Micro biosensor and method for reducing measurement interference by using micro biosensor

Abstract

The present disclosure at least relates to a microbiosensor and a method thereof for reducing measurement interference. The invention provides a micro biosensor for being implanted under the skin surface of a living body to monitor a target analyte. The micro biosensor comprises a substrate; the first working electrode is arranged on the surface of the substrate and comprises a first sensing section; the second working electrode is arranged on the surface of the substrate and comprises a second sensing section, and the second sensing section is adjacent to at least one side edge of the first sensing section; and a chemical reagent at least covering a portion of the first sensing section, where the first sensing section is configured to measure a physiological signal associated with the target analyte when the first working electrode is driven, and the second sensing section is configured to directly consume interferents in the organism when the second working electrode is driven, thereby continuously reducing interference of the interferents to the measurement.

Description

[0001] This application is a divisional application of the invention patent application "Microbiosensor and method for reducing measurement interference" with application date of August 3, 2020 and application number 202010769134.5. Technical Field

[0002] The present invention relates to a micro biosensor, and more particularly to a micro biosensor capable of reducing measurement interference when measuring a target analyte in a biological fluid and a method for reducing the measurement interference. Background Art

[0003] Due to the rapid growth of the population of patients with chronic diseases, the detection of analytes in biological fluids in vivo is very important for the diagnosis and monitoring of patients, especially the effective monitoring of glucose concentration in the body is the key to the treatment of diabetes. Therefore, in recent years, continuous glucose monitoring (CGM) systems have become increasingly important. The system has many advantages over traditional biosensors, such as reducing the pain of finger blood sampling and continuously monitoring the physiological parameters of one or more target analytes in biological fluids.

[0004] Continuous glucose monitoring systems include enzyme-based biosensors that measure physiological signals corresponding to glucose concentration in the body, and most of them use electrochemical methods, that is, glucose oxidase (GOx) catalyzes the glucose reaction to generate gluconolactone and reduced enzymes, and then the reduced enzymes will transfer electrons with oxygen in the biological fluid in the body to generate hydrogen peroxide (H2O2) as a byproduct, and finally quantify the glucose concentration by catalyzing the oxidation reaction of the byproduct H2O2. However, if there are interfering substances such as ascorbic acid (AA), the main component of vitamin C, acetaminophen (AM), uric acid (UA), proteins or glucose analogs in the blood or tissue fluid, and their oxidation potential is close to the oxidation potential of H2O2, electrochemical signals unrelated to the target analyte will be generated. Such interfering signals must be reduced to ensure accurate measurement of physiological parameters. Summary of the invention

[0005] The micro biosensor of the present invention can be implanted subcutaneously in a living body to measure physiological parameters of a target analyte in a biological fluid. The micro biosensor of the present invention includes two working electrodes composed of different conductive materials, one of which can consume interfering substances in the biological fluid that affect the measurement, so that the other working electrode can obtain more accurate measurement results during measurement.

[0006] Therefore, the present invention provides a micro biosensor for implantation under the skin of a living body to measure a glucose concentration in a biological fluid and reduce the interference of at least one interferent in the biological fluid on the measurement, comprising: a substrate having a first surface and a second surface arranged opposite to each other; a first working electrode comprising at least a first sensing segment arranged on the first surface of the substrate, wherein the material of the first sensing segment of the first working electrode at least includes a first conductive material; a chemical reagent covering at least a portion of the first conductive material of the first sensing segment, and being used to react with glucose in the biological fluid and produce hydrogen peroxide; and at least one second working electrode arranged on the first surface of the substrate and comprising a second sensing segment, and the second sensing segment is adjacent to at least one side of the first sensing segment, wherein the material of the second sensing segment of the second working electrode at least includes a conductive material different from the first conductive material. A second conductive material, wherein: when the first working electrode is driven by a first working voltage so that the first sensing segment has a first sensitivity to the hydrogen peroxide and forms a measurement range, the first conductive material reacts with the hydrogen peroxide to generate a current signal, and a physiological signal is obtained when the value of the current signal corresponds to the glucose concentration; when the first working electrode is driven by the first working voltage so that the first conductive material reacts with the interferent to generate an interference current signal, the interference current signal is mixed with the current signal and output to interfere with the physiological signal; and when the second working electrode is driven by a second working voltage, the second conductive material has a second sensitivity to the hydrogen peroxide that is less than the first sensitivity, and the second sensing segment generates a de-interference range that contacts the periphery of the first sensing segment and can at least partially overlap with the measurement range, so as to consume the interferent and thereby reduce the generation of the interference current signal.

[0007] The present invention further proposes a micro biosensor for implantation under the skin of a biological body to measure a physiological parameter of a target analyte in a biological fluid and reduce the interference of an interferent in the biological fluid on the measurement, comprising: a substrate having a surface; a first working electrode comprising a first sensing segment disposed on the surface, wherein the first sensing segment comprises a first conductive material; at least one second working electrode disposed on the surface and comprising a second sensing segment adjacent to at least one side of the first sensing segment, wherein the second sensing segment comprises a second conductive material; and a chemical reagent covering at least a portion of the first conductive material, for reacting with the target analyte in the biological fluid to produce a product, wherein: when the first working electrode is driven by a first working voltage, the first conductive material reacts with the product to output a physiological signal corresponding to the physiological parameter of the target analyte; and when the second working electrode is driven by a second working voltage, the second conductive material consumes the interferent to reduce the interference of the interferent on the physiological signal.

[0008] The present invention further proposes a method for reducing interference in the measurement of a target analyte, comprising: providing a micro-biosensor for measuring a physiological parameter of a target analyte in a biological fluid, wherein the micro-biosensor comprises: a substrate having a surface; a first working electrode, comprising a first sensing segment disposed on the surface, and the first sensing segment comprises a first conductive material; at least one second working electrode, disposed on the surface and comprising a second sensing segment, and the second sensing segment comprises a second conductive material; and a chemical reagent, at least covering a portion of the first conductive material, for reacting with the target analyte in the biological fluid to produce a product; performing a de-interference action, wherein the de-interference action is to drive the second working electrode with a second working voltage so that the second conductive material consumes an interferent in the biological fluid to reduce the interference of the interferent on the measurement; and performing a measurement action, wherein the measurement action is to drive the first working electrode with a first working voltage so that the first conductive material reacts with the product to output a physiological signal corresponding to the physiological parameter of the target analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The above objects and advantages of the present invention will become more immediately apparent to those having ordinary knowledge in the art after referring to the following detailed description and the accompanying drawings.

[0010] FIG. 1(A) is a front view schematically showing a first embodiment of a micro biosensor according to the present invention.

[0011] FIG. 1(B) is a schematic diagram showing the configuration of the first working electrode and the second working electrode of the first embodiment of the micro biosensor of the present invention.

[0012] FIG. 2(A) is a schematic cross-sectional view along line AA' in FIG. 1(A) of the present invention.

[0013] FIG. 2(B) is a schematic cross-sectional view along line BB' in FIG. 1(A) of the present invention.

[0014] FIG. 2(C) is a schematic cross-sectional view along line CC' in FIG. 1(A) of the present invention.

[0015] FIG. 2(D) is a schematic cross-sectional view of the sensing region of a micro biosensor obtained by another process of the present invention.

[0016] FIG. 3(A) is a front view schematically showing a second embodiment of the micro biosensor of the present invention.

[0017] FIG. 3(B) is a schematic diagram showing the configuration of the first working electrode and the second working electrode of the second embodiment of the micro biosensor of the present invention.

[0018] Figure 4It is a schematic cross-sectional view along line AA' in FIG. 3(A) of the present invention.

[0019] FIG. 5(A) is a front view schematically showing a third embodiment of a micro biosensor according to the present invention.

[0020] FIG. 5(B) is a schematic cross-sectional view along line AA' in FIG. 5(A) of the present invention.

[0021] Figure 6(A)-6(C) Schematic diagram of other configurations of the first sensing segment and the second sensing segment of the present invention.

[0022] FIG6(D) is a schematic cross-sectional view along line II' in FIG6(C).

[0023] Figure 7 Schematic diagram of other configurations of the first sensing segment and the second sensing segment of the present invention.

[0024] Figure 8(A)-8(C) Schematic diagram of other configurations of the first sensing segment and the second sensing segment of the present invention.

[0025] FIG. 9(A) is a schematic cross-sectional view of the sensing region of the micro-biosensor of the present invention.

[0026] FIG9(B) is a schematic cross-sectional view of the sensing region of the micro-biosensor of the present invention.

[0027] Fig.10 It is a schematic diagram of the measuring range of the first sensing segment and the interference removal range of the second sensing segment of the micro biosensor of the present invention after being driven.

[0028] Fig.11 Schematic diagram of a circuit for controlling the voltage and measuring the current of the micro biosensor of the present invention.

[0029] Fig.12 The flowchart is a method for reducing interference generated by a micro biosensor during measurement according to the present invention.

[0030] Figure 13(A)-13(C) 13(A) is a schematic diagram of the time relationship between the interference removal action and the measurement action when the micro-biosensor is measured, wherein FIG13(B) is a diagram showing that the interference removal action and the measurement action are partially overlapped, and FIG13(C) is a diagram showing that the interference removal action and the measurement action are completely overlapped.

[0031] Fig.14 It is a schematic diagram of the time relationship between the interference removal action and the measurement action when the micro biosensor is measuring according to the present invention.

[0032] Fig.15It is a schematic diagram of the time relationship between the interference removal action and the measurement action when the micro biosensor is measuring according to the present invention.

[0033] Fig.16 FIG. 1 is a schematic diagram of the measurement range of the micro biosensor of the present invention when only the first sensing segment is driven.

[0034] Fig.17 It is a measurement curve diagram illustrating the results of applying the test examples of the present invention and the comparative test examples to an in vitro interference removal test, wherein when the interference removal function of the second working electrode is turned on, the current signal measured in the first sensing segment is curve C1, and the current signal measured in the second sensing segment is curve C2; ​​and when the interference removal function of the second working electrode is not turned on, the current signal measured in the first sensing segment is curve C3.

[0035] Figure 18(A)-18(B) 18(A) is a measurement curve without a de-interference mechanism, and FIG. 18(B) is a measurement curve with a de-interference mechanism. DETAILED DESCRIPTION

[0036] The detailed features and advantages of the present invention are described in detail in the following embodiments, and the contents are sufficient to enable anyone familiar with the relevant technology to understand the technical content of the present invention and implement it accordingly, and according to the contents, claims and drawings disclosed in this specification, anyone familiar with the relevant technology can easily understand the relevant purposes and advantages of the present invention. The following examples are to further illustrate the viewpoints of the present invention in detail, but are not intended to limit the scope of the present invention in any viewpoint.

[0037] The micro biosensor of the present invention can be a sensor of a continuous glucose monitoring system, which is implanted under the skin of a living body to continuously measure the physiological parameters of a target analyte in a biological fluid. In addition, the "target analyte" mentioned herein refers to any substance to be measured that exists in a living body, such as but not limited to glucose, lactose, uric acid, etc. "Biological fluid" can be but not limited to blood or interstitial fluid (ISF) of human tissues, and "physiological parameter" can be but not limited to concentration.

[0038] Please refer to FIG. 1(A), which is a front view of a first embodiment of a micro biosensor of the present invention. The micro biosensor 10 of the present invention comprises a substrate 110 having a surface 111, a first working electrode 120 and a second working electrode 130 disposed on the surface 111 of the substrate 110, and an insulating layer 140 covering a portion of the surface 111, a portion of the first working electrode 120, and a portion of the second working electrode 130. The substrate 110 of the present invention is an insulator. Please refer to Figure 1(B), in which removing the insulating layer 140 can more clearly illustrate the configuration of the first working electrode 120 and the second working electrode 130 on the surface 111 of the substrate 110, wherein the substrate 110 includes the surface 111, the opposite surface 112 (see Figures 2(A), 9(A) and 9(B)), the first end 113 and the second end 114, and the area on the substrate 110 close to the first end 113 is the signal output area 115, the section close to the second end 114 is the sensing area 116, and the area between the signal output area 115 and the sensing area 116 and covered by the insulating layer 140 is the insulating area 117. The first working electrode 120 and the second working electrode 130 extend from the first end 113 to the second end 114 of the substrate 110. The portion of the first working electrode 120 in the sensing region 116 and including the first conductive material 1C is the first sensing segment 121, the portion in the signal output region 115 is the first signal output segment 122 (as shown in FIG. 1(A)), and the portion connecting the first sensing segment 121 to the first signal output segment 122 and at least located in the insulating region 117 is the first signal connection segment 123 (as shown in FIG. 1(B)). The portion of the second working electrode 130 in the sensing region 116 and including the second conductive material 2C is the second sensing segment 131, the portion in the signal output region 115 is the second signal output segment 132 (as shown in FIG. 1(A)), and the portion connecting the second sensing segment 131 to the second signal output segment 132 and at least located in the insulating region 117 is the second signal connection segment 133 (as shown in FIG. 1(B)). The second sensing segment 131 of the present invention is disposed adjacent to at least one side of the first sensing segment 121 and extends along at least one side of the first sensing segment 121 with one side thereof. In the first embodiment, the second sensing segment 131 extends along three sides of the first sensing segment 121 and is in a U-shape. Therefore, the first sensing segment 121 and the second sensing segment 131 of the present invention maintain their relative positions only through the surface 111, and the first sensing segment 121 and the second sensing segment 131 are directly adjacent to each other without any intermediate objects such as electrodes or connecting wires.

[0039] In order to achieve such structures, during the manufacturing process, the second conductive material 2C may be first formed on the surface 111 of the substrate 110 and patterned into a pattern as shown in FIG. 1(B), that is, the second conductive material 2C is defined into two mutually separated blocks, wherein one block extending from the first end 113 of the substrate 110 toward the second end 114 and bending at the second end 114 to form a U-shape is preset as the second working electrode 130, and the other block extending from the first end 113 of the substrate 110 toward the second end 114 and surrounded by the aforementioned U-shaped structure at the second end 114 is preset as the first working electrode 120. After the insulating layer 140 is then formed on the substrate 110 to expose the signal output region 115 and the sensing region 116, the first conductive material 1C may be formed on the second conductive material 2C of the first working electrode 120 in the sensing region 116 to complete the first sensing segment 121 of the first working electrode 120. However, although not shown in the figure, the first conductive material 1C may also be formed only on a portion of the second conductive material 2C of the first working electrode 120 within the sensing region 116. Therefore, the cross-sectional schematic diagrams along the lines AA', BB' and CC' in FIG. 1(A) of the present invention are respectively Figure 2(A) , 2(B) 2(C). In FIG. 2(A), the first sensing segment 121 of the first embodiment of the present invention has the first conductive material 1C covering the second conductive material 2C, and the second sensing segment 131 has the second conductive material 2C. In FIG. 2(B), the U-shaped bottom area of ​​the second sensing segment 131 is shown, so only the second conductive material 2C is present on the surface 111 of the substrate 110. In FIG. 2(C), since the first conductive material 1C is only formed in the sensing area 116, the portion of the first working electrode 120 located in the insulating area 117 has only the second conductive material 2C and is covered by the insulating layer 140.

[0040] In other embodiments, the step of covering the insulating glue 140 can also be performed after forming the first conductive material 1C, so the first conductive material 1C can be substantially formed on all the second conductive materials 2C in the first working electrode 120. In addition, in the present invention, the position, size and shape of the second conductive material 2C retained after the patterning step can be changed as required, so in other embodiments, the second conductive material 2C can be defined as a pattern as shown in FIG. 1 (B) but without the first sensing segment 121 in the patterning step, that is, the second conductive material 2C of the first working electrode 120 is only formed in the signal output area 115 and the insulating area 117, or at most extends to part of the sensing area 116, and then the first conductive material 1C is directly formed on the original surface 111 in the area where the first sensing segment 121 is expected to be formed, and the first conductive material 1C is electrically connected to the other parts of the first working electrode 120 (i.e., the second conductive material 2C) to complete the configuration of the first sensing segment 121. At this time, the cross-sectional view of the sensing area 116 of the micro biosensor 10 is shown in FIG. 2 (D). In other embodiments, the position of the first working electrode 120 may be left empty during the patterning step, and then the first working electrode 120 may be formed with the first conductive material 1C before the covering insulating layer 140 is formed.

[0041] In the micro biosensor 10 of the present invention, the gap between the second sensing segment 131 and the first sensing segment 121 in the sensing region 116 is not greater than 0.2 mm. More preferably, the gap ranges from 0.01 mm to 0.2 mm; more preferably, the gap ranges from 0.01 mm to 0.1 mm; and even more preferably, the gap ranges from 0.02 mm to 0.05 mm. Specifically, referring to FIG. 2 (A), in the first embodiment, the gap distances S3 and S5 between the first sensing segment 121 and the second sensing segment 131 are 0.04 mm.

[0042] In addition, the first conductive material 1C of the present invention can be any one of carbon, platinum, aluminum, gallium, gold, indium, iridium, iron, lead, magnesium, nickel, molybdenum, osmium, palladium, rhodium, silver, tin, titanium, zinc, silicon, zirconium, their derivatives (such as alloys, oxides or metal compounds, etc.) or combinations thereof, and the second conductive material 2C can also use the elements or their derivatives as exemplified in the first conductive material 1C. The material of the insulating layer 140 of the present invention can be any material that can achieve an insulating effect, such as but not limited to Parylene, polyimide, polydimethylsiloxane (PDMS), liquid crystal polymer material (LCP) or MicroChem's SU-8 photoresist, etc.

[0043] Please refer to FIG. 3(A), which is a front view of the second embodiment of the micro biosensor 10 of the present invention, and the insulating layer 140 is removed in FIG. 3(B) to more clearly illustrate the configuration of the first working electrode 120 and the second working electrode 130 on the surface 111 of the substrate 110. In the second embodiment, the first working electrode 120 and the second working electrode 130 extend from the first end 113 to the second end 114 of the substrate 110, and the portion of the first working electrode 120 in the sensing area 116 and covered by the first conductive material 1C is the first sensing segment 121, and the portion of the second working electrode 130 in the sensing area 116 and containing the second conductive material 2C is the second sensing segment 131 (as shown in FIG. 3(A)). In the second embodiment, the second sensing segment 131 extends along one side of the first sensing segment 121 without being bent, so that the second sensing segment 131 is only adjacent to one side of the first sensing segment 121. Therefore, the cross-sectional schematic diagram along the A-A' line in FIG. 3(A) is as follows. Figure 4 As shown, the first sensing segment 121 of the second embodiment of the present invention has the first conductive material 1C covering the second conductive material 2C, and the second sensing segment 131 has the second conductive material 2C and is only disposed adjacent to one side of the first sensing segment 121 .

[0044] Please refer to FIG. 5(A), which is a front view of the third embodiment of the micro biosensor 10 of the present invention. In the third embodiment, the micro biosensor 10 has two second working electrodes 130, the first working electrode 120 and the two second working electrodes 130 extend from the first end 113 to the second end 114 of the substrate 110, and the two second working electrodes 130 extend along the opposite two sides of the first working electrode 120 respectively. The portion of the first working electrode 120 in the sensing region 116 and covered by the first conductive material 1C is the first sensing segment 121, and the portions of the two second working electrodes 130 in the sensing region 116 and containing the second conductive material 2C are the second sensing segments 131. In the third embodiment, the two second sensing segments 131 are respectively disposed adjacent to the opposite two sides of the first sensing segment 121. Therefore, the cross-sectional schematic diagram along the AA' line in Figure 5(A) is shown in Figure 5(B), where the first sensing segment 121 of the third embodiment of the present invention has a first conductive material 1C covering the second conductive material 2C, and the two second sensing segments 131 have the second conductive material 2C and are respectively disposed only adjacent to the opposite two sides of the first sensing segment 121.

[0045] Although the configuration between the first sensing segment 121 and the second sensing segment 131 of the present invention has been disclosed as described in the first to third embodiments, other configurations are also possible. For example, in the first embodiment, the second sensing segment 131 extends along three adjacent sides of the first sensing segment 121 and is U-shaped, but in a variation, the length of the second sensing segment 131 extending along the three sides of the first sensing segment 121 can also be adjusted as shown in FIG. 6(A), or the second sensing segment 131 can also be arranged only along the two adjacent sides of the first sensing segment 121 to be L-shaped, as shown in FIG. 6(B). In another variation of the first embodiment, the first signal connection segment 123 of the first working electrode 120 can be penetrated to the opposite surface 112 of the substrate 110 by drilling a hole 118 on the substrate 110. At this time, the second sensing segment 131 can surround the four sides of the first sensing segment 121, as shown in FIG. Figure 6(C)-6(D) Next, whether in the second embodiment or the third embodiment, the length of the second sensing segment 131 can be adjusted relative to the first sensing segment 121 as shown. Figure 7 to Figure 8 (C) Therefore, the aforementioned “the second sensing segment 131 is adjacent to at least one side of the first sensing segment 121 ” specifically refers to the portion of the periphery of the first sensing segment 121 of the present invention where the second sensing segment 131 is adjacent to accounts for 30%-100% of the total periphery of the first sensing segment 121 .

[0046] Furthermore, as shown in FIG. 1(A), FIG. 2(A), FIG. 3(A), Figure 4 5(A) and 5(B), the micro biosensor 10 of the present invention further comprises a chemical reagent layer 150. The chemical reagent layer 150 covers at least the first conductive material 1C of the first sensing section 121. Specifically, in the manufacturing process of the micro biosensor 10, the substrate 110 having electrodes formed on the surface 111 and / or the opposite surface 112 can be immersed in a solution containing a chemical reagent and the depth of the immersion of the substrate 110 can be adjusted so that the chemical reagent layer 150 formed subsequently can cover at least the sensing area 116 of the micro biosensor 10 at one time, that is, simultaneously cover the first conductive material 1C of the first sensing section 121 and the second conductive material 2C covering the second sensing section 131. Alternatively, the chemical reagent layer 150 can further cover part of the insulating area 117, as shown in FIG1(A). Thereby, the chemical reagent layer 150 covering the first conductive material 1C can react with the target analyte in the biological fluid to generate a product, and then the first conductive material 1C reacts with the product to output a physiological signal corresponding to the target analyte.

[0047] In any of the above embodiments, the configuration of the dual working electrodes of the present invention can be applied to a two-electrode system and a three-electrode system. In a two-electrode system, the micro biosensor 10 of the present invention further includes at least one pair of electrodes 160 disposed on the opposite surface 112 of the substrate 110, as shown in FIG. 9(A), which is a cross-sectional schematic diagram of the sensing area 116 of the micro biosensor 10, and the counter electrode 160 is used to cooperate with the first working electrode 120 or the second working electrode 130. The counter electrode 160 in the two-electrode system can also function as a reference electrode based on the selected material, that is, the counter electrode 160 of the present invention can form an electronic circuit with the first working electrode 120, so that the current on the first working electrode 120 is unobstructed, so as to ensure that the electrochemical reaction occurs on the first working electrode 120, and can also provide a stable relative potential as a reference potential. The counter electrode 160 is coupled to the first working electrode 120 and / or the second working electrode 130. In other embodiments, the counter electrode 160 can also be disposed on the surface 111 of the substrate 110 (not shown). In the three-electrode system, the micro biosensor 10 of the present invention includes a reference electrode 170 in addition to the counter electrode 160, as shown in FIG9(B), which is a cross-sectional schematic diagram of the sensing area 116 of the micro biosensor 10, and the reference electrode 170 is used to provide a reference potential. Specifically, the counter electrode 160 and the reference electrode 170 are separated and not electrically connected, and the counter electrode 160 is coupled to the first working electrode 120 and / or the second working electrode 130. The counter electrode 160 and the reference electrode 170 can also be disposed on the surface 111 of the substrate 110 (not shown), or respectively disposed on different surfaces of the substrate 110. In addition, as shown in the figure, in one embodiment of the present invention, the chemical reagent layer 150 is substantially covered on the counter electrode 160 and / or the reference electrode 170.

[0048] It should be noted that in the present invention, the so-called "driving" is to apply a voltage so that the potential of one electrode is higher than the potential of the other electrode so that the electrode with the higher potential starts an oxidation reaction. Therefore, the potential difference between the first working electrode 120 and the counter electrode 160 that causes the first working electrode 120 to be driven is the first working voltage, and the potential difference between the second working electrode 130 and the counter electrode 160 that causes the second working electrode 130 to be driven is the second working voltage.

[0049] Further cooperation Fig.10To illustrate, the first working electrode 120 of the micro biosensor 10 of the present invention is driven to measure the physiological parameters of the target analyte in the biological fluid, and when the first working electrode 120 of the micro biosensor 10 is driven by the first working voltage, the first sensing section 121 will generate a measurement range 1S and have a first sensitivity to the product, so that the first conductive material 1C reacts with the product to generate a current signal, and the current signal is transmitted to the first signal output section 122 via the first signal connection section 123, and when the value of the current signal is proportional to the concentration of the product, a physiological signal corresponding to the physiological parameter is obtained. Therefore, when the first working electrode 120 is driven by the first working voltage, the action of the first conductive material 1C reacting with the product to output a physiological signal corresponding to the physiological parameter of the target analyte is the measurement action. However, if the biological fluid contains interfering substances, the first conductive material 1C will also react with the interfering substances to generate an interfering current signal, and the interfering current signal will be mixed with the current signal and output to interfere with the physiological signal.

[0050] The second working electrode 130 of the micro biosensor 10 of the present invention can be used to consume interference. When the second working electrode 130 of the micro biosensor 10 is driven by the second working voltage, the second conductive material 2C of the second sensing segment 131 has a second sensitivity to the product, and each second sensing segment 131 will generate an interference removal range 2S. At this time, because the second sensing segment 131 is quite close to the first sensing segment 121, the interference removal range 2S contacts the periphery of the first sensing segment 121 and can at least partially overlap the measurement range 1S of the first sensing segment 121, whereby the second conductive material 2C will undergo an oxidation reaction with the interference to directly and continuously consume the interference, reduce the generation of interference current signals, and further reduce the influence of the interference on the measurement action. Therefore, when the second working electrode 130 is driven by the second working voltage, the action of causing the second conductive material 2C to consume the interference in the organism is a interference removal action.

[0051] Furthermore, when the second working electrode 130 is driven by the second working voltage, the second conductive material 2C also has the opportunity to react with the product to generate another current signal, but consumes the product that should be measured by the first working electrode 120 and used to infer the physiological parameters of the target analyte, thereby affecting the actual measured physiological parameters. Therefore, when the target analyte is glucose, the product is hydrogen peroxide, and the physiological parameter is the glucose concentration in one embodiment, the first conductive material 1C is more preferably selected from a material having a first sensitivity to hydrogen peroxide after being driven by the first working voltage, and more preferably, the first conductive material 1C can be selected from gold, platinum, palladium, iridium or a combination of the foregoing. The second conductive material 2C needs to be different from the first conductive material 1C, and specifically, it is necessary to select a material having a second sensitivity to hydrogen peroxide that is less than the first sensitivity after being driven by the second working voltage, or even a material that is almost insensitive to hydrogen peroxide after being driven by the second working voltage (that is, the second sensitivity is close to 0 or equal to 0). More specifically, in one embodiment of the present invention, the first conductive material 1C is platinum, and the first operating voltage range is recommended to be 0.2 volts (V) to 0.8 volts (V), and preferably 0.4 V to 0.7 V, and the second conductive material 2C is carbon, and the second operating voltage range is recommended to be 0.2 V to 0.8 V, and preferably 0.4 V to 0.7 V. However, in another embodiment of the present invention, the first conductive material 1C is platinum, and the second conductive material 2C is gold. It must be noted that the above-mentioned platinum can be in the form of platinum metal, platinum black, platinum paste, other platinum-containing materials or a combination thereof. In addition, the value of the first operating voltage can be the same as the value of the second operating voltage, but the present invention is not limited thereto.

[0052] Next, see Fig.11 and Fig.12 Further explanation of how to operate the micro biosensor 10 of the present invention, wherein Fig.11 9 (A) of the present invention is a schematic diagram of an example of a circuit for controlling the voltage and measuring the current of the micro biosensor 10, and Fig.12 This is a flow chart of a method for reducing interference generated by the micro biosensor 10 during measurement according to the present invention. Fig.11In the figure, the current sensing unit 201 is connected to the first working electrode 120 of the micro biosensor 10, and the other current sensing unit 202 is connected to the counter electrode 160. The current sensing units 201 and 202 measure the current signals i1 and i3 from the first working electrode 120 and the counter electrode 160 respectively. i2 is the current signal of the second working electrode 130, which can also be measured by other current sensing units (not shown). In this example, the first working voltage is the difference between the potential V1 of the first working electrode 120 and the potential V3 of the counter electrode 160, and the second working voltage is the difference between the potential V2 of the second working electrode 130 and the potential V3 of the counter electrode 160. Switches S1 and S2 can respectively determine whether the first working electrode 120 and the second working electrode 130 are connected to voltage. The method of reducing measurement interference of the present invention is as follows. Fig.12 The method includes providing a micro biosensor (step 101), performing a de-interference action (step 102), and performing a measurement action (step 103). The de-interference action and the measurement action of the present invention are in a time relationship, and the possible operation timings are as follows:

[0053] The first time relationship: The micro biosensor of the present invention will be measured within a period T (such as 2 weeks), and the period T includes multiple first sub-time (T1) segments and / or multiple second sub-time (T2) segments. The interference removal action is performed in the first sub-time (T1) segment, and the measurement action is performed in the second sub-time (T2) segment, and the interference removal action and the measurement action are performed at intervals. In other words, the execution method is to perform the first interference removal action in the first T1 segment to consume the interference, perform the first measurement action in the first T2 segment to output the first physiological signal corresponding to the physiological parameter at that time, perform the second interference removal action in the second T1 segment to consume the interference, and then perform the second measurement action in the second T2 segment to output the second physiological signal corresponding to the physiological parameter at that time, and so on, to obtain all the performances of the physiological parameters in each T2 segment in the period T. Please refer to Figure 13(A) to Figure 13(C), where the horizontal axis is time and the vertical axis is current, wherein the measurement sequence lines represent the application and removal of the first working voltage, and the interference removal sequence lines represent the application and removal of the second working voltage. In the first time relationship, the T1 segment and the T2 segment can partially overlap (as shown in FIG. 13 (A)), not overlap (as shown in FIG. 13 (B)), or completely overlap (as shown in FIG. 13 (C)). During the period T, the interference removal action can be stopped between any two T1 segments by removing the second working voltage as a partition, and the measurement action can also be stopped between any two T2 segments by removing the first working voltage as a partition between two T2 segments. In the first time relationship, the length of the T2 segment is based on the condition that the current signal corresponds to the concentration of the product and is proportional to the physiological parameter, and the length of the T1 segment can be the same as or longer than the length of the T2 segment, so as to achieve the goal of effectively removing interference.

[0054] In addition, as shown in FIG. 13(A) or FIG. 13(B), the first interference removal action may preferably be performed earlier than or simultaneously with the first measurement action to effectively avoid interference of the interference object in the body with the measurement. That is, when there are multiple measurement actions, the interference removal action is performed at least once and is preferably started no later than the start of the first measurement action of each of the multiple measurement actions.

[0055] The second time relationship: The micro biosensor of the present invention will perform measurements within a period T (such as 2 weeks), and the period T includes multiple sub-time segments. The interference removal action is performed during the entire period T, and the measurement action is performed in each sub-time segment, and the measurement action is performed at intervals. In other words, please refer to Fig.14 , the second time relationship is to continuously perform the interference removal action during the entire period T to consume the interference until the end of the period, and at the same time perform the first measurement action in the first sub-time segment to output the first physiological signal corresponding to the physiological parameter at that time, and perform the second measurement action in the second sub-time segment after a period of time after the first measurement action to output the second physiological signal corresponding to the physiological parameter at that time, and so on, to obtain all the performances of the physiological parameters in each sub-time segment during the period T. In the period T, the measurement action can be stopped by removing the first working voltage between any two sub-time segments as an interval. In the second time relationship, the lengths of each sub-time segment can be the same or different, and their lengths are also based on the condition that the current signal can correspond to the concentration of the product and be proportional to the physiological parameter.

[0056] The third time relationship: Although not shown in the figure, the third time relationship is different from the second time relationship in that the third time relationship is to perform the measurement action uninterruptedly during the entire period T, and to perform the interference removal action in each sub-time segment, that is, the interference removal action is performed at intervals.

[0057] The fourth time relationship: please refer to Fig.15 The micro biosensor of the present invention will perform measurements within a period T (such as 2 weeks), and will continuously perform measurement actions during the entire period T, and will also continuously perform interference removal actions until the end of the period, so as to continuously remove interferences and measure physiological parameters.

[0058] In vitro interference assay

[0059] [Test example]

[0060] In the test example, a micro biosensor having a dual working electrode structure of the first embodiment was used for measurement, wherein the first sensing segment was a carbon electrode covered with platinum black, the second sensing segment was a carbon electrode, the first working voltage was 0.5 V, and the second working voltage was 0.5 V, and the interfering substance was acetaminophen.

[0061] [Comparative test example]

[0062] The micro biosensor used in the comparative test example is the same as that in the test example, but the second working voltage is not applied. Since the second working voltage is not applied, the second sensing segment 131 is not driven, so only the measurement range of the first sensing segment 121 is 1S. Fig.16 shown.

[0063] Next, the in vitro interference removal test was performed using the micro biosensor of the present invention as follows. First, the micro biosensors of the test example and the comparative test example were immersed in phosphate buffered saline (PBS) solution, 40 mg / dL glucose solution, 100 mg / dL glucose solution, 300 mg / dL glucose solution, 500 mg / dL glucose solution, 100 mg / dL glucose solution, 100 mg / dL glucose solution mixed with 2.5 mg / dL acetaminophen, 100 mg / dL glucose solution and 100 mg / dL glucose solution mixed with 5 mg / dL acetaminophen at different time intervals (P1 to P9). The test results are as follows. Fig.17 As shown, in the test example, the current signal measured in the first sensing section 121 is a curve C1, the current signal measured in the second sensing section 131 is a curve C2, and in the comparative test example, the current signal measured in the first sensing section 121 is a curve C3.

[0064] from Fig.16It can be seen from the time intervals P1 to P5 that the first sensing segment of both the test example and the comparative test example will generate current signals of different strengths in different time segments with different concentrations of glucose, that is, there is a proportional relationship between the current signal of the first sensing segment and the physiological parameter, but no current signal is generated in the second sensing segment of the test example, indicating that the reactivity of the second sensing segment to the byproduct hydrogen peroxide derived from glucose catalyzed by enzymes is very low, close to or equal to 0. Next, it can be seen from curve C3 that when the micro-biosensor of the comparative test example is immersed in a 100 mg / dL glucose solution containing 2.5 mg / dL acetaminophen in time segment P7, compared with its current signal in time segment P3, the current signal measured by the first sensing segment 121 in time segment P7 is obviously disturbed by acetaminophen and floats higher, and the degree of interference in the measurement is more obvious when the micro-biosensor is immersed in a 100 mg / dL glucose solution containing 5 mg / dL acetaminophen in time segment P9. On the contrary, it can be seen from curves C1 and C2 that when the micro biosensor of the test example is immersed in a 100 mg / dL glucose solution containing 2.5 mg / dL acetaminophen in time segment P7, its current signal is consistent with the current performance of time segment P3, that is, when the second working electrode 130 is driven by the second working voltage to perform the interference removal action, the degree to which the first sensing segment 121 is affected by acetaminophen can be reduced, even if the concentration of acetaminophen is increased. From another perspective, since the second sensing segment 131 of the second working electrode 130 is used to consume acetaminophen, it has no current signal in both the PBS solution and the glucose solution, but will generate a current signal when acetaminophen is present. It can be seen from this that when acetaminophen is present in the measurement environment (i.e., the aforementioned measurement range), the second sensing segment 131 will consume acetaminophen, reducing the possibility that the measurement of the first sensing segment 121 is interfered by acetaminophen. In this way, the micro biosensor of the present invention can measure more accurate physiological parameters.

[0065] In vivo interference removal assay

[0066] In the in vivo interference removal test, the micro biosensor with the dual working electrode structure of the first embodiment of the present invention was also used for measurement, wherein the first sensing segment was a carbon electrode covered with platinum black, the second sensing segment was a carbon electrode, the first working voltage was 0.5V, and the second working voltage was 0.5V. The micro biosensor was actually implanted subcutaneously in the human body to continuously monitor the glucose concentration in the interstitial fluid, and 1g of Panadol (whose main ingredient is acetaminophen) was taken at the 86th hour, and the data with and without the interference removal mechanism were measured, and compared with the traditional blood glucose meter (Blood Glucose Meter, BGM), and the experimental results are as follows: Figure 18(A)-18(B)As shown, FIG. 18(A) is a measurement curve without a de-interference mechanism, and FIG. 18(B) is a measurement curve with a de-interference mechanism.

[0067] exist Figure 18(A)-18(B) In the figure, the black dots are the values ​​measured by the conventional blood glucose meter, the dotted line is the measurement curve of the first working electrode in the micro biosensor of the present invention, and the solid line is the measurement curve of the second working electrode in the micro biosensor of the present invention. As can be seen from FIG18(A) when the interference removal function is not turned on, the values ​​measured by the first working electrode of the micro biosensor of the present invention are all high around the 90th to 96th hour (i.e., the reaction time is 4-6 hours after taking 1g of Panadol). On the contrary, as can be seen from FIG18(B) when the interference removal function is turned on, the second working electrode of the micro biosensor of the present invention will measure the corresponding current value, and the measurement value of the first working electrode will not be high and can match the value of the conventional blood glucose meter.

[0068] In addition, when the micro-biosensor has the interference removal function turned on, its average error value during the period without drug interference is 0.1 mg / dL, the average error value during the drug interference period is -2.1 mg / dL, the total error value is -1.1 mg / dL, and the mean absolute relative deviation (MARD) during the drug interference period is 4.6. When the micro-biosensor does not have the interference removal function turned on, its average error value during the period without drug interference is -0.2 mg / dL, but the average error value during the drug interference period is 12.6 mg / dL, the total error value is 6.7 mg / dL, and the mean absolute relative deviation value during the drug interference period is 10.6. It can be seen that the action of consuming the interferent by the second sensing segment 131 of the second working electrode 130 can indeed reduce the influence of the interferent on the physiological signal measured by the first sensing segment 121 to less than or equal to an error range, such as 20%, and more specifically 10%. In summary, the present invention disposes the second sensing segment in the micro biosensor adjacent to at least one side of the first sensing segment, so that the second sensing segment can directly and continuously consume the interference around the first sensing segment, thereby reducing the interference of the interference on the measurement of the first sensing segment, so that the micro biosensor can measure more accurate data.

[0069] The present invention is a rare innovative invention with great industrial value, and the application is filed in accordance with the law. In addition, the present invention can be modified by a person with ordinary knowledge in the technical field, but it does not deviate from the scope of protection as shown in the attached patent application.

[0070]

Explanation of symbols

[0071] 10: Micro biosensors

[0072] 110:Substrate

[0073] 111: Surface

[0074] 112: Opposite surface

[0075] 113: First End

[0076] 114: Second End

[0077] 115:Signal output area

[0078] 116: Sensing area

[0079] 117: Insulation area

[0080] 118: Holes

[0081] 120: first working electrode

[0082] 121: First sensing section

[0083] 122: First signal output section

[0084] 123: First signal connection section

[0085] 130: second working electrode

[0086] 131: Second sensing segment

[0087] 132: Second signal output section

[0088] 133: Second signal connection section

[0089] 140: Insulation glue

[0090] 150: Chemical reagent layer

[0091] 160: Counter electrode

[0092] 170: Reference electrode

[0093] S101-S103: Steps

[0094] 1C: First conductive material

[0095] 2C: Second conductive material

[0096] 1S: Measuring range

[0097] 2S: Interference removal range

Claims

1. A micro biosensor for implantation under the skin of a living body to measure a physiological parameter of a target analyte in a biological fluid and reduce the interference of an interfering substance in the biological fluid on the measurement, comprising: a substrate having a surface; a first working electrode, comprising a first sensing segment disposed on the surface, wherein the first sensing segment comprises a first conductive material; At least one second working electrode is disposed on the surface and includes a second sensing segment, wherein the second sensing segment is disposed with one side thereof extending along the periphery of the first sensing segment, and a portion of the periphery of the first sensing segment adjacent to the second sensing segment accounts for 30%-100% of the total periphery of the first sensing segment, and the second sensing segment comprises a second conductive material; as well as a chemical reagent, covering at least a portion of the first conductive material, for reacting with a target analyte in the biological fluid to produce a product, wherein: When the first working electrode is driven by a first working voltage to perform a measurement operation, the first conductive material reacts with the product to output a physiological signal corresponding to the physiological parameter of the target analyte; as well as When the second working electrode is driven by a second working voltage to perform an interference removal operation, the second conductive material consumes the interferer to reduce the interference of the interferer on the physiological signal.

2. The micro biosensor as claimed in claim 1, wherein the second conductive material is different from the first conductive material, and when the first working electrode is driven by the first working voltage, the first conductive material has a first sensitivity to the product, and when the second working electrode is driven by the second working voltage, the second conductive material has a second sensitivity to the product that is smaller than the first sensitivity. 3 . The micro biosensor as claimed in claim 2 , wherein the second sensitivity is close to or equal to 0. 4 . The micro biosensor as claimed in claim 1 , wherein the first operating voltage and the second operating voltage have the same value. 5 . The micro biosensor as claimed in claim 1 , wherein the chemical reagent further covers a portion of the second conductive material of the second sensing segment of the second working electrode. 6 . The micro biosensor as claimed in claim 1 , wherein the second sensing section is adjacent to the periphery of the first sensing section with a gap therebetween, and the gap is no greater than 0.2 mm. 7 . The micro biosensor as claimed in claim 1 , wherein the number of the second working electrodes is two, and the two second sensing segments of the two second working electrodes are respectively disposed adjacent to two opposite sides of the first sensing segment.

8. The micro biosensor as claimed in claim 1 further comprises at least one pair of electrodes, and the substrate further comprises a pair of side surfaces opposite to the surface, wherein the pair of electrodes are disposed on the pair of side surfaces and coupled to at least one of the first working electrode and the second working electrode.

9. The micro biosensor as claimed in claim 1, wherein the micro biosensor is used to perform the measurement within a period, wherein the measurement action and the interference removal action are performed simultaneously, and the micro biosensor performs the measurement action and the interference removal action uninterruptedly within the period. 10 . The micro biosensor as claimed in claim 9 , wherein the interference elimination operation is performed no later than the measurement operation.

11. The micro biosensor as claimed in claim 1, wherein the micro biosensor is used to perform the measurement within a period, wherein the micro biosensor performs the measurement action at intervals, and the micro biosensor continuously performs the interference removal action within the period. 12 . The micro biosensor as claimed in claim 11 , wherein when there are multiple measurement actions, the interference elimination action is started at least earlier than the start of the first measurement action of each of the multiple measurement actions.

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