Enzyme dripping method of microsensor and microsensor

Abstract

The method comprises the following steps: forming a working electrode substrate on a substrate, and forming an enzyme spot sensing layer on a working electrode of the working electrode substrate by adopting a trace spot coating process of enzyme spot scribing; limiting an effective area on the enzyme spot sensing layer in a laser cutting mode to obtain a target enzyme sensing layer; the working electrode is uniformly coated with an enzyme line or a plurality of enzyme lines close to each other to form an enzyme spot sensing layer in a whole area, and irregular parts at the edge of the enzyme spot sensing layer are removed in a laser cutting manner, so that the consistency of the area size of the target enzyme sensing layer is ensured, the requirement on micro spot coating process equipment is low, the operation is convenient, and the cost is low. Meanwhile, the total area of enzyme spots can be accurately controlled, the final consistency of products is also ensured, the yield of the products is improved, the requirement for an accurate ultramicro sample application solution is greatly reduced, and the applicability of the enzyme point scribing process is expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensors, and particularly relates to a dot-enzyming method for a micro sensor and a micro sensor. Background Art

[0002] At present, for micro analyte detection sensors on the market, such as continuous glucose monitoring system (CGMS) sensors, the development trend is to achieve unified factory calibration, and to achieve unified factory calibration, good process consistency must be achieved. In the dot-enzyming production process of traditional CGMS sensor products, the traditional process mode usually uses an ultra-micro sampling system (a picoliter piezoelectric jet sampling device) to dot enzymes in a dotting manner, that is, one or more circular enzyme spots are evenly dotted in the middle part of the working electrode of the sensor. This method has high requirements for the sampling device. On the one hand, it requires the device to stably control picoliter-level droplets, with a single droplet below 1 nL, preferably below 300 pL, even below 100 pL and smaller droplet volumes, and the droplet volume control accuracy requirement is extremely high; on the other hand, it has high requirements for the enzyme preparation liquid for dotting enzymes, requiring extremely small particle size or even no particles, and extremely low viscosity (such as below 20 cps). In addition, the positioning accuracy is required to be below 20 um. Generally speaking, to achieve dot-enzyming consistency, the existing ultra-micro sampling technology has high requirements for equipment and processes, greatly increasing the R & D and production costs. Summary of the Invention

[0003] In view of this, the present invention provides a dot-enzyming method for a micro sensor and a micro sensor. By using the dot-enzyming scribing method to fabricate an enzyme sensing layer, without a high-precision sampling device and an enzyme solution with extremely low viscosity, the total area of the enzyme spots can be accurately controlled while ensuring the final consistency of the product, reducing the production cost, and specifically adopting the following technical solutions to achieve.

[0004] In a first aspect, the present invention provides a dot-enzyming method for a micro sensor, including 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 dot coating process of dot-enzyming scribing; Defining an effective area on the enzyme spot sensing layer by using a laser cutting method to obtain a target enzyme sensing layer.

[0005] As a preference of the above technical solution, setting a working electrode on the working electrode substrate, and forming an enzyme spot sensing layer on the working electrode by using a micro dot coating process of dot-enzyming scribing, includes: Coating an enzyme layer solution on the working electrode by using a dot-enzyming scribing method of continuous scribing or intermittent scribing to form an enzyme spot sensing layer; When the dot enzyme scribing method is continuous scribing, the enzyme layer solution is uniformly coated 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 together form an enzyme spot sensing layer in an integral area.

[0006] As a preference of the above technical solution, when the dot enzyme scribing method is intermittent scribing, the enzyme layer solution is intermittently and uniformly coated on the working electrode in sequence to form the first enzyme line, the second enzyme line and the third enzyme line arranged at intervals; After the enzyme layer solution on each enzyme line volatilizes and dries for the second time, the enzyme layer solution 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 in an integral area.

[0007] As a preference of the above technical solution, the dot enzyme scribing method of intermittent scribing is adopted to coat the enzyme layer solution on the working electrode to form an enzyme spot sensing layer, including: A strip of enzyme layer solution is uniformly coated on the working electrode according to the first scribing movement position to form the fourth enzyme line; 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 scribing movement position; A strip of enzyme layer solution is uniformly coated along the starting point and parallel to the fourth enzyme line to form the fifth enzyme line, and a strip of enzyme layer solution is uniformly coated in sequence according to the second scribing movement position and the first scribing movement position to form the sixth enzyme line; According to the first scribing movement position, the enzyme layer solution is uniformly 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 an integral area.

[0008] As a preference of the above technical solution, the first scribing movement position is the dot enzyme movement position, the second scribing movement position is the needle-lifting and empty-running movement position, and the first scribing movement position and the second scribing movement position form a preset angle. Among them, the dot enzyme scribing method is adopted on the working electrode by using a micro-dotting system for the micro-dotting process, and the micro-dotting process adopts the way of screw propulsion or piston injection valve for ultra-micro dot enzyme.

[0009] As a preference of the above technical solution, when the preset angle is 90 degrees and the first scribing movement position 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 movement position form a "ji" character movement position or an inverted "ji" character movement position; When the preset angle is 90 degrees and the first scribing movement position 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 movement position form an inverted "ji" character movement position or an inverted "ji" character movement position in the opposite direction.

[0010] As an optimization of the above technical solution, when the preset angle is less than 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 "Z" - shaped path or an inverted "Z" - shaped path; When the preset angle is less than 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 "Z" - shaped path or an inverted "Z" - shaped path in the opposite direction.

[0011] In a second aspect, the present invention also provides a micro - sensor, comprising a working electrode substrate, a working electrode disposed on the working electrode substrate, and a sensing element disposed on the working electrode. The sensing element includes a target enzyme sensing layer prepared by the dot - enzyme method of the above - mentioned micro - sensor and a film layer on the target enzyme sensing layer.

[0012] As an optimization 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 width of the electrode tip of the working electrode is less than 400 μm.

[0013] As an optimization of the above technical solution, the micro - sensor is an analyte sensor for detecting analytes including glucose, blood ketone, uric acid, lactic acid, blood lipid, cholesterol, nitrogen oxides or inorganic salts.

[0014] The present invention provides a dot - enzyme method for a micro - sensor and the micro - sensor. By forming a working electrode substrate on a substrate, a dot - enzyme scribing micro - dot coating process is used to form an enzyme spot sensing layer on the working electrode of the working electrode substrate, and a laser cutting method is used to define an effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer. Coating a single enzyme line or multiple closely - adjacent enzyme lines evenly on the working electrode can form an enzyme spot sensing layer for an entire area. The laser cutting method is used to remove the uneven parts at the edge of the enzyme spot sensing layer, thereby ensuring the consistency of the area size of the enzyme spot sensing layer. The dot - enzyme scribing method has low requirements for the micro - dot coating process equipment and the enzyme layer solution itself, is easy to operate, can accurately control the total area of the enzyme spots, ensures the final consistency of the product, improves the product yield, greatly reduces the requirements for a precise ultra - micro - sample solution, and expands the applicability of the dot - enzyme scribing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0016] Figure 1 Flow chart of the dot enzyme method for the micro sensor provided by the present invention; Figure 2 Schematic diagram of the dot enzyme effect of three sensing layers horizontally and three sensing layers vertically provided by the present invention; Figure 3 Schematic diagram of the dot enzyme effect of five sensing layers horizontally and five sensing layers vertically provided by the present invention; Figure 4 Schematic diagram of the dot enzyme effect of the "ji" character walking position and the reverse "ji" character walking position provided by the present invention; Figure 5 Schematic diagram of the dot enzyme effect of the "Z" character walking position and the reverse "Z" character walking position provided by the present invention; Figure 6 Schematic diagram of the dot enzyme effect of the inverted "ji" character walking position and the reverse inverted "ji" character walking position provided by the present invention; Figure 7 Schematic diagram of the dot enzyme effect of the inverted "Z" character walking position and the reverse inverted "Z" character walking position provided by the present invention; Figure 8 Schematic diagram of the structure of the micro sensor provided by the present invention.

[0017] The main element symbols are explained as follows: 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. Specific embodiments

[0018] The following will describe in detail the embodiments of the present invention. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0019] It should be noted that when an element is referred to as "fixed to" another element, it can be directly on the other element or there may also be an intermediate element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intermediate element at the same time. On the contrary, 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.

[0020] Referring to Figure 1 , the present invention provides a dot enzyme method for a micro sensor, comprising the following steps: S1: Provide a substrate and form at least one working electrode substrate on the substrate; 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.

[0021] S2: Set a working electrode on the working electrode substrate and form an enzyme spot sensing layer on the working electrode by a micro dot coating process of dot enzyme scribing; In this embodiment, a working electrode and a conductive line are provided on each working electrode substrate, the working electrode is connected to the conductive line, and dot enzyme is applied to the working electrode (i.e., the upper surface of the working electrode) by a micro dispensing system. Among them, the printed working electrode area can be larger than its actual use area to facilitate subsequent dot enzyme application. After dot enzyme application, the excess working electrode and the irregularly shaped part of the dot enzyme are removed by laser. A micro dot coating process (also called micro dispensing) is carried out on the working electrode by dot enzyme scribing using a micro dispensing system. The micro dispensing system uses a relatively low-cost screw propulsion or piston-type injection valve to perform ultra-micro dot enzyme application. Since the droplets sprayed by the screw propulsion or piston-type injection valve are large and continuous, the granularity of the enzyme layer liquid or enzyme preparation solution in this embodiment does not require an extremely low viscosity and extremely small volume particles. Dot enzyme scribing means uniformly coating one or more mutually adjacent enzyme lines on the working electrode (the upper surface of the working electrode) to form an integral enzyme spot sensing layer.

[0022] It should be noted that an enzyme spot sensing layer is formed on the working electrode by a micro dot coating process of dot enzyme scribing, including: coating an enzyme layer solution on the working electrode by a dot enzyme scribing method of continuous scribing or spaced scribing respectively to form an enzyme spot sensing layer; when the dot enzyme scribing method is continuous scribing, the enzyme layer solution is uniformly coated 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 together form an enzyme spot sensing layer in an entire area. In other words, the dot enzyme scribing method mainly includes continuous scribing and spaced scribing. Continuous scribing means that the positions of the successively scribed enzyme lines are close to each other or partially overlapping, and there needs to be a certain waiting time between scribing each enzyme line, that is, after the enzyme layer solution of a single enzyme line has volatilized and dried for a certain time, the next enzyme line is scribed continuously in position. Spaced scribing means that the enzyme lines are scribed at spaced positions first, and after the enzyme layer solution has volatilized and dried for a certain time, the gaps (the gaps between two enzyme lines) are partially scribed with enzyme layer solution lines to be filled, so as to accurately control the total area of the enzyme spots and also improve the dot enzyme efficiency.

[0023] It can be understood that by means of screw propulsion or a piston-type injection valve, the enzyme layer solution can be output in a strip shape (forming an enzyme line). Through continuous scribing with the positions of each enzyme layer solution being continuously close to each other or partially overlapping (waiting for the first time when scribing each enzyme layer solution enzyme line), or through spaced scribing where the positions of each enzyme layer solution are first scribed with intervals and then the gaps between each enzyme layer solution are filled (waiting for the second time when filling the enzyme layer solution into the gaps), a continuous and single enzyme spot sensing layer can be formed on the working electrode. The cost of screw propulsion or a piston-type injection valve is low, and the liquid particle size of the enzyme layer solution (enzyme line) output in a strip shape is large, so the requirements for the enzyme layer solution, also known as the enzyme preparation solution, are low, and the cost is also reduced. However, the detection accuracy of the finally formed enzyme spot sensing layer will not be reduced. Among them, the first time and the second time can be the same or different, and no limitation is made here.

[0024] It is further worth noting that the substrate is a plastic film, and one or more working electrode substrates are included on the plastic film substrate. A working electrode substrate includes conductive lines and a working electrode. Multiple working electrode substrates can be arranged side by side in multiple columns. In the working electrode substrates in the same row, for the dot enzyme scribing method of continuous scribing, the first enzyme line on multiple parallel working electrodes can be scribed first, and then after waiting for a first period of time (starting from the time when the first enzyme line on the first working electrode is scribed), the second enzyme line on multiple parallel working electrodes can be scribed, and so on, until an enzyme spot sensing layer covering an entire area is formed on each working electrode. For the dot enzyme scribing method of spaced scribing, the first enzyme line on multiple parallel working electrodes can also be scribed first, then the second enzyme line and the third enzyme line on multiple parallel working electrodes can be scribed in sequence. Then, after waiting for a second period of time (starting from the time when the first enzyme line on the first working electrode is scribed), the gap between the first enzyme line and the second enzyme line can be filled, and so on, until an enzyme spot sensing layer covering an entire area is formed on each working electrode. Thereby, the working efficiency of the dot enzyme method for the micro-sensor is further improved.

[0025] S3: Define an effective area on the enzyme spot sensing layer by laser cutting to obtain a target enzyme sensing layer.

[0026] In this embodiment, laser cutting means irradiating the material to be cut with a high-power density laser beam, so that the material is quickly heated to the vaporization temperature and evaporated to form holes. As the beam moves relative to the material, the holes continuously form a very narrow slit (such as about 0.01 mm wide), completing the cutting of the material. During the dot enzyme scribing process, the edges of the enzyme spot sensing layer will be in close contact or partially overlap with each other, resulting in uneven parts at the dot enzyme edges. Using the laser cutting method to remove the uneven parts at the dot enzyme edges can ensure the consistency of the enzyme spot area size. Among them, the target enzyme sensing layer is the enzyme spot sensing layer after cutting in a certain area, that is, the entire area.

[0027] It should be noted that the effective area refers to the area on the working electrode for sensing chemical substances. The parameters or characteristics of the effective area are reproducible, so that the coefficient of variation (CV) of the effective area between micro-sensors is less than about 5%, such as less than about 3% or 1%, etc. This can be achieved through the manufacturing process control and definition procedures for defining the effective area during the manufacture of micro-sensors. In addition, by making the size (width, length, diameter, and thickness) of the effective area of the working electrode in the micro-sensor remain basically constant, the reproducibility of the effective area of the micro-sensor minimizes the variation sensitivity between sensors. For example, before the parameter-related values that affect the manufacturing accuracy (and thus the reproducibility) of the sensor batch number based on the sensor batch number change, the effective area of the working electrode can be not defined.

[0028] It should be understood that by forming a working electrode substrate on a substrate, disposing a working electrode on the working electrode substrate, and performing a micro-drop coating process on the working electrode by means of dot enzyme scribing to form an enzyme spot sensing layer, a laser cutting method is used to define an effective area on the enzyme spot sensing layer, and a target enzyme sensing layer corresponding to the enzyme spot sensing layer is fabricated. A whole area of the enzyme spot sensing layer can be formed by uniformly coating one enzyme line or multiple closely adjacent enzyme lines on the working electrode (i.e., the upper surface or the working electrode area of the working electrode). The uneven parts at the edge of the enzyme spot sensing layer are removed by laser cutting, and finally a target enzyme sensing layer with a whole target area is formed on the working electrode, thereby ensuring the consistency of the enzyme spot area size, having low requirements for the micro-drop coating process equipment, being convenient to operate, accurately controlling the total area of the target enzyme sensing layer at the same time, ensuring the final consistency of the product, improving the product yield, greatly reducing the requirements for the precise ultra-micro sample solution, and expanding the applicability of the dot enzyme scribing process.

[0029] Optionally, referring to Figure 2 and Figure 3 , when the dot enzyme scribing method is spaced scribing, enzyme layer solution is sequentially and evenly coated on the working electrode at intervals to form first enzyme line 3, second enzyme line 4, and third enzyme line 5 arranged at intervals; After the enzyme layer solution of each enzyme line has volatilized and dried 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 respectively to form a whole area of the enzyme spot sensing layer 2.

[0030] In this embodiment, enzyme layer solution is uniformly coated on the working electrode to form first enzyme line 3, second enzyme line 4, and third enzyme line 5 arranged at position intervals, and there are gaps between every two enzyme lines, that is, the enzyme layer solution is uniformly coated parallel to the working electrode substrate 1 direction or perpendicular to the working electrode substrate 1 direction. The enzyme layer solution can be glucose oxidase and enzyme mediator solution, and can be specifically prepared according to the actual situation. The above process can be understood as: first, three parallel enzyme lines, namely first enzyme line 3, second enzyme line 4, and third enzyme line 5, are continuously scribed horizontally (horizontally), and after the enzyme layer solution corresponding to each enzyme line has volatilized and dried (i.e., after the second time), the gaps between every two enzyme lines are then filled horizontally. The gaps between two enzyme lines can be filled and complemented by one or more enzyme lines or a certain amount of enzyme layer solution to finally form a whole area of the enzyme spot sensing layer 2. Similarly, three parallel enzyme lines can be continuously scribed vertically (vertically) to obtain the enzyme spot sensing layer 2. Finally, the uneven parts at the dot enzyme edge on the enzyme spot sensing layer 2 are removed by laser cutting to define the effective area.

[0031] It should be noted that the dot-dash enzyme line method can be divided into continuous scribing and spaced scribing. In continuous scribing, the sequentially scribed enzyme lines are close to each other or partially overlapped, and there needs to be a certain time interval, that is, the first time, between scribing each line. After waiting for a certain time for a single enzyme solution to volatilize and dry, the next enzyme line is scribed; in spaced scribing, enzyme lines are scribed at intervals first, and after waiting for a certain time for the enzyme solution to volatilize and dry, that is, the second time, the gap parts between the enzyme lines are scribed to complete and form a whole enzyme spot sensing layer 2 with a certain area. The scribing method and the moving position include but are not limited to Figure 2 or Figure 3 the scribing method and the moving position in

[0032] Optionally, referring to Figure 4 、 Figure 5 、 Figure 6 and Figure 7 , using the dot enzyme scribing method of spaced scribing to coat the enzyme layer solution on the working electrode to form an enzyme spot sensing layer, including: Uniformly coating a strip of enzyme layer solution on the working electrode according to the first scribing moving position to form a fourth enzyme line 6; Adjusting the starting point of the next enzyme line of the fourth enzyme line 6 according to the second scribing moving position at the end point of the fourth enzyme line 6; Uniformly coating a strip of enzyme layer solution along the starting point and parallel to the fourth enzyme line 6 to form a fifth enzyme line 7, and sequentially uniformly coating a strip of enzyme layer solution according to the second scribing moving position and the first scribing moving position to form a sixth enzyme line 8; According to the first scribing moving position, uniformly coating enzyme layer solution 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 to form the enzyme spot sensing layer 2 of a whole area.

[0033] In this embodiment, the first scribing moving position is the dot enzyme moving position, the second scribing moving position is the needle-lifting and empty-running moving position, and the first scribing moving position and the second scribing moving position form a preset angle. Among them, using a micro-dotting system to perform a micro-dotting process (micro-dotting) by the dot enzyme scribing method on the working electrode 9 of the working electrode substrate 1, the micro-dotting system uses the method of screw propulsion or piston-type injection valve for ultra-micro dot enzyme.

[0034] Optionally, as shown in Figure 4 and Figure 6 , when the preset angle is 90 degrees and the first scribing moving position 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 moving position form a "ji" character moving position or an inverted "ji" character moving position; When the preset angle is 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 are moved in the reverse direction of the Chinese character "己" or in the opposite direction of the Chinese character "己".

[0035] In this embodiment, the first stroke movement can be regarded as the solid line enzyme dot direction, and its direction can be the same or opposite. The second stroke movement can be regarded as the dotted line needle lift empty running movement, and the stroke can be understood as dot enzyme in one direction or uniformly coating enzyme layer liquid. First, draw a solid line (arrowed horizontal line) horizontally (horizontally) to the right, that is, the fourth enzyme line 6, and draw a dotted line vertically downward, that is, lift the needle and run empty (do not execute the dot enzyme stroke, resulting in a gap); then draw the fifth enzyme line 7 (solid line) parallel to the fourth enzyme line 6, and the stroke direction is opposite to the stroke direction of the fourth enzyme line 6; adjust the starting point of the next enzyme line of the fifth enzyme line 7, and finally draw the sixth enzyme line 8 parallel to the fifth enzyme line 7 from the starting point, the direction is the same as the direction of the fourth enzyme line 6, and the stroke direction of the fifth enzyme line 7 is opposite. The stroke path composed of the fourth enzyme line 6, the second stroke movement (arrowed dotted line), the fifth enzyme line 7, the second stroke movement and the sixth enzyme line 8 is in the direction of "self". Similarly, the enzyme line is first drawn horizontally to the left, and the line path, that is, the point enzyme line path, is also obtained in the reverse direction of the word "己". In addition, the fourth enzyme line 6 is first formed by uniformly coating according to the first line movement vertically downward (perpendicular to the working electrode substrate), and then the starting point of the fifth enzyme line 7 is determined according to the second line movement at the end of the fourth enzyme line 6, and the fifth enzyme line 7 is drawn parallel to the fourth enzyme line 6, and the line direction 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, and 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 reversed "己" or reversed "己" in the opposite direction.

[0036] Alternatively, if 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; 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" shape or an inverted "Z" shape in the opposite direction.

[0037] In this embodiment, horizontally (horizontally), that is, the enzyme layer liquid is evenly coated in accordance with the first scribing movement to form the fourth enzyme line 6, and the starting point of the fifth enzyme line 7 is determined in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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 in accordance with 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, the scribing of 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 supplement to form the enzyme spot sensing layer 2.

[0038] Specifically, three lines are scribed horizontally and 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. 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 the present invention.

[0039] 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, thereby 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 should be noted that for any other movement methods of the lines, such as the scribing methods of reverse "ji", "Z", reverse "Z" - shaped movements, etc. (such asFigure 4 in (b) of Figure 5 as shown, are all within the protection scope of the present invention.

[0040] Specifically, first draw three lines vertically 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

[0041] 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 requirements. Any number of enzyme spotting methods are within the protection scope of the present invention.

[0041] It should be noted that the interval width (gap or clearance) between the lines (enzyme lines) is 0.01 to 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, 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 evaporation and drying time of the enzyme solution, and the longest time is determined according to the enzyme spotting efficiency; the scribing method adopts a reverse "ji" - shaped walking position. The specific walking position method is as shown in (a) of Figure 6 It should be noted that for any other walking position methods of the scribing line, such as reverse - direction reverse "ji", reverse "Z", reverse - direction reverse "Z" - shaped scribing methods (such as shown in (b) of Figure 6 in Figure 7 as shown), are all within the protection scope of the present invention.

[0042] As described above, it can first scribe horizontally and then vertically, or first scribe vertically and then horizontally, or adjust device parameters such as the character - shaped walking position (such as "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. 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.

[0043] 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.

[0044] In this embodiment, there is a sensing element on the sensing surface of a single micro-sensor. 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, and 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 width of the electrode tip of the working electrode 9 is less than 400 μm. The micro-sensor is an analyte sensor for detecting glucose, blood ketone, uric acid, lactic acid, blood lipid, cholesterol, nitrogen oxide or inorganic salt. In other words, the types of micro-sensors are not limited to continuous glucose monitoring sensors, but also include other micro-sensors, such as analyte sensors for blood ketone, uric acid, lactic acid, blood lipid, cholesterol, nitrogen oxide, inorganic salt, cancer and other disease markers, and other chemical small molecules in the body.

[0045] It should be noted that the continuous glucose monitoring device (system) includes the above-mentioned micro-sensor, an electronic system, and an inserter. The micro-sensor includes a flexible electrode substrate, a flexible electrode, and a sensing element for painless minimally invasive implantation. The electronic system can be a mobile terminal for receiving analyte data, and the micro-sensor is arranged on the inserter. The flexible electrode is prepared by a low-cost printing process, with a thickness less than 300 μm and the width of the tip of the flexible electrode less than 400 μm. Due to the small width and thickness, the tip of the micro-sensor is very minimally invasive during implantation, and there is basically no obvious pain. The micro-sensor achieves response consistency after sensor implantation through production process consistency control and the principle of using a low working voltage, including the consistency of the current-glucose concentration response sensitivity and the background current under a low working voltage. To achieve the consistency of the response sensitivity, a micro-drop enzyme process is required to precisely control the volume and thickness of the target enzyme sensing layer of the micro-sensor, and it is suitable for mass production.

[0046] Specifically, the enzyme layer solution can be a glucose-responsive enzyme (such as glucose oxidase, glucose dehydrogenase, etc.) or a lactic acid-responsive enzyme (such as lactic acid oxidase). In other embodiments, the enzyme layer solution also includes a redox mediator and other enzyme compositions, such as quinone compounds or transition metal complexes.

[0047] Specifically, a non-conductive material and a conductive layer are formed on the working electrode substrate of the micro-object sensor. 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 variation coefficient 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 spot and independent of the thickness of the enzyme spot. Therefore, there is partial overlap and different thicknesses in the enzyme spot sensing layer formed by scribing, and the CV deviation of the final sensitivity of the entire batch of sensors can still be controlled within an acceptable range, ensuring the consistency of the working electrode 9.

[0048] It should be noted that the sensitivity of the micro-sensor depends on the area of the sensing element, that is, the total area of the target enzyme sensing layer. For example, a layer disposed on the surface of the working electrode (on the working electrode) including a sensing preparation with an analyte-reactive enzyme, or a redox mediator or a redox mediator covalently or non-covalently linked to a polymer. The sensitivity of the micro-sensor depends on the area of the sensing element but does not significantly depend on the edge effect of the sensing element. Additionally, the sensitivity of the micro-sensor can also depend on the analyte flow rate flowing to the surface of the working electrode (such as a flat surface) through a flow-limiting membrane (such as a permeation membrane control layer or a membrane layer) disposed on the sensing element in a two-dimensional manner. The sensitivity of the micro-sensor can also depend on the analyte flow rate through a flow-limiting membrane disposed on the sensing element in a three-dimensional manner, which is not defined herein.

[0049] It can be understood that the dot-enzyme method of the present invention has the characteristics of convenient and simple operation and low requirements for equipment. At the same time, it can accurately control the total area of the enzyme spots, thereby ensuring the final consistency of the product, improving the product yield, greatly reducing the requirements for accurate ultra-trace spotting solutions, and also expanding the applicability of this process.

[0050] In all the examples shown and described herein, any specific value should be construed as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values.

[0051] It should be noted that like reference numerals and letters denote like items in the following figures. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0052] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A microsensor spot enzyme method, characterized in that: It includes the following steps: Provide a substrate, and form at least one working electrode substrate on the substrate; Set a working electrode on the working electrode substrate, and form an enzyme spot sensing layer on the working electrode by using a micro-drop coating process of dot enzyme scribing; Use a laser cutting method to define an effective area on the enzyme spot sensing layer to obtain a target enzyme sensing layer.

2. The microsensor spot enzyme method according to claim 1, characterized in that: Set a working electrode on the working electrode substrate, and form an enzyme spot sensing layer on the working electrode by using a micro-drop coating process of dot enzyme scribing, including: Use a dot enzyme scribing method of continuous scribing or spaced scribing to coat an enzyme layer solution on the working electrode to form an enzyme spot sensing layer; When the dot enzyme scribing method is continuous scribing, sequentially and uniformly coat the enzyme layer solution on the working electrode to form multiple enzyme lines arranged closely or partially overlapping, wherein the time interval between the coating of every two enzyme lines is a first time, and the multiple enzyme lines together form an enzyme spot sensing layer for an entire area.

3. The microsensor spot enzyme method according to claim 2, characterized in that: It further includes: When the dot enzyme scribing method is spaced scribing, sequentially and evenly coat the enzyme layer solution on the working electrode at intervals to form a first enzyme line, a second enzyme line, and a third enzyme line arranged at intervals; After the enzyme layer solution of each enzyme line has volatilized and dried for a second time, fill 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 for an entire area.

4. The microsensor spot enzyme method according to claim 2, characterized in that: Use a dot enzyme scribing method of spaced scribing to coat an enzyme layer solution on the working electrode to form an enzyme spot sensing layer, including: Evenly coat an enzyme layer solution on the working electrode according to a first scribing path to form a fourth enzyme line; Adjust the starting point of the next enzyme line of the fourth enzyme line at the end point of the fourth enzyme line according to a second scribing path; Evenly coat an enzyme layer solution along the starting point and parallel to the fourth enzyme line to form a fifth enzyme line, and sequentially and evenly coat an enzyme layer solution according to the second scribing path and the first scribing path to form a sixth enzyme line; Evenly coat 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 for the entire area.

5. The microsensor spot enzyme method according to claim 4, characterized in that: 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.

6. The microsensor spot enzyme method according to claim 5, 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 "ji" character path in the opposite direction.

7. The microsensor spot enzyme method according to claim 5, characterized in that: 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; 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.

8. 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 point enzyme method of the microsensor as described in any one of claims 1 to 7 and a membrane layer on the target enzyme sensing layer.

9. The microsensor according to claim 8, 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. The electrode tip width of the working electrode is smaller than 400 μm.

10. The microsensor according to claim 8, characterized in that: The micro sensor is an analyte sensor for detecting glucose, blood ketone, uric acid, lactic acid, blood lipid, cholesterol, nitrogen oxide or inorganic salt.

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