TWI475221B - Method of a test strip detecting concentration of an analyte of a sample, three-electrode test strip, and method of utilizing a test strip detecting diffusion factor of a mediator of a sample - Google Patents

Description

Method for detecting the concentration of a sample to be tested in a sample, a test piece for a three electrode, and a profit Method for detecting medium diffusion factor in a sample by using a test piece

The invention relates to a method for detecting the concentration of a sample to be tested in a sample, a test piece of a three-electrode and a method for detecting a medium diffusion factor in a sample by using a test piece, in particular, an electric device having different polarities at different time periods. The signal detects the concentration of the analyte in the sample and the method of detecting the medium diffusion factor in the sample, and the test piece for detecting the concentration of the analyte in the sample and detecting the medium diffusion factor in the sample.

Electrochemical biosensors have been widely used in test samples to determine the concentration of different analytes (eg, glucose, uric acid, and cholesterol in biological fluids). For example, in a biological sample test, a test piece can be inserted into a blood glucose meter, and a single liquid sample can be dropped into the test piece and the body fluid sample is directed to the same chamber to determine the concentration of the analyte in the biological sample.

In recent years, the number of people with diabetes has gradually increased, so monitoring blood glucose levels is very important for the daily life of diabetic patients. Routine testing blood glucose 3-4 times a day and controlling fixed blood glucose levels can reduce the risk of serious damage, such as vision loss and kidney failure. Therefore, accurate blood glucose measurements must be expected for the daily life of diabetic patients.

However, the biosensor test results will contain a lot of analysis errors. When test sample In the case of whole blood, the source of the analytical error may be derived from the physical characteristics of the whole blood (eg, interferents), environmental factors (eg, temperature), and operational conditions (eg, unfilled), where the physical properties of the whole blood contain interferents, For example, blood volume ratio (volume ratio of red blood cells to whole blood), ascorbic acid, uric acid, cholesterol, and the like.

For example, a typical person has a normal blood volume ratio ranging from about 35% to 55%. However, in some special cases, the blood volume ratio can range from 10% to 70%, resulting in large errors in blood glucose measurements. In high blood volume ratios, red blood cells can block the reaction of enzymes and electron transport media, and even reduce the rate of diffusion of electron transport media to the working electrode, resulting in hypoglycemia readings. Conversely, a low blood volume ratio produces a high blood glucose reading.

The prior art provides a number of methods to reduce the analytical error of the hematocrit effect. For example, U.S. Patent No. 5,591,836 discloses a reagent formulation for filtering red blood cells using silicone particles. U.S. Patent No. 5,628,890 discloses the effect of reducing blood volume ratio effects by dispersing blood samples at a wide interval in combination with a mesh layer. U.S. Patent No. 8,388,821 discloses a method of providing a plurality of microelectrodes on a working electrode for blood volume ratio related measurements. U.S. Patent No. 2011/0139634 discloses the measurement of analyte concentration after measurement of blood volume ratio by using a two-electrode group (application of a direct current signal and an alternating current signal, respectively). However, the methods provided by the prior art have a number of disadvantages, such as high manufacturing costs, complicated processes, and the need for large test sample volumes.

In addition, temperature is another source of analytical error during blood glucose measurement. Because the enzyme reaction is a temperature-dependent reaction, temperature changes during blood glucose measurement will affect the accuracy of blood glucose measurements.

In summary, the methods provided by the prior art are not a good choice for the user.

An embodiment of the present invention provides a method for detecting a concentration of a test object in a sample, wherein the test piece includes a substrate and a reaction zone, and the reaction zone includes a working electrode, a reference electrode, and an auxiliary electrode, and Apply an enzyme. The method comprises: placing the sample into the reaction zone, wherein the analyte and the enzyme react to generate a plurality of electrons, and transmitting the plurality of electrons to the working electrode through a medium; applying an electrical signal to the working electrode; Measuring a first current through the working electrode in a first period; the medium generates an intermediate product according to the electrical signal in a second period; and measuring a second current through the working electrode in a third period, wherein the current The second polarity of the signal in the second period is opposite to the first polarity in the first period and the third polarity in the third period; according to the first current, an initial concentration of the object to be tested is calculated; A second current is calculated, and a diffusion factor of the intermediate product in the sample is calculated; according to the diffusion factor, the initial concentration is corrected to generate a new concentration of the analyte.

Another embodiment of the present invention provides a three-electrode test piece. The test piece comprises a substrate and a reaction zone. The reaction zone is formed at a first end of the substrate, and the reaction zone is coated with an enzyme, wherein when the reaction zone is placed in the reaction zone, the analyte in the sample reacts with the enzyme to generate a plurality of electrons, and A medium transfers the plurality of electrons. The reaction zone comprises a working electrode, a reference electrode and an auxiliary electrode. The working electrode is configured to receive an electrical signal when the sample is placed in the reaction zone, generate a first current according to the electrical signal during a first time period, and a third time period after a second time period, Generating a second current according to the electrical signal, wherein the second polarity of the electrical signal during the second time period is opposite to a first polarity of the first time period and a third polarity of the third time period, wherein the medium Forming an intermediate product according to the electrical signal during the second time period; the reference electrode is configured to receive a reference voltage when the sample is placed in the reaction zone; and the auxiliary electrode is configured to insert the sample into the reaction zone Receiving a floating voltage to satisfy a current generated by the working electrode in the first period, the second period, and the third period; the first current is used to calculate the object to be tested The initial concentration, the second current is a diffusion factor used to calculate the intermediate product, and the diffusion factor is a new concentration used to correct the initial concentration to produce the analyte.

An embodiment of the invention provides a method for detecting a concentration of a sample in a sample by a test piece. The method comprises a substrate and a reaction zone, wherein the reaction zone comprises a working electrode, a reference electrode and an auxiliary electrode, and an enzyme is coated. The method comprises: placing the sample into the reaction zone, wherein the analyte and the enzyme react to generate a plurality of electrons, and transmitting the plurality of electrons to the working electrode through a medium; applying an electrical signal to the working electrode; Measuring a first current through the working electrode in a first period; the medium generates an intermediate product according to the electrical signal in a second period; and measuring a second current through the working electrode in a third period, wherein the current The signal has a second polarity and a polarity in the second period, and the second polarity is opposite to a first polarity of the electrical signal in the first period and a third polarity of the electrical signal in the third period; Calculating an initial concentration of the analyte according to the first current; calculating a diffusion factor of the intermediate product in the sample according to the second current; and correcting the initial concentration according to the diffusion factor to generate a new sample concentration.

Another embodiment of the present invention provides a method for detecting a medium diffusion factor in a sample using a test piece, wherein the test piece includes a reaction zone including a working electrode, a reference electrode, and an auxiliary electrode. The method includes placing an identical portion into the reaction zone; applying an electrical signal to the working electrode; the medium generating an intermediate product according to the electrical signal during a first time period; and passing the second time period after the first time period The working electrode measures a first current, wherein the second polarity of the electrical signal during the second period is opposite to the first polarity of the first period; and the diffusion factor of the intermediate product in the sample is estimated based on the first current .

Another embodiment of the present invention provides a method for detecting a medium diffusion factor in a sample using a test piece, wherein the test piece includes a reaction zone including a working electrode, a reference electrode, and an auxiliary electrode. The method includes placing the same in the reaction zone; applying an electrical signal to the a working electrode; the medium generates an intermediate product according to the electrical signal during a first time period; and measuring a first current through the working electrode during a second time period after the first time period, wherein the electrical signal has the first time period A first polarity and a non-polarity, and the first polarity is opposite to a second polarity of the electrical signal during the second period; and based on the first current, a diffusion factor of the intermediate product in the sample is estimated.

The invention provides a method for detecting the concentration of a sample to be tested in a sample, a test piece for three electrodes, and a method for detecting a medium diffusion factor in a sample by using a test piece. The method and the test piece use a working electrode to generate a first current according to an electrical signal provided by a measuring circuit in a first period of time to estimate an initial concentration of the analyte in the sample, using the working electrode. The medium in the sample is reacted according to the electrical signal during a second period, and the working electrode is used to generate a diffusion factor of the medium in the sample according to the electrical signal for a third period of time. Two currents. After the diffusion factor is generated, the measuring circuit can correct the initial concentration of the analyte in the sample according to the diffusion factor to generate a new concentration of the analyte. Therefore, the present invention can accurately correct the initial concentration of the analyte in the sample compared to the prior art. In addition, the present invention further provides a method for detecting a medium diffusion factor in a sample by using a test piece by detecting a sample intermediate by using an electrical signal of a first polarity in a first period and a second polarity in a second period. Mass diffusion factor. Therefore, the present invention can quickly, simply and accurately detect the medium diffusion factor in a sample compared to the prior art.

100‧‧‧ test piece

110, 601‧‧‧ substrate

120‧‧‧electrode layer

121, WE‧‧‧ working electrode

122, CE‧‧‧ auxiliary electrode

123, RE‧‧‧ reference electrode

124, 125, 126, WP, RP, CP, WP1, WP2‧‧‧ pads

127, 128, 129‧‧ ‧ electrode track

130‧‧‧Insulation

131‧‧‧ Groove

140‧‧‧Reagent layer

150‧‧‧Baffle

151‧‧‧T-channel

160‧‧‧First cover

170‧‧‧second cover

201‧‧‧ lateral vents

300, 600, 1100‧‧‧ three-electrode test piece

302, 602‧‧‧Reaction zone

304, 604, 704, 804, 904‧‧‧ measuring circuits

IW, IW1, IW2, IW3, IW4, IWP‧‧‧ current

OP1, OP2‧‧‧Operational Amplifier

RW‧‧‧ equivalent resistance

T1, T2, T3‧‧‧

VG‧‧‧ ground voltage

VCE‧‧‧Floating voltage

VWE‧‧‧ actual voltage

VRE, VRP, VWP, V2‧‧‧ voltage

V1‧‧‧ reference voltage

2800-2818, 2900-2912‧‧‧ steps

Fig. 1 is a schematic view showing the explosion of a test piece according to an embodiment of the present invention.

Fig. 2 is a schematic view showing a cross section of the test piece.

Figure 3 is a schematic diagram showing a three-electrode test piece, a reaction zone, and a measuring circuit.

Fig. 4 is a schematic view showing a three-electrode test piece, a reaction zone, and a measuring circuit according to an embodiment of the present invention.

Fig. 5 is a schematic view showing a three-electrode test piece, a reaction zone, and a measuring circuit according to another embodiment of the present invention.

Figure 6 is a schematic view showing a three-electrode test piece, a reaction zone, and a measuring circuit according to another embodiment of the present invention.

Figure 7 is a schematic view showing a three-electrode test piece, a reaction zone, and a measuring circuit according to another embodiment of the present invention.

Figure 8 is a schematic view showing the structure of a three-electrode test piece

Figure 9 is a schematic view showing a three-electrode test piece according to another embodiment of the present invention.

Fig. 10 is a schematic view illustrating the pressure difference between the working electrode and the reference electrode in the first period, the second period, and the third period.

11 to 14 are schematic diagrams showing changes in the concentration distribution of the medium and the intermediate product in the reaction zone as a function of the pressure difference between the working electrode and the reference electrode when the reaction zone is placed in the sample.

Fig. 15 to Fig. 17 are diagrams showing the relationship between the diffusion factor of the medium and the current generated by the working electrode under different interferers.

18 to 24 are schematic views illustrating the pressure difference between the working electrode and the reference electrode in the first period, the second period, and the third period, in accordance with various embodiments of the present invention.

Fig. 25 is a view showing the current generated by the working electrode corresponding to different samples in the case of the pressure difference between the working electrode and the reference electrode in Fig. 10.

Figure 26 is a diagram illustrating the relationship between blood volume ratio and deviation before correction by the present invention.

Figure 27 is a graph showing the relationship between blood volume ratio and deviation after correction by the present invention.

Figure 28 is a flow chart showing a method of detecting the concentration of a sample in a sample by a test piece according to another embodiment of the present invention.

Figure 29 is a flow chart showing a method of detecting a medium diffusion factor in a sample using a test piece according to another embodiment of the present invention.

The invention is illustrated by the following examples in which it is understood that the following examples are intended to illustrate the invention and not to limit the scope of the invention.

Please refer to FIG. 1. FIG. 1 is a schematic view showing the explosion of a test piece 100 according to an embodiment of the present invention. As shown in FIG. 1 , the test piece 100 can include a substrate 110 , an electrode layer 120 , an insulating layer 130 , a reagent layer 140 , a spacer 150 , a first cover layer 160 , and a second cover layer 170 . The substrate 110 is made of a plastic material (for example, polyethylene terephthalates (PET), vinyl polymers, polyimides, or polyesters). Composition.

The substrate 110 supports an electrode layer 120. The electrode layer 120 has a working electrode 121, an auxiliary electrode 122 and a reference electrode 123. The working electrode 121, the auxiliary electrode 122 and the reference electrode 123 are formed at the first end of the electrode layer 120. The working electrode 121, the auxiliary electrode 122, and the reference electrode 123 may be laser-engraved on the electrode layer 120 or printed on the substrate 110 by screen printing. The second end of the electrode layer 120 can provide a plurality of pads 124, 125, 126, wherein the plurality of pads 124, 125, 126 are electrically connected to a measuring circuit. An electrode track 127 provides a continuous conductive path from the working electrode 121 to the pad 124. Similarly, an electrode track 128 provides a continuous conductive path from the auxiliary electrode 122 to the pad 125, and the electrode track 129 provides a continuous conductive path from the reference electrode 123 to the pad 126. The electrode layer 120 may be composed of a conductive material (for example, gold, platinum, silver, carbon or carbon/silver composite material) provided by the prior art.

An insulating layer 130 can be used to protect the electrode tracks described above and to define an effective area of a reaction zone. A recess 131 is located in a front half of the insulating layer 130 for exposing portions of the working electrode 121, the auxiliary electrode 122, and the reference electrode 123, wherein the exposed portions of the working electrode 121, the auxiliary electrode 122, and the reference electrode 123 are bonded to the reagent layer 140. A reaction zone is formed. The insulating layer 130 may be composed of an ink or a photopolymer and may be screen printed on the electrode layer 120.

The reagent layer 140 may be located at the working electrode 121 exposed by the insulating layer groove 131, and assisted Above the portions of the electrode 122 and the reference electrode 123. Those skilled in the art will recognize that the components of the reagents within the reagent layer 140 can be selected based on the particular analyte. In an embodiment of the invention, the reagent layer 140 is a glucose used to measure a blood sample of a human. A reagent may include, but is not limited to, an enzyme, an electron transport medium, a stabilizer, and a binder, wherein the enzyme is glucose oxidase or glucose dehydrogenase. The electron transport medium is an electron acceptor and can transfer electrons between the enzyme and the working electrode 121. In general, the electron transporting medium is a derivative of ferrocene, potassium ferricyanide or other ferrocene. In an embodiment of the invention, in the reaction zone of the reagent layer 140, glucose and enzyme reactions of the human blood sample, electrons are transferred to the working electrode 121 through the electron transport medium, and an electrical reaction is generated.

A spacer 150 covers the height above the substrate 110 to define a sample-receiving chamber. In an embodiment of the invention, the spacer 150 has a T-shaped channel 151 in the front half of the spacer 150.

The first cover layer 160 may be attached to a portion of the spacer 150 to form an upper surface of the sample receiving chamber. The bottom layer of the first cover layer 160 comprises a hydrophilic coating (not shown in Figure 1). When a human blood sample enters the sample receiving chamber, the hydrophilic coating can help capillary action and increase the speed of movement of the human blood sample. As shown in FIG. 1, the last layer of the test piece 100 is the second cover layer 170. The second cover layer 170 includes a transparent window, wherein the transparent window allows a user to visually confirm whether the human blood sample enters the sample receiving chamber. As shown in FIG. 2, the first cover layer 160, the second cover layer 170, and the spacer 150 form a lateral vent 201, wherein the lateral vent 201 allows air to pass from the sample when the human blood sample enters the sample receiving chamber. The inside of the receiving chamber escapes.

In general biochemical measurement, the measuring circuit applies an electrical signal to the working electrode of the test piece, and then the measuring circuit reads the current on the working electrode for subsequent measurement. Please refer to FIG. 3, which is a schematic diagram illustrating a three-electrode test strip 300, a reaction zone 302, and a metrology circuit 304. In the figure, the measuring circuit 304 is connected through the pads WP, RP, CP and the working electrode WE of the reaction zone 302, the reference electrode RE and the auxiliary electrode CE, respectively, and OP1 and OP2 are operational amplifiers. As shown in FIG. 3, when the reaction region 302 of the three-electrode test strip 300 coupled to the measuring circuit 304 is placed in the same state, the measuring circuit 304 applies a fixed voltage (for example, a ground voltage VG) to the operational amplifier. OP2 to read the current IW on the working electrode WE. Since the conduction path between the working electrode WE and the pad WP has an equivalent resistance RW, the actual voltage VWE on the working electrode WE is determined by the equation (1): VWP = VG VWE = VWP - IW × RW = VG -IW×RW (1)

In addition, the voltage difference VWR between the working electrode WE and the reference electrode RE (equal to the electrical signal applied to the working electrode WE) is determined by the equation (2): VWR = VWE - VRE = VG - IW × RW - VRE =VG-VRP-IW×RW=VG-V1-IW×RW (2)

Where VRE is the voltage on the reference electrode RE, VRP is the voltage on the pad RP, and V1 is the reference voltage input to the operational amplifier OP1. As can be seen from equation (2), the voltage difference VWR between the working electrode WE and the reference electrode RE is affected by the current IW. Since the voltage difference VWR between the working electrode WE and the reference electrode RE is affected by the current IW, the measuring circuit 304 can provide a stable voltage of the reference electrode RE, but cannot provide a stable voltage of the working electrode WE for subsequent accurate amount. For measurement purposes. Therefore, the test piece with three electrodes provided by the prior art (such as the three-electrode test piece 300 shown in FIG. 3) cannot meet the needs of the user.

Please refer to FIG. 4, which is a third electrode test piece according to an embodiment of the present invention. 600, a reaction zone 602 and a schematic diagram of a measurement circuit 604, wherein the measurement circuit 604 is connected through the pads WP1, WP2 and the working electrode WE of the reaction zone 602, the pad RP and the reference electrode RE of the reaction zone 602, and the lining The pad CP is connected to the auxiliary electrode CE of the reaction zone 602, the OP1 and OP2 in the measuring circuit 604 are operational amplifiers, and the reaction zone 602 is coated with an enzyme. As shown in FIG. 4, when the reaction region 602 of the three-electrode test strip 600 coupled to the measuring circuit 604 is placed in the same state, the measuring circuit 604 applies a voltage V2 to the operational amplifier OP2 to read the working electrode WE. The current IW on the sample, wherein the sample at least covers the working electrode WE. Since the conduction path between the working electrode WE and the pad WP2 has an equivalent resistance RW, the actual voltage VWE on the working electrode WE is determined by the equation (3): VWE=VWP-IWP×RW=VWP (∵IWP) =0)=V2 (3)

Where VWP is the voltage across pad WP2 and IWP is the current flowing through pad WP2. Further, in Fig. 4, the voltage difference VWR between the working electrode WE and the reference electrode RE (equal to the electric signal applied to the working electrode WE) is determined by the equation (4): VWR = VWE - VRE = VWP - VRP =V2-V1 (4)

Where VRE is the voltage on the reference electrode RE, VRP is the voltage on the pad RP, and V1 is the reference voltage input to the operational amplifier OP1. As is clear from the equation (4), the voltage difference VWR between the working electrode WE and the reference electrode RE (equal to the electric signal applied to the working electrode WE) is not affected by the current IW. Since the voltage difference VWR between the working electrode WE and the reference electrode RE (equal to the electrical signal applied to the working electrode WE) is not affected by the current IW, the measuring circuit 604 can provide work in addition to the voltage stable to the reference electrode RE. The electrode WE stabilizes the voltage for subsequent accurate quantities For measurement purposes. Therefore, the test piece of the three-pad three-electrode provided by the present invention (such as the three-electrode test piece 600 shown in FIG. 4) can satisfy the needs of the user.

Please refer to FIG. 5. FIG. 5 is a schematic diagram showing a three-electrode test strip 600, a reaction zone 602 and a measuring circuit 704 according to another embodiment of the present invention, wherein the measuring circuit 704 passes through the pads WP1, WP2 and The working electrode WE connection of the reaction zone 602, the pad electrode RP and the reference electrode RE connection of the reaction zone 602, and the pad CP and the auxiliary electrode CE of the reaction zone 602 are connected, and OP1, OP2 in the measurement circuit 704 are operational amplifiers. As shown in FIG. 5, the difference between the measuring circuit 704 and the measuring circuit 604 of FIG. 4 is that the voltage V2 applied by the measuring circuit 704 to the operational amplifier OP2 is a variable voltage and the measuring circuit 704 is applied to the operational amplifier OP1. The voltage V1 is a fixed voltage. In addition, the remaining operating principles of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 704 of FIG. 5 are the same as those of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 604 of FIG. This will not be repeated here.

Please refer to FIG. 6. FIG. 6 is a schematic diagram showing a three-electrode test strip 600, a reaction zone 602 and a measuring circuit 804 according to another embodiment of the present invention, wherein the measuring circuit 804 passes through the pads WP1, WP2 and The working electrode WE connection of the reaction zone 602, the pad electrode RP and the reference electrode RE connection of the reaction zone 602, and the pad CP and the auxiliary electrode CE of the reaction zone 602 are connected, and OP1, OP2 in the measurement circuit 804 are operational amplifiers. As shown in FIG. 5, the difference between the measuring circuit 804 and the measuring circuit 604 of FIG. 4 is that the voltage V2 applied by the measuring circuit 804 to the operational amplifier OP2 is a fixed voltage and the measuring circuit 804 is applied to the operational amplifier OP1. Voltage V1 is a variable voltage. In addition, the remaining operating principles of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 804 of FIG. 6 are the same as those of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 604 of FIG. This will not be repeated here.

Please refer to FIG. 7. FIG. 7 is a diagram showing another three-electrode test according to another embodiment of the present invention. Schematic diagram of a sheet 600, a reaction zone 602, and a metrology circuit 904, wherein the metrology circuit 904 is connected through the pads WP1, WP2 and the working electrode WE of the reaction zone 602, the pad RP and the reference electrode RE of the reaction zone 602, and The pad CP is connected to the auxiliary electrode CE of the reaction region 602, and OP1 and OP2 in the measuring circuit 904 are operational amplifiers. As shown in FIG. 5, the difference between the measuring circuit 904 and the measuring circuit 604 of FIG. 4 is that the voltage V2 applied by the measuring circuit 904 to the operational amplifier OP2 is a variable voltage and the measuring circuit 904 is applied to the operational amplifier OP1. The voltage V1 is a variable voltage. In addition, the remaining operating principles of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 904 of FIG. 7 are the same as those of the three-electrode test strip 600, the reaction region 602, and the measuring circuit 604 of FIG. This will not be repeated here.

Please refer to FIG. 8. FIG. 8 is a schematic view showing the structure of the three-electrode test piece 600. As shown in FIG. 8, the three-electrode test piece 600 includes a substrate 601, a reaction zone 602, a working electrode WE, a reference electrode RE, and an auxiliary electrode CE, wherein the working electrode WE is connected to the pads WP1, WP2, and the reference electrode RE is connected to the pad. The RP and the auxiliary electrode CE are connected to the pad CP. In addition, the substrate 601 is an insulating material (for example, polyethylene terephthalate (PET) or a similar insulating material). As shown in FIG. 8, the working electrode WE, the reference electrode RE, and the auxiliary electrode CE are formed on the substrate 601 and form a reaction region 602 at the first end of the substrate 601, wherein the working electrode WE, the reference electrode RE, and the auxiliary electrode CE It is a conductive material, including but not limited to gold, platinum, silver or graphite. In addition, the pads WP1, WP2, RP, CP are formed at the second end of the substrate 601, wherein the second end of the substrate 601 is opposite to the first end of the substrate 601. As shown in Fig. 8, the pads WP1, WP2 are formed on the left side of the substrate 601 (where the positions of the pads WP1, WP2 are interchangeable), the pads RP, CP are formed on the right side of the substrate 601, and the spacer RP is located Between the pad WP2 and the pad CP. In addition, in another embodiment of the present invention, the pads WP1, WP2 are formed on the right side of the substrate 601, the pads RP and the pads CP are formed on the left side of the substrate 601, and the pads CP are located on the pads WP2 and the linings. Between the pads RP. Further, in the reaction zone 602, the reference electrode RE is located between the auxiliary electrode CE and the working electrode WE.

In addition, please refer to FIG. 9. FIG. 9 is a schematic view showing a three-electrode test piece 1100 according to another embodiment of the present invention. As shown in FIG. 9, the difference between the three-electrode test piece 1100 and the three-electrode test piece 600 of FIG. 8 is that the pad WP1 and the pad WP2 coupled to the working electrode WE are formed on the left and right sides of the substrate 601, respectively. The pad RP coupled to the reference electrode RE and the pad CP coupled to the auxiliary electrode CE are formed in the middle of the substrate 601, and the pad RP is located between the pad WP1 and the pad CP. In addition, the operation principle of the three-electrode test piece 1100 is the same as that of the three-electrode test piece 600, and details are not described herein again.

In addition, as shown in FIG. 4, when the reaction region 602 of the three-electrode test piece 600 coupled to the measuring circuit 604 through the pads WP1, WP2, RP, CP is placed into a sample (for example, blood), the measuring circuit 604 applies a voltage V2 to the operational amplifier OP2 and applies a reference voltage V1 to the operational amplifier OP1 to read the current IW on the working electrode WE. Since, as shown in the formula (4), when the reaction region 602 of the three-electrode test piece 600 is placed in the sample, the voltage difference VWR between the working electrode WE and the reference electrode RE (equal to the electric signal applied to the working electrode WE) Without being affected by the current IW, the measuring circuit 604 can provide a stable reaction voltage VWR and accurately read the current IW generated by the working electrode WE through the pad WP1 for subsequent calculation. In addition, the operation principles of the measurement circuit 704, the measurement circuit 804, and the measurement circuit 904 are the same as those of the measurement circuit 604, and are not described herein again.

Referring to FIGS. 10 to 14, FIG. 10 is a diagram illustrating a voltage difference VWR between the working electrode WE and the reference electrode RE (equal to an electrical signal applied to the working electrode WE) in a first period T1 and a second period. A schematic diagram of T2 and a third time period T3, and Figures 11 through 14 illustrate the concentration distribution of a medium and intermediate product in the reaction zone 602 with the working electrode WE and when the reaction zone 602 is placed in a sample (e.g., blood). A schematic diagram of the change in pressure differential VWR between reference electrodes RE, wherein the medium can be pre-coated in reaction zone 602 or added to reaction zone 602 when the sample is placed in reaction zone 602. For example, if the medium is potassium ferricyanide, it is applied in the second period T2. The negative polarity signal on the working electrode WE can reduce potassium ferricyanide which is not reacted with the analyte (for example, blood glucose) into potassium ferrocyanide, wherein potassium ferrocyanide is the middle of the invention. The product, and the concentration of potassium ferrocyanide, is not affected by the blood glucose of the test substance. As shown in FIG. 10 and FIG. 11 , when the reaction zone 602 is placed in a sample (including a test object (eg, blood glucose)), the medium in the sample can directly or indirectly capture electrons from the analyte to become a reduced state medium. The concentration of the medium is much larger than the concentration of the analyte (for example, the concentration of the medium is equal to 2-4 times the concentration of the analyte). Therefore, as shown in FIG. 10 and FIG. 12, in the first period T1, the electrical signal applied to the working electrode WE is a positive polarity signal, so that the reduced medium transmits electrons to the working electrode WE through a diffusion action, That is, in the first period T1, the working electrode WE generates a current IW (first current) through the reduced state medium, wherein the current IW (first current) in the first period T1 can be used to calculate the initial concentration of the analyte. For example, if the sample is blood and the test substance is blood sugar, the medium in the reaction zone 602 may be potassium ferricyanide, and the potassium ferricyanide may indirectly react with blood sugar (through the enzyme) to produce reduced ferrocyanide. Potassium ferrocyanide. In the first time period T1, the positive polarity signal applied to the working electrode can diffuse potassium ferrocyanide to the working electrode to generate a current IW (first current), and the current IW (first current) can be used to calculate blood sugar. The initial concentration. However, the present invention is not limited to the medium in the sample directly or indirectly taking electrons from the object to be tested into a reduced state medium, that is, the medium in the sample may directly or indirectly transfer electrons to the object to be tested into a medium of oxidation state.

As shown in FIGS. 10 and 13, since the concentration of the medium is much larger than the concentration of the object to be tested, in the second period T2, when the electrical signal applied to the working electrode WE is a negative polarity signal, a large amount is not The medium in which the analyte reacts produces a reduction reaction on the surface of the working electrode WE, resulting in accumulation on the surface of the working electrode WE to produce a high concentration of reduced state medium (ie, an intermediate product), which is accumulated on the surface of the working electrode WE. The concentration of the reduced medium is not affected by the concentration of the analyte (eg, blood glucose). However, in another embodiment of the present invention, the electrical signal applied to the working electrode WE in the second time period T2 is a positive polarity signal, resulting in a large amount of medium not reacting with the object to be tested being generated on the surface of the working electrode WE. An oxidation reaction, that is, a high concentration of oxygen on the surface of the working electrode WE Chemical medium. In addition, the present invention is not limited to the electrical signal applied to the working electrode WE in the second time period T2 is a voltage signal, that is, in the second time period T2, the electrical signal applied to the working electrode WE may also be a current. signal.

The diffusion behavior of the medium in the sample is related to the diffusion factor of the sample. The diffusion factor is a function of, but not limited to, temperature, interference concentration, and sample viscosity. When the diffusion factor of the medium in the sample is low, the reduced medium generated in the second period T2 is less likely to diffuse (as shown in FIG. 14); conversely, when the diffusion factor of the medium in the sample is high, The reduced state medium produced in the period T2 is more likely to diffuse. Therefore, in the third period T3 of FIG. 10, when the electrical signal applied to the working electrode WE is a positive polarity signal, the working electrode WE can generate a larger current IW in the sample having a lower diffusion factor medium. (second current) (because more reduced medium accumulates on the surface of the working electrode WE, the working electrode WE can receive more electrons, resulting in a larger current IW of the working electrode WE), and the working electrode WE has A smaller current IW (second current) can be generated in the sample of the higher diffusion factor medium (because less reduced medium accumulates on the surface of the working electrode WE, the working electrode WE can receive less electrons, resulting in a working electrode WE can generate a small current IW). In addition, in another embodiment of the present invention, the electrical signal applied to the working electrode WE in the third period T3 is a negative polarity signal, and the polarity is opposite to the positive polarity electrical signal in the second period T2.

In addition, in the first time period T1, the second time period T2, and the third time period T3, the auxiliary electrode CE is configured to receive a floating voltage VCE provided by the operational amplifier OP1 to satisfy the working electrode WE in the first time period T1 and the second time period. The current IW generated by T2 and the third period T3. Therefore, a reactive substance may be coated on the surface of the auxiliary electrode CE (or the auxiliary electrode CE may directly react to an electrical signal applied to the working electrode WE) to avoid in the first time period T1, the second time period T2, and the third time period T3. The voltage rise of the auxiliary electrode CE is too large, wherein the reactive substance coated on the surface of the auxiliary electrode CE may be the opposite state of the original redox state of the medium in the sample.

In addition, as shown in FIG. 4 to FIG. 7, the reference electrode RE is coupled to the negative input terminal of the operational amplifier OP1 (ie, no current flows through the reference electrode RE), and the reference electrode RE is interposed between the working electrode WE and the auxiliary. Between the electrodes CE, the reference electrode RE can prevent the reaction material or product coated on the surface of the auxiliary electrode CE from diffusing to the working electrode WE, that is, the reference electrode RE can avoid the auxiliary electrode CE from affecting to the first period T1 and the second period. The current IW generated by the working electrode WE in the period T2 and the third period T3.

Referring to FIGS. 15 to 17 , FIGS. 15 to 17 are diagrams illustrating the relationship between the diffusion factor of the medium and the current IW (second current) generated by the working electrode WE under different interferers. As shown in Fig. 15, when the blood volume in the sample is higher than the HCT (the medium has a lower diffusion factor), the current IW generated by the working electrode WE is larger. For example, the current IW generated when the working electrode WE is 70% of the blood volume ratio HCT in the sample is greater than the current IW generated when the working electrode WE is 40% of the blood volume ratio HCT in the sample. As shown in Fig. 16, when the temperature of the sample is low (the medium has a low diffusion factor), the current IW generated by the working electrode WE is large. For example, the current IW generated when the working electrode WE is at a temperature of 20 ° C is greater than the current IW generated when the working electrode WE is at a temperature of 30 ° C. As shown in Fig. 17, when the concentration of the oil (triglyceride) of the sample is high (the medium has a low diffusion factor), the current IW generated by the working electrode WE is large. For example, the current IW generated when the working electrode WE is at a grease concentration of 750 mg/dL of the sample is larger than the current IW generated when the working electrode WE is at a concentration of 500 mg/dL of the oil. Thus, in the third time period T3 of FIG. 10, the measuring circuit 604 can calculate the diffusion factor of the intermediate product in the sample by the current IW (second current) generated by the working electrode WE according to the above principle, wherein the second The current is the diffusion current of the intermediate product in the third period T3.

Since the concentration of the reduced medium on the surface of the working electrode WE is not affected by the concentration of the analyte (eg, blood glucose), the measurement circuit 604 is generated after the diffusion factor of the medium in the sample is generated. The error of the concentration of the analyte in the sample may be corrected according to the diffusion factor of the medium in the sample to generate a new concentration of the analyte, wherein the factor causing the error of the concentration of the analyte may be related to the temperature of the sample, A combination of viscosity, hematocrit ratio, oil (triglyceride) and ionic strength.

In addition, when the electrical signal applied to the working electrode WE is a voltage, the range of the electrical signal in the first period T1 and the range of the electrical signal in the third period T3 are approximately 50 to 1000 mV, preferably about 200 to 500mV. The electrical signal during the second time period T2 ranges from about -50 to -1000 mV, preferably from about -100 to -500 mV. And the range of the second time period T2 is about 0.5 second to 10 seconds, and preferably about 1 second to 8 seconds. Additionally, in another embodiment of the invention, the electrical signal during the second time period T2 may be a predetermined current.

Referring to FIGS. 18 to 24, FIGS. 18 to 24 are diagrams illustrating different embodiments of the present invention illustrating a voltage difference VWR between the working electrode WE and the reference electrode RE (equal to an electrical signal applied to the working electrode WE). A schematic diagram of a first time period T1, a second time period T2, and a third time period T3, wherein in FIGS. 18 to 24, a voltage difference VWR between the working electrode WE and the reference electrode RE is equal to being applied to the working electrode The operating principle of the electrical signal of WE in the first time period T1, the second time period T2, and the third time period T3 is the same as the voltage difference VWR between the working electrode WE and the reference electrode RE in FIG. 10 in the first time period T1, the second time The operation principle of the time period T2 and the third time period T3 is the same, and details are not described herein again. Further, as shown in FIGS. 18, 19, 20, 21, 22, 23, and 24, in the second period T2, between the working electrode WE and the reference electrode RE The voltage difference VWR can be 0 (no polarity) and a negative polarity signal, that is, the electrical number applied to the operating voltage WE is 0 and a negative polarity signal.

Referring to FIG. 25, FIG. 25 is a diagram illustrating currents IW1, IW2, IW3, and IW4 corresponding to different samples generated by the working electrode WE in the case of the voltage difference VWR between the working electrode WE and the reference electrode RE in FIG. Schematic diagram. Because of other causes of errors in the concentration of the analyte The principle of the child (such as temperature, viscosity, grease and ionic strength of the sample) is the same as the blood volume ratio, and will not be described here. As shown in Fig. 11 and Fig. 12, when the reaction zone 602 is placed in a sample (including blood glucose), the medium in the sample directly or indirectly captures electrons from the analyte to become a reduced medium, so the working electrode WE can be reduced. The medium of the medium generates a current IW, wherein the current IW (first current) in the period T1 can be used to represent the estimated concentration of the object to be tested. In addition, before the high concentration of the reduced state medium has been accumulated on the surface of the working electrode WE, the working electrode WE can generate a large current IW when the blood volume of the sample is relatively low. Therefore, as shown in FIG. 25, in the period T1, the current IW1 corresponding to the sample 1 > the current IW2 corresponding to the sample 2 > the current IW3 corresponding to the sample 3 > the current IW4 corresponding to the sample 4, wherein the sample 1 has a blood glucose concentration (200 mg) /dL) and blood volume ratio HCT (10%), sample 2 has blood glucose concentration (200mg/dL) and blood volume ratio HCT (70%), sample 3 has blood glucose concentration (100mg/dL) and blood volume ratio HCT (10 %), and sample 4 had a blood glucose concentration (100 mg/dL) and a hematocrit ratio HCT (70%). As shown in FIG. 13, in the period T2, the electrical signal applied to the working electrode WE is a negative polarity signal, causing a large amount of the medium not reacting with the analyte to cause a reduction reaction on the surface of the working electrode WE, that is, A high concentration of reduced medium (intermediate product) is accumulated on the surface of the working electrode WE. It is worth noting that the concentration of the reduced medium accumulated on the surface of the working electrode WE is not affected by the concentration of the analyte (for example, blood glucose), so when the diffusion factor of the medium in the sample is high, the working electrode WE is generated. The current IW (second current) is low, and when the diffusion factor of the medium in the sample is low, the current IW (second current) generated by the working electrode WE is high. Therefore, as shown in FIG. 25, in the period T3, the difference between the second current IW1 corresponding to the sample 1 and the second current IW3 of the corresponding sample 3 is small (because the sample 1 and the sample 3 have the same blood volume ratio HCT (10) %)) and the second current IW2 corresponding to the sample 2 and the second current IW4 of the corresponding sample 4 are not significantly different (because the sample 1 and the sample 3 have the same blood volume ratio HCT (70%)), and correspond to the sample 1 The two currents IW1 and the second current IW3 of the corresponding sample 3 are smaller than the second current IW2 of the corresponding sample 2 and the second current IW4 of the corresponding sample 4. Thus, in the period T3 of FIG. 25, the measuring circuit 604 can calculate the medium in the samples 1, 2, 3, 4 by the second currents IW1, IW2, IW3, and IW4 generated by the working electrode WE according to the above principle. The diffusion factor in . When the medium spreads in samples 1, 2, 3, 4 After the factor is generated, the measurement circuit 604 can correct the error of the concentration of the analyte in the samples 1, 2, 3, 4 according to the diffusion factor of the medium in the samples 1, 2, 3, 4 to generate the sample 1. 2, 3, 4 new concentration of the analyte.

Further, the present invention is not limited to the redox state of the medium in Figs. 11 to 14, that is, in another embodiment of the present invention, a new medium has the opposite of those in Figs. 11 to 14 The redox state, and the electrical signal applied to the working electrode WE is a new electrical signal that is reversed from FIG.

In addition, the three-electrode test strips 600 and 1100 provided by the present invention can also be integrated with the measurement circuit 604 to form a biometric measurement system. The operation principle of the biometric measurement system can be referred to the three-electrode test strips 600, 1100 and the measurement circuit 604. So I won't go into details here.

Referring to Figure 26, Figure 26 is a schematic diagram showing the relationship between blood volume ratio and deviation before correction by the present invention. As shown in Fig. 26, the analyte concentration having a blood volume ratio of 41% is used as a standard, wherein the deviation is the difference between the test analyte concentration and the standard for each blood volume ratio, and the glucose concentration in the sample is 100 and 350 mg/dl. As shown in Figure 26, a large deviation will exist when the blood volume ratio within the sample is offset. Referring to Fig. 27, Fig. 27 is a view showing the relationship between the blood volume ratio and the deviation after correction by the present invention. As shown in Fig. 27, by using the method and apparatus provided by the present invention described above, the deviation in the entire blood volume ratio range can be greatly reduced, and the deviation value can be controlled within plus or minus 10%.

Referring to FIG. 4, FIG. 10-14 and FIG. 28, FIG. 28 is a flow chart showing a method for detecting the concentration of a test object in a sample by a test piece according to another embodiment of the present invention. The method of Fig. 28 is illustrated by the three-electrode test piece 600 of Fig. 4, and the detailed steps are as follows: Step 2800: Start; Step 2802: Place the same into the reaction area 602; Step 2804: The measurement circuit 604 applies an electrical signal to the working electrode WE; Step 2806: The measurement circuit 604 passes the working electrode WE during a first time period T1. Measuring a first current; step 2808: the medium generates an intermediate product according to the electrical signal in a second time period T2; step 2810: the measuring circuit 604 measures a second current through the working electrode WE in a third time period T3; step 2812 The measuring circuit 604 calculates the initial concentration of the object to be tested according to the first current; step 2814: the measuring circuit 604 calculates the diffusion factor of the intermediate product in the sample according to the second current; step 2816: the measuring circuit 604 according to the diffusion factor , correcting the initial concentration of the analyte to generate a new concentration of the analyte; step 2818: ending.

In step 2802, the sample contains a test object (eg, blood glucose). In step 2804, the electrical signal applied to the working electrode WE is equal to the voltage difference VWR between the working electrode WE and the reference electrode RE. As shown in FIG. 4, FIG. 10 and FIG. 11, when the reaction zone 602 is placed, the medium in the sample can directly or indirectly capture electrons from the object to be tested into a reduced state medium, wherein the concentration of the medium is much larger than The concentration of the analyte (for example, the concentration of the medium is equal to 2-4 times the concentration of the analyte). Therefore, in step 2806, as shown in FIG. 10 and FIG. 12, in the first period T1, the electrical signal applied to the working electrode WE is a positive polarity signal, so the reduced medium transmits electrons through a diffusion action. The working electrode WE, that is, in the first time period T1, the working electrode WE generates a first current through the reduced state medium. However, the present invention is not limited to the medium in the sample directly or indirectly taking electrons from the object to be tested into a reduced state medium, that is, the medium in the sample can directly or indirectly transfer electrons to the object to be tested into a medium of oxidation state.

In step 2808, as shown in FIGS. 10 and 13, since the concentration of the medium is much larger than the concentration of the object to be tested, in the second period T2, when the electrical signal applied to the working electrode WE is a negative polarity signal At this time, a large amount of the medium which does not react with the analyte reacts on the surface of the working electrode WE, resulting in accumulation of a high concentration of reduced medium (ie, an intermediate product) on the surface of the working electrode WE, wherein the working electrode WE The concentration of the reduced medium accumulated on the surface is not affected by the concentration of the analyte (e.g., blood glucose). However, in another embodiment of the present invention, the electrical signal applied to the working electrode WE in the second time period T2 is a positive polarity signal, resulting in a large amount of medium not reacting with the object to be tested being generated on the surface of the working electrode WE. The oxidation reaction, that is, the production of a high concentration of the oxidized medium on the surface of the working electrode WE. In addition, the present invention is not limited to the electrical signal applied to the working electrode WE in the second time period T2 is a voltage signal, that is, in the second time period T2, the electrical signal applied to the working electrode WE may also be a current. signal.

In step 2810, the diffusion behavior of the medium in the sample is related to the diffusion factor of the sample, and the diffusion factor is a function including, but not limited to, temperature, interference concentration, and sample viscosity. When the diffusion factor of the medium in the sample is low, the reduced medium generated in the second period T2 is less likely to diffuse (as shown in FIG. 14); conversely, when the diffusion factor of the medium in the sample is high, The reduced state medium produced in the period T2 is more likely to diffuse. Therefore, in the third period T3 of FIG. 10, when the electrical signal applied to the working electrode WE is a positive polarity signal, the working electrode WE can be generated to a larger second in the sample having a lower diffusion factor medium. Current (because more reduced medium accumulates on the surface of the working electrode WE, the working electrode WE can receive more electrons, resulting in a larger second current of the working electrode WE), and the working electrode WE has a higher diffusion A smaller second current can be generated in the sample of the factor medium (since less reduced medium accumulates on the surface of the working electrode WE, the working electrode WE can receive less electrons, resulting in a smaller working electrode WE Two currents). In addition, in another embodiment of the present invention, the electrical signal applied to the working electrode WE in the third period T3 is a negative polarity signal, and the positive polarity in the second period T2 Electrical signal, the opposite polarity.

In step 2812, a first current in the first time period T1 may be used to calculate an initial concentration of the analyte. Additionally, in step 2814, because the diffusion behavior of the medium in the sample is related to the diffusion factor of the sample, measurement circuit 604 can calculate the diffusion factor of the intermediate product in the sample based on the second current. Finally, in step 2816, after the diffusion factor of the intermediate product in the sample is generated, the measurement circuit 604 corrects the initial concentration of the analyte to generate a new concentration of the analyte based on the diffusion factor.

In addition, in the first time period T1, the second time period T2, and the third time period T3, the auxiliary electrode CE is configured to receive a floating voltage VCE provided by the operational amplifier OP1 to satisfy the working electrode WE in the first time period T1 and the second time period. The current IW generated by T2 and the third period T3. Therefore, a reactive substance may be coated on the surface of the auxiliary electrode CE (or the auxiliary electrode CE may directly react to an electrical signal applied to the working electrode WE) to avoid in the first time period T1, the second time period T2, and the third time period T3. The voltage rise of the auxiliary electrode CE is too large, wherein the reactive substance coated on the surface of the auxiliary electrode CE may be the opposite state of the original redox state of the medium in the sample.

Referring to FIG. 4, FIG. 10 and FIG. 29, FIG. 29 is a flow chart showing a method for detecting a medium diffusion factor in a sample using a test piece according to another embodiment of the present invention. The method of Figure 29 is illustrated using the three-electrode test strip 600 of Figure 4, the detailed steps are as follows: Step 2900: Start; Step 2902: Place the same in the reaction zone 602; Step 2904: The measurement circuit 604 applies an electrical signal Step 2906: The medium generates an intermediate product according to the electrical signal in a first period; step 2908: the measuring circuit 604 measures a first current through the working electrode WE in a second period, wherein the electrical signal is in the first The second polarity of the second period is opposite to the first polarity of the first period; Step 2910: The measurement circuit 604 calculates a diffusion factor of the intermediate product in the sample according to the first current, and step 2912: ends.

The difference between the embodiment of FIG. 29 and the embodiment of FIG. 28 is that in step 2906, the medium generates an intermediate product according to the electrical signal during the first time period (corresponding to the second time period T2 of FIG. 10); in step 2908 The measuring circuit 604 measures the first current (corresponding to the second current of the embodiment of FIG. 28) through the working electrode WE in the second period (corresponding to the third period T3 of FIG. 10); in step 2910, measuring Circuit 604 calculates the diffusion factor of the intermediate product in the sample based on the first current (corresponding to the second current of the embodiment of Figure 28). Therefore, it is within the scope of the present invention to detect the medium diffusion factor in the sample by using an electrical signal having a second polarity in the second period opposite to the first polarity in the first period.

In summary, the method for detecting the concentration of the sample in the sample and the test piece of the three electrodes provided by the test piece are generated by the working electrode according to the electrical signal provided by the measuring circuit in the first time period, and are generated for estimation. a first current of an initial concentration of the analyte in the sample, using the working electrode to generate a reaction in the sample according to an electrical signal provided by the measuring circuit in the second period, and using the working electrode in the third period according to the measuring circuit The electrical signal is provided to generate a second current that is used to calculate a diffusion factor of the medium in the sample. After the diffusion factor of the medium in the sample is generated, the measurement circuit can correct the initial concentration of the analyte in the sample according to the diffusion factor of the medium in the sample to generate a new concentration of the analyte. Therefore, the present invention can accurately correct the initial concentration of the analyte in the sample compared to the prior art. In addition, another method for detecting a medium diffusion factor in a sample by using a test piece is to detect a medium diffusion factor in a sample by using an electrical signal having a first polarity in a first period and a second polarity in a second period. . Therefore, the present invention can quickly, simply and accurately detect the medium diffusion factor in a sample compared to the prior art.

First time of T1‧‧

T2‧‧‧ second period

T3‧‧‧ third period

Claims (23)

A method for detecting a concentration of a sample in a sample, wherein the test piece comprises a substrate and a reaction zone, the reaction zone comprising a working electrode, a reference electrode and an auxiliary electrode, and coating an enzyme, the method The method comprises: placing the sample into the reaction zone, wherein the analyte and the enzyme react to generate a plurality of electrons, and transmit the plurality of electrons to the working electrode through a medium; applying an electrical signal to the working electrode; Measuring a first current through the working electrode in a first period; the medium generates an intermediate product according to the electrical signal in a second period; and measuring a second current through the working electrode in a third period, wherein the electrical signal The second polarity in the second period is opposite to the first polarity in the first period and the third polarity in the third period; according to the first current, an initial concentration of the object to be tested is calculated; a second current, calculating a diffusion factor of the intermediate product in the sample; and correcting the initial concentration according to the diffusion factor to generate a new concentration of the analyte. The method of claim 1, wherein when the electrical signal is a voltage, the range of the first period electrical signal and the electrical signal of the third period are between 50 and 1000 mV, and the electrical signal during the second period The range is from -50 to -1000 mV, and the second period of time ranges from 0.5 seconds to 10 seconds. The method of claim 1, wherein the sample covers at least the working electrode. The method of claim 1, wherein the reference electrode receives a reference voltage when the sample is placed in the reaction zone, and when the electrical signal is a voltage, the magnitude of the electrical signal is equal to the voltage of the working electrode and the The voltage difference of the reference voltage. The method of claim 1, wherein the auxiliary electrode receives a floating voltage when the sample is placed in the reaction zone, to satisfy that the working electrode is generated in the first time period, the second time period, and the third time period Current. The method of claim 1, wherein the intermediate product is a reduced state medium or a mono-oxidized medium. The method of claim 1, wherein the second current is a diffusion current of the intermediate product during the third period. The method of claim 1, wherein the second time period is after the first time period, and the third time period is after the second time period. The method of claim 1, wherein the electrical signal during the second time period is a predetermined current. The method of claim 1, wherein the diffusion factor is a combination of temperature, viscosity, hematocrit ratio, oil and ionic strength with respect to the sample. The method of claim 1 wherein the medium is applied to the reaction zone. The method of claim 1, wherein the medium is added to the reaction zone when the sample is placed in the reaction zone. A three-electrode test piece comprising: a substrate; and a reaction zone formed at a first end of the substrate, the reaction zone being coated with an enzyme, wherein When the reaction zone is placed in the reaction zone, the analyte in the sample reacts with the enzyme to generate a plurality of electrons, and the plurality of electrons are transmitted through a medium, the reaction zone includes: a working electrode for when the sample is placed When entering the reaction zone, receiving an electrical signal, generating a first current according to the electrical signal during a first time period, and generating a second current according to the electrical signal during a third time period after a second time period And wherein the second polarity of the electrical signal during the second time period is opposite to the first polarity of the first time period and the third polarity of the third time period, wherein the medium is based on the electrical signal during the second time period, Generating an intermediate product; a reference electrode for receiving a reference voltage when the sample is placed in the reaction zone; and an auxiliary electrode for receiving a floating voltage when the sample is placed in the reaction zone to satisfy a current generated by the working electrode during the first time period, the second time period, and the third time period; wherein the first current is used to calculate an initial concentration of the object to be tested, and the second current is used to calculate the middle Product diffusion Promoter, and the diffusion coefficient is used to correct the initial concentration to generate new concentration of the analyte. The test piece of claim 13, wherein the electrical signal of the second time period is a predetermined current. The test piece of claim 13, wherein the working electrode is connected to a first pad and a second pad, wherein the second pad is configured to transmit the electrical signal to the working electrode, and An electrical signal that stabilizes the working electrode at a corresponding voltage, wherein the first pad is configured to transmit a current generated by the working electrode during the first time period, the second time period, and the third time period, wherein the first A pad and the second pad are formed at the second end of the substrate, and the second end of the substrate is opposite the first end of the substrate. The test piece of claim 15, wherein the reference electrode is connected to a third pad, and the auxiliary The auxiliary electrode is connected to a fourth pad, wherein the third pad is configured to transmit the reference voltage to the reference electrode, and the fourth pad is configured to transmit the floating voltage to the auxiliary electrode, wherein the third pad is A pad and the fourth pad are formed at the second end of the substrate. A method for detecting a concentration of a sample in a sample, wherein the test piece comprises a substrate and a reaction zone, the reaction zone comprising a working electrode, a reference electrode and an auxiliary electrode, and coating an enzyme, the method The method comprises: placing the sample into the reaction zone, wherein the analyte and the enzyme react to generate a plurality of electrons, and transmit the plurality of electrons to the working electrode through a medium; applying an electrical signal to the working electrode; Measuring a first current through the working electrode in a first period; the medium generates an intermediate product according to the electrical signal in a second period; and measuring a second current through the working electrode in a third period, wherein the electrical signal Having a second polarity and a polarity in the second period, and the second polarity is opposite to a first polarity of the electrical signal in the first period and a third polarity of the electrical signal in the third period; Calculating an initial concentration of the analyte according to the first current; calculating a diffusion factor of the intermediate product in the sample according to the second current; and correcting the initial concentration according to the diffusion factor A new concentration of the analyte. A method for detecting a medium diffusion factor in a sample by using a test piece, wherein the test piece comprises a reaction zone comprising a working electrode, a reference electrode and an auxiliary electrode, the method comprising: placing the same into the reaction And applying an electrical signal to the working electrode; the medium generates an intermediate product according to the electrical signal during a first time period; and measuring a first current through the working electrode during a second time period after the first time period, wherein the Electricity The second polarity of the signal during the second time period is opposite to the first polarity of the first time period; and based on the first current, the diffusion factor of the intermediate product in the sample is estimated. The method of claim 18, wherein when the electrical signal is a voltage, the electrical signal of the second time period ranges from 50 to 1000 mV, and the electrical signal of the first time period ranges from -50 to -1000 mV, And the range of the first time period is between 0.5 seconds and 10 seconds. The method of claim 18, wherein the reference electrode receives a reference voltage when the sample is placed in the reaction zone, and when the sample is placed in the reaction zone, the auxiliary electrode receives a floating voltage to satisfy the The first current generated by the working electrode during the first time period and the second time period. The method of claim 18, wherein the first current is a diffusion current of the intermediate product during the second time period. The method of claim 18, wherein the electrical signal of the first time period is a predetermined current. A method for detecting a medium diffusion factor in a sample by using a test piece, wherein the test piece comprises a reaction zone comprising a working electrode, a reference electrode and an auxiliary electrode, the method comprising: placing the same into the reaction And applying an electrical signal to the working electrode; the medium generates an intermediate product according to the electrical signal during a first time period; and measuring a first current through the working electrode during a second time period after the first time period, wherein the The electrical signal has a first polarity and a non-polarity during the first time period, and the first polarity is opposite to the second polarity of the electrical signal during the second time period; and estimating the presence of the intermediate product based on the first current The diffusion factor in the sample.