch-to-batch variations affect the manufacturing accuracy (and thus reproducibility) of the sensor batch.

[0051] It should be understood that an enzyme plaque sensing layer is formed by forming a working electrode base on a substrate, arranging a working electrode on the working electrode base, and performing a micro-dot coating process on the working electrode by a dot enzyme streaking method, and an effective area is defined on the enzyme plaque sensing layer by a laser cutting method, and a target enzyme sensing layer corresponding to the enzyme plaque sensing layer is produced. An enzyme line or multiple enzyme lines close to each other are uniformly coated on the working electrode (i.e., the upper surface of the working electrode or the working electrode area) to form an enzyme plaque sensing layer of an entire area, and the laser cutting method is used to remove the irregular parts of the edge of the enzyme plaque sensing layer, and finally a target enzyme sensing layer of an entire target area is formed on the working electrode, thereby ensuring the consistency of the size of the enzyme plaque area, having low requirements for the micro-dot coating process equipment, and convenient operation. At the same time, the total area of the target enzyme sensing layer can be accurately controlled, which also ensures the final consistency of the product, improves the product yield, greatly reduces the requirements for precise ultra-micro spotting solution, and expands the applicability of the dot enzyme streaking process.

[0052] Optionally, see Figure 2 and Figure 3 When the dot enzyme streaking method is spaced streaking, the enzyme layer liquid is uniformly coated on the working electrode in sequence to form a first enzyme line 3, a second enzyme line 4 and a third enzyme line 5 arranged at intervals;

[0053] After the enzyme layer liquid of each enzyme line evaporates and dries for a second time, the gaps between the first enzyme line 3 and the second enzyme line 4 and between the second enzyme line 4 and the third enzyme line 5 are filled to form an enzyme spot sensing layer 2 of a whole area.

[0054] In this embodiment, the enzyme layer liquid is evenly applied to the working electrode to form the first enzyme line 3, the second enzyme line 4 and the third enzyme line 5 arranged at intervals, and there is a gap between each two enzyme lines, that is, the enzyme layer liquid is evenly applied in the direction parallel to the working electrode substrate 1 or perpendicular to the working electrode substrate 1. The enzyme layer liquid can be glucose oxidase and an enzyme medium liquid, which can be prepared according to actual conditions. The above process can be understood as follows: first, three parallel enzyme lines are continuously drawn horizontally (horizontally), namely the first enzyme line 3, the second enzyme line 4 and the third enzyme line 5. After the enzyme layer liquid corresponding to each enzyme line evaporates and dries (i.e., after the second time), the gap (gap) between each two enzyme lines is filled horizontally, and the enzyme plaque is formed by uniformly applying the enzyme layer liquid to the gap. The gap between the two enzyme lines can be filled and completed with one or more enzyme lines or a certain amount of enzyme layer liquid, and finally an enzyme plaque sensing layer 2 of an entire area is formed. Similarly, three parallel enzyme lines are continuously drawn vertically (vertically) to obtain an enzyme plaque sensing layer 2. Finally, laser cutting is used to remove the irregular parts of the enzyme spot edge on the enzyme spot sensing layer 2 to define the effective area.

[0055] It should be noted that the dot-dash enzyme line method can be divided into continuous and spaced lines. Continuous lines are lines drawn in sequence close to each other or partially overlapping, and there needs to be a certain time interval between each line, that is, the first time, waiting for a single enzyme liquid to evaporate and dry for a certain time before drawing the next enzyme line; spaced lines are first drawn at intervals, waiting for the enzyme liquid to evaporate and dry for a certain time, that is, the second time, and then the gaps between the enzyme lines are filled with lines to form a whole piece of enzyme spot sensing layer 2 of a certain area. The marking method and the movement include but are not limited to Figure 2 or Figure 3 The way of drawing lines and the movement in .

[0056] Optionally, see Figure 4 、 Figure 5 、 Figure 6 and Figure 7 , coating the enzyme layer liquid on the working electrode by using an enzyme streaking method with spaced lines to form an enzyme spot sensing layer, comprising:

[0057] Evenly coating a line of enzyme coating liquid on the working electrode along the first line to form a fourth enzyme line 6;

[0058] At the end point of the fourth enzyme line 6, the starting point of the next enzyme line of the fourth enzyme line 6 is adjusted according to the second stroke line;

[0059] A line of enzyme coating liquid is uniformly applied along the starting point and parallel to the fourth enzyme line 6 to form a fifth enzyme line 7, and a line of enzyme coating liquid is uniformly applied in accordance with the second marking line and the first marking line to form a sixth enzyme line 8;

[0060] Apply the enzyme layer solution evenly between the fourth enzyme line 6 and the fifth enzyme line 7, and between the fifth enzyme line 7 and the sixth enzyme line 8 according to the first scribing path to form the enzyme spot sensing layer 2 of a whole area.

[0061] In this embodiment, the first scribing path is a dot enzyme path, the second scribing path is a needle-lifting empty running path, and the first scribing path and the second scribing path form a preset angle. Among them, a micro dotting process (micro dispensing) is carried out by using a dot enzyme scribing method on the working electrode 9 of the working electrode substrate 1 by using a micro dispensing system, and the micro dispensing system uses a screw propulsion or a piston-type injection valve to perform ultra-micro dot enzyme.

[0062] Optionally, as Figure 4 and Figure 6 shown, when the preset angle is 90 degrees and the first scribing path is parallel to the direction of the working electrode substrate 1, the fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8 and the second scribing path form a "ji" character path or an inverted "ji" character path;

[0063] When the preset angle is 90 degrees and the first scribing path is perpendicular to the direction of the working electrode substrate 1, the fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8 and the second scribing path form an inverted "ji" character path or an inverted "ji" character path in the opposite direction.

[0064] In this embodiment, the first stroke can be regarded as the direction of the solid line enzyme application, and its direction can be the same or opposite. The second stroke can be regarded as the dotted line needle lift empty run position, and the stroke can be understood as the direction of enzyme application or uniform application of enzyme layer liquid. First, a solid line (with an arrowed horizontal line) is drawn horizontally (horizontally) to the right, which is the fourth enzyme line 6. A dotted line is drawn vertically downward, which is the needle lift empty run (no enzyme application is performed, resulting in a gap); then, a fifth enzyme line 7 (solid line) is drawn parallel to the fourth enzyme line 6, and the stroke direction is opposite to that of the fourth enzyme line 6; the starting point of the next enzyme line of the fifth enzyme line 7 is adjusted, and finally, a sixth enzyme line 8 is drawn parallel to the fifth enzyme line 7 from the starting point, in the same direction as the movement direction of the fourth enzyme line 6 and opposite to the movement direction of the fifth enzyme line 7. The stroke path formed by the fourth enzyme line 6, the second stroke position (with an arrowed dotted line), the fifth enzyme line 7, the second stroke position, and the sixth enzyme line 8 is in the shape of a "self" (self) shape. Similarly, by first drawing an enzyme line horizontally to the left, a line path can also be obtained, that is, a point enzyme line path that is in the reverse direction of the "self" character. In addition, first vertically downward (perpendicular to the working electrode substrate) according to the first line movement uniformly coats to form a fourth enzyme line 6, then at the end of the fourth enzyme line 6, the starting point of the fifth enzyme line 7 is determined according to the second line movement, and the fifth enzyme line 7 is drawn parallel to the fourth enzyme line 6, and the direction of the line movement is opposite to the direction of the fourth enzyme line 6. At this time, the angle between the first line movement and the second line movement is 90 degrees (vertical), and then the starting point of the sixth enzyme line 8 is determined according to the second line movement, and the direction is the same as the direction of the fourth enzyme line 6. The sixth enzyme line 8 is drawn in the opposite direction of the line movement of the fifth enzyme line 7. The fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8 and the second line movement are in the reverse direction of the "self" character movement or the reverse direction of the "self" character movement.

[0065] Alternatively, as Figure 5 and Figure 7 As shown, when the preset angle is less than 90 degrees and the first scribing line is parallel to the direction of the working electrode substrate 1, the fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8 and the second scribing line form a "Z" shape or a reverse "Z" shape;

[0066] When the preset angle is less than 90 degrees and the first marking line is perpendicular to the direction of the working electrode substrate 1, the fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8 and the second marking line form an inverted "Z" movement or an inverted "Z" movement in the opposite direction.

[0067] In this embodiment, horizontally (horizontally), that is, the enzyme layer liquid is evenly coated according to the first scribing movement to form the fourth enzyme line 6, and the starting point of the fifth enzyme line 7 is determined according to the second scribing movement. The preset angle between the first scribing movement and the second scribing movement can be 45 degrees. The fifth enzyme line 7 is scribed from the starting point of the fifth enzyme line 7 according to the first scribing movement (parallel to the same direction as the fourth enzyme line), and the direction is opposite to the movement direction of the fourth enzyme line 6. And the sixth enzyme line 8 is formed according to the second scribing movement and the first scribing movement, and the direction is the same as the movement direction of the fourth enzyme line 6 and opposite to the scribing direction of the fifth enzyme line 7. The fourth enzyme line 6, the second movement direction, the fifth enzyme line 7, the second movement direction, and the sixth enzyme line 8 are in a "Z" - shaped movement. Similarly, vertically (vertically), that is, the enzyme layer liquid is evenly coated according to the first scribing movement to form the fourth enzyme line 6, and then the fifth enzyme line 7 and the sixth enzyme line 8 are formed according to the scribing method and movement of scribing enzyme lines horizontally. The fourth enzyme line 6, the second scribing movement, the fifth enzyme line 7, the second scribing movement, and the sixth enzyme line 8 are in a "Z" - shaped movement. When the preset angle is less than 90 degrees and the first scribing movement is perpendicular to the direction of the working electrode substrate 1, the fourth enzyme line 6, the fifth enzyme line 7, the sixth enzyme line 8, and the second scribing movement form an inverted "Z" - shaped movement or an inverted "Z" - shaped movement in the opposite direction. Among them, scribing enzyme lines can be regarded as vectors, the three enzyme lines are parallel, and there are gaps (intervals) between every two enzyme lines, and then the gaps are continuously scribed to form the enzyme spot sensing layer 2.

[0068] Specifically, three horizontal lines are scribed at intervals on the working electrode 9 of the working electrode substrate 1, as shown in (a) of Figure 2 . After waiting for a certain drying time, which can be waiting for the second time, two lines are scribed in the interval part to form a whole enzyme spot sensing layer as shown in (a) of Figure 3 . The lengths of the five horizontal lines are the same and can be any length (not exceeding the length of the working electrode), and are determined according to the final sensitivity value of the sensor. The number of horizontal lines is not limited to five, and the specific number is determined according to the enzyme - dotting parameters of the enzyme - dotting machine (which determine the scribing width) and the width of the working electrode. Any number of enzyme - dotting methods are within the protection scope of this invention.

[0069] It should be noted that the interval width between the lines is 0.01 - 10 times the width of a single line, and more preferably 0.5 - 1 times. However, it should be noted that the gap should be as small as possible compared to the line width to avoid leaving gaps during splicing, thus affecting the enzyme spot area and the final electrode sensitivity; the interval time, that is, the second time, between the first three horizontal lines and the last two horizontal lines ranges from dozens of seconds to several minutes. The shortest time is determined according to the time for the enzyme solution to volatilize and dry, and the longest time is determined according to the enzyme - dotting efficiency. The scribing method adopts a "ji" - shaped movement, and the specific movement method is as shown in (a) of Figure 4 . It is worth noting that any other movement methods, such as reverse "ji", "Z", reverse "Z" - shaped movement, etc. (such as Figure 4 in (b) of Figure 5 as shown), are all within the protection scope of the present invention.

[0070] Specifically, first draw three vertical lines at intervals on the working electrode 9 of the working electrode substrate 1, as Figure 2 shown in (b) of Figure 3 After waiting for a certain drying time, which can be waiting for the second time, then draw two lines in the interval part to form a whole enzyme spot sensing layer as shown in (b) of

[0071] ; The lengths of the five vertical lines are the same and can be any length (not less than the width of the working electrode substrate), which is determined according to the final sensitivity value of the sensor; The number of vertical lines is not limited to five, and the specific number is determined according to the enzyme spotting parameters of the enzyme spotting machine (which determines the line width) and the final sensitivity requirement. Any number of enzyme spotting methods are within the protection scope of the present invention. Figure 6 It should be noted that the interval width (gap or clearance) between the lines (enzyme lines) is 0.01 - 10 times the width of a single line, and more preferably 0.5 - 1 times, but it should be noted that the gap should be as small as possible compared to the line width to avoid leaving gaps during splicing, thereby affecting the enzyme spot area and the final electrode sensitivity; The interval time, that is, the second time, between the first three vertical lines and the last two vertical lines can range from dozens of seconds to several minutes. The shortest time is determined according to the time for the enzyme solution to volatilize and dry, and the longest time is determined according to the enzyme spotting efficiency; The scribing method adopts the inverted "ji" - shaped walking position. The specific walking position method is as shown in (a) of Figure 6 in (b) of Figure 7 as shown), are all within the protection scope of the present invention.

[0072] For the above, it can first draw lines horizontally and then vertically, or first draw lines vertically and then horizontally, or adjust device parameters such as the character walking position (such as the "ji" - shaped), or other scribing methods that meet the requirements, or set the starting point, ending point, line length, width, thickness, enzyme spot area, etc. of the enzyme spotting scribing to adjust the enzyme spotting scribing method, and finally fill and complete the gaps between every two enzyme lines to form an enzyme spot sensing layer with a certain area, which is not limited here.

[0073] Referring to Figure 8 , the present invention also provides a micro - sensor, including a working electrode substrate 1, a working electrode 9 formed on the working electrode substrate 1, and a sensing element on the working electrode 9. The sensing element includes a target enzyme sensing layer 10 prepared by the enzyme spotting method of the above - mentioned micro - sensor and a film layer 11 on the target enzyme sensing layer 10.

[0074] In this embodiment, there is one sensing element on the sensing surface of a single microsensor, and the area of the target enzyme sensing layer 10 is less than or equal to the area of the working electrode on the working electrode substrate 1. The working electrode substrate 1 can be a flexible electrode substrate, and the corresponding working electrode 9 can be a flexible electrode. The working electrode 9 is prepared by a printing process, the thickness of the working electrode 9 is less than 300 μm, and the electrode tip width of the working electrode 9 is less than 400 μm. The microsensor is an analyte sensor for detecting glucose, blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides or inorganic salts. In other words, the types of microsensors are not limited to continuous glucose monitoring sensors, but also include other microsensors, such as analyte sensors for blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides, inorganic salts, cancer and other disease markers, other chemical small molecules in the body, and the like.

[0075] It should be noted that the continuous glucose monitoring device (system) includes the aforementioned microsensor, an electronic system, and an injection device. The microsensor comprises a flexible electrode substrate, a flexible electrode, and a sensing element for painless and minimally invasive implantation. The electronic system can be a mobile terminal that receives analyte data. The microsensor is mounted on the injection device. The flexible electrode is produced using a low-cost printing process and is less than 300 μm thick. The tip width of the flexible electrode is less than 400 μm. Due to its small width and thickness, the tip of the microsensor is minimally invasive during implantation, with virtually no noticeable pain. The microsensor achieves consistent response after implantation through consistent production process control and the use of a low operating voltage principle, including consistent current-glucose concentration response sensitivity and consistent background current at low operating voltage. To achieve consistent response sensitivity, a micro-dot enzyme process is required to precisely control the volume and thickness of the target enzyme sensing layer in the microsensor, making it suitable for mass production.

[0076] Specifically, the enzyme layer liquid can be a glucose-responsive enzyme (such as glucose oxidase, glucose dehydrogenase, etc.) or a lactate-responsive enzyme (such as lactate oxidase). In other embodiments, the enzyme layer liquid also includes redox mediators and other enzyme compositions, such as quinone compounds or transition metal complexes.

[0077] Specifically, the working electrode substrate of the micro-object sensor comprises a non-conductive material and a conductive layer. The conductive layer is disposed on and in contact with the non-conductive material. A working electrode is defined on the conductive layer, and a sensing element is disposed on the working electrode. The working electrode has an incomplete boundary surrounding the sensing element. A reagent, such as an analyte-responsive enzyme, is located within the boundary of the sensing element. The sensitivity coefficient of variation of the working electrode is less than or equal to 8%. When the thickness of the enzyme layer (enzyme line) reaches a certain level, the sensitivity of the micro-sensor is related to the surface area of the enzyme plaque and not to its thickness. Therefore, even if the enzyme plaque sensing layer formed by the marking has some overlap and varying thickness, the final sensitivity CV deviation of the entire batch of sensors can still be controlled within an acceptable range, ensuring the consistency of the working electrode 9.

[0078] It is worth noting that the sensitivity of the microsensor depends on the area of the sensing element, that is, the total area of the target enzyme sensing layer, such as a layer disposed on the surface of a working electrode (on the working electrode) comprising a sensing agent having an analyte-reactive enzyme, or a redox mediator or a redox mediator covalently or non-covalently linked to a polymer. The sensitivity of the microsensor depends on the area of the sensing element, but is not significantly dependent on the edge effect of the sensing element. In addition, the sensitivity of the microsensor may also depend on the analyte flux flowing to the working electrode surface (e.g., to a flat surface) through a flux-limiting membrane (e.g., a permeable membrane control layer or membrane layer) disposed in a two-dimensional manner on the sensing element. The sensitivity of the microsensor may also depend on the analyte flux through a flux-limiting membrane disposed in a three-dimensional manner on the sensing element, but this is not limited here.

[0079] It can be understood that the enzyme spotting method of the present invention has the characteristics of easy and convenient operation and low equipment requirements. At the same time, the total area of the enzyme spot can be accurately controlled, thereby ensuring the final consistency of the product, improving the product yield, greatly reducing the requirements for precise ultra-micro spotting solution, and expanding the applicability of this process.

[0080] In all examples shown and described herein, any specific values should be interpreted as merely exemplary and not limiting, and thus other examples of the exemplary embodiments may have different values.

[0081] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0082] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible without departing from the scope of the present invention, and such variations and modifications are fully within the scope of protection of the present invention.

Claims

1. A microsensor enzyme method, characterized in that: Comprising the following steps: Providing a substrate, and forming at least one working electrode substrate on the substrate; Setting a working electrode on the working electrode substrate, and forming an enzyme spot sensing layer on the working electrode by using a micro-drop coating process of dot enzyme scribing; Defining an effective area on the enzyme spot sensing layer by laser cutting to obtain a target enzyme sensing layer; Setting a working electrode on the working electrode substrate, and forming an enzyme spot sensing layer on the working electrode by using a micro-drop coating process of dot enzyme scribing, including: Coating an enzyme layer solution on the working electrode by using a dot enzyme scribing method of continuous scribing or spaced scribing to form an enzyme spot sensing layer; When the dot enzyme scribing method is continuous scribing, uniformly coating the enzyme layer solution on the working electrode in sequence to form multiple enzyme lines arranged closely or partially overlapping, wherein the time interval between the coating of every two enzyme lines is the first time, and the multiple enzyme lines jointly form an enzyme spot sensing layer of a whole area; When the dot enzyme scribing method is spaced scribing, uniformly coating the enzyme layer solution on the working electrode at intervals in sequence to form first, second and third enzyme lines arranged at intervals; After the enzyme layer solution of each enzyme line volatilizes and dries for the second time, filling the enzyme layer solution into the gaps between the first enzyme line and the second enzyme line, and between the second enzyme line and the third enzyme line respectively to form an enzyme spot sensing layer of a whole area; Coating an enzyme layer solution on the working electrode by using a dot enzyme scribing method of spaced scribing to form an enzyme spot sensing layer, including: Uniformly coating an enzyme layer solution on the working electrode according to a first scribing path to form a fourth enzyme line; adjusting the starting point of the next enzyme line of the fourth enzyme line according to a second scribing path at the end point of the fourth enzyme line; Uniformly coating an enzyme layer solution along the starting point and parallel to the fourth enzyme line to form a fifth enzyme line, and uniformly coating an enzyme layer solution in sequence according to the second scribing path and the first scribing path to form a sixth enzyme line; Uniformly coating the enzyme layer solution between the fourth enzyme line and the fifth enzyme line, and between the fifth enzyme line and the sixth enzyme line according to the first scribing path to form the enzyme spot sensing layer of the whole area; The first scribing path is a dot enzyme path, the second scribing path is a needle-lifting empty running path, and the first scribing path and the second scribing path form a preset angle. Among them, a micro-drop coating process of dot enzyme scribing is carried out on the working electrode by using a micro-drop dispensing system, and the micro-drop coating process is a super-micro dot enzyme by using a screw propulsion or piston injection valve method.

2. The microsensor spot enzyme method according to claim 1, characterized in that: When the preset angle is 90 degrees and the first scribing path is parallel to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second scribing path form a "ji" character path or an inverted "ji" character path; When the preset angle is 90 degrees and the first scribing path is perpendicular to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second scribing path form an inverted "ji" character path or an inverted direction inverted "ji" character path.

3. The microsensor enzyme method according to claim 1, characterized in that: When the preset angle is less than 90 degrees and the first scribing line is parallel to the substrate direction of the working electrode, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second scribing line form a "Z" shape or a reverse "Z" shape; When the preset angle is less than 90 degrees and the first marking line is perpendicular to the direction of the working electrode substrate, the fourth enzyme line, the fifth enzyme line, the sixth enzyme line and the second marking line form an inverted "Z" shape or an inverted "Z" shape in the opposite direction.

4. A micro sensor, characterized in that: It comprises a working electrode substrate, a working electrode arranged on the working electrode substrate and a sensing element arranged on the working electrode, wherein the sensing element comprises a target enzyme sensing layer prepared by the dot enzyme method of the microsensor according to any one of claims 1 to 3 and a membrane layer on the target enzyme sensing layer.

5. The microsensor according to claim 4, characterized in that: The area of the target enzyme sensing layer is smaller than or equal to the area of the working electrode. The working electrode is prepared by a printing process. The thickness of the working electrode is smaller than 300 μm, and the electrode tip width of the working electrode is smaller than 400 μm.

6. The microsensor according to claim 4, characterized in that The micro sensor is an analyte sensor for detecting glucose, blood ketones, uric acid, lactic acid, blood lipids, cholesterol, nitrogen oxides or inorganic salts.

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