Method for testing analyte in biological sample, sensor, and system

Abstract

A method for testing an analyte in a biological sample, a sensor, and a system. The method comprises: bringing a first working electrode (D) and a second working electrode (A) into contact with a biological sample, and inputting a first excitation signal and a second excitation signal to the first working electrode (D); between the first excitation signal and the second excitation signal, inputting an excitation signal sequence to the second working electrode (A); measuring at least one first output signal responsive to the first excitation signal, at least one second output signal responsive to the second excitation signal, and at least one output signal sequence responsive to the excitation signal sequence, on the first working electrode (D); and on the basis of the first output signal, the second output signal, and the output signal sequence, obtaining the concentration of the analyte in the biological sample. The first output signal, the second output signal, and the output signal sequence are used as parameters for calculating the concentration of the analyte in the biological sample, so that the impact of interferents can be eliminated, thereby improving the measurement accuracy of the test method.

Description

Method, sensor and system for testing analytes in biological samples

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 28, 2023, with application number "202311851595.7" and invention name "A method, sensor and system for testing analytes in biological samples", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to a method, a sensor and a system for testing an analyte in a biological sample, and belongs to the technical field of biological sample analysis. Background Art

[0003] When performing an analyte detection analysis on a biological sample, an input signal is applied to the working electrode of the sensor through a measuring device. The working electrode transmits the input signal to the biological sample, and the analyte in the biological sample undergoes an oxidation-reduction reaction to generate an output signal responsive to the input signal, thereby determining the analyte concentration in the biological sample based on the output signal. The analytes include substances such as blood glucose, blood ketones, blood lactate, cholesterol, uric acid, triglycerides, coagulation factors, and anticoagulant factors. The test results of the above analytes can be used for medical diagnosis and treatment of physiological abnormalities. For example, a diabetic individual can use a biosensor system to measure the glucose level in the blood to adjust diet and / or medication.

[0004] Traditional biosensors achieve test selectivity by catalyzing a single reaction with biomolecules, based on a two-step chain reaction. In the second reaction (the comb flow reaction), the reduced state of the mediator diffuses to the electrode and is then oxidized, generating a corresponding current signal. The catalytic process for the analyte can be described in three steps:

[0005] (1) Analyte + enzyme 氧化态 = biochemical reaction product + enzyme 还原态

[0006] (2) Mediator 氧化态 +Enzyme 还原态 =Mesophysique 还原态 +Enzyme 氧化态

[0007] (3) Mediator 还原态 (Electrode) = Medium 氧化态 +e -

[0008] The enzyme biomolecules in the glucose detection sensor system catalyze the oxidation of glucose molecules in the blood. The reaction between the added mediator and the enzyme converts the enzyme from the reduced state back to the oxidized state, thus playing a combing role. Glucose enzymes can be oxidases or dehydrogenases. Since oxidases are oxidized by the dissolved oxygen in the blood sample, while dehydrogenases are not, there is no "dissolved oxygen effect". Dehydrogenases have been the first choice of enzymes in biosensors for the past two decades. The catalytic enzymes of other analytes may be oxidases or dehydrogenases, and the biosensor principles are basically the same. However, during the test, although the enzyme-catalyzed reaction is basically single, there may be other interfering substances that can be oxidized in the blood sample. These interfering substances may undergo oxidation reactions, resulting in interference signals. Generally speaking, the higher the standard potential of the mediator of the combing reaction, such as ferric cyanide (Fe(CN)6 -3 ), it is easier to generate interference signals, because the higher the electrode oxidation potential, the more interference substances will be oxidized.

[0009] In addition to the chemical interferents mentioned above, biosensors also experience other test interferences. For example, the hematocrit percentage (%-HCT) is a relatively significant interfering factor. This interference occurs when the medium after the comb flow reaction diffuses to the electrode, where its diffusion is hindered by blood cells, causing the diffusion coefficient to vary depending on the %-HCT. The current in the electrochemical reaction is affected by varying %-HCT. Therefore, factory batch calibration of sensor strips is typically centered around 42% HCT. If the user's %HCT is higher than 42%, the test will experience a negative bias. If the user's %HCT is lower than 42%, the test will experience a positive bias. Furthermore, the aging effects of the test strips during storage can interfere with test results. Specifically, as storage time increases, the sensitivity of the sensor test decreases, or more oxidizable substances are generated that can contribute to background signals. Another interfering factor is the test temperature.

[0010] Traditional biosensors generally apply voltage to electrodes in contact with the analyte sample, oxidizing the reduced mediator produced by the comb flow reaction, thereby generating a current signal in response to the analyte. Using appropriate calculation methods, the current signal is converted into the analyte concentration. Existing methods generally use different single parameters to compensate for interference factors during the test process, eliminating or reducing the interference, thereby improving the accuracy and precision of the test. However, these single-parameter interference compensation methods often ignore the interplay between multiple interfering factors. For example, the interaction between temperature and HCT, the interaction between temperature and test strip aging, the interaction between HCT and test strip aging, and so on.

[0011] For a long time, traditional biosensors have used a single excitation to generate an output signal, and this single output signal was used to determine the analyte concentration. Later developments in multi-pulse excitation methods, such as the method disclosed in CN109690304A, aim to improve the accuracy and precision of sensors, overcome the effects of chemical interferences, and provide multivariate parameters for distinguishing whole blood samples from quality control solutions, significantly improving the performance of biosensors. However, this method requires alternating input pulse signals between the two working electrodes, which can easily cause test vibrations that affect the continuity of subsequent input signals, resulting in erroneous signals that are difficult to identify, causing the test results to be less accurate due to excessive uncertainties.

[0012] Therefore, in order to avoid the generation of interference signals, there is still much room for improvement in the testing methods of analytes in biological samples in terms of multi-pulse excitation and multi-parameter compensation, so as to further improve the accuracy and precision of testing analytes in biological samples.

[0013] Summary of the Invention

[0014] To address the above-mentioned issues, the inventors have disclosed a device, system, and method for testing analytes in biological samples. The disclosed device, system, and method are related to testing the concentration of one or more analytes in a biological sample. The method inputs at least two excitation signals to a first working electrode and inputs a sequence of excitation signals to a second working electrode between the at least two excitation signals to the first working electrode. The input signals to the first and second working electrodes do not overlap in time. That is, after the excitation signal to the first working electrode is completed, the system controls the first working electrode in an open-circuit state and then initiates the input of the excitation signal sequence to the second working electrode. After the excitation signal sequence to the second working electrode is completed, the system controls the second working electrode in an open-circuit state and then initiates another excitation signal to the first working electrode. The excitation signals input to the first and second working electrodes are defined as "sandwiched pulsing excitation." The first working electrode is coated with a first chemical that reacts chemically with a predetermined analyte. The first chemical includes an enzyme that catalyzes the analyte reaction, one or more mediators that generate a combing effect between the enzyme reaction and the electrode reaction, and an inert substance that assists the chemical. The chemical coating the first counterelectrode corresponding to the first working electrode includes at least a mediator or is composed of the same components as the first chemical. The second working electrode is a blank electrode or coated with an inert substance that does not chemically react with the analyte. Corresponding to the sandwich excitation method described above, the sensor system measures the output signals generated by each input excitation signal, namely, the output signals generated by the first and second excitation signals input to the first electrode, and the output signal series generated by the series of excitation signals input to the second electrode. Furthermore, the measured signals also include the residual attenuation signal generated by the corresponding electrode that reacts conjugately with the first electrode after the first excitation signal ceases, as well as the residual attenuation signal generated by the first electrode after the second excitation signal ceases, and so on.

[0015] One aspect of the sandwich excitation method disclosed herein is to be used to determine one or more analytes in a biological sample, and to determine the analyte concentration from one or more signal values ​​in the first and second output signals of the first electrode, one or more signal values ​​in the output signal series of the second electrode, and the remaining attenuated signal values ​​of the first and second input signals of the first electrode.

[0016] Another aspect of the sandwich excitation method disclosed herein, particularly the output signal series values ​​generated by the sandwich input excitation signal series, is used to measure other parameters in a biological sample, including the hematocrit value (%-HCT) of a whole blood sample. The method also provides appropriate compensation and correction for analyte determination using parameters related to the hematocrit value, thereby eliminating or reducing the influence of blood cells on analyte determination, thereby improving the accuracy and precision of analyte determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a schematic diagram illustrating a sensor for testing an analyte in a biological sample according to the present invention.

[0018] FIG2 is a schematic diagram of the main excitation signals of the sandwich excitation method in this embodiment.

[0019] FIG3 is a schematic diagram of different step waves in the sandwich excitation method.

[0020] FIG4 shows the output current signals of different step waves at different blood glucose concentrations.

[0021] FIG5A is a schematic diagram of another embodiment of the multi-pulse sandwich excitation method.

[0022] FIG5B is a schematic diagram of another embodiment of the multi-pulse sandwich excitation method.

[0023] FIG5C is a schematic diagram of another embodiment of the multi-pulse sandwich excitation method.

[0024] FIG6A is a graph showing the output current of the sandwich excitation method at different blood glucose concentrations.

[0025] FIG6B is a diagram showing the linear relationship between the output current of the excitation signal to the first working electrode and the blood glucose concentration in the sandwich excitation method.

[0026] FIG6C is a graph showing the output current of the multi-pulse sandwich excitation method at different blood glucose concentrations.

[0027] FIG6D is a diagram showing the linear relationship between the output current of the excitation signal of the first working electrode and the blood glucose concentration using the multi-pulse sandwich excitation method.

[0028] FIG6E is a graph showing the output current of different step waves at different blood glucose concentrations.

[0029] FIG7A is a potential decay curve at different blood glucose concentrations after the first excitation wave applied to the first working electrode is completed.

[0030] FIG7B is a graph showing the relationship between the relative attenuation coefficient of the attenuation potential of the first working electrode and the reference concentration of the analyte.

[0031] FIG7C is a potential decay curve at different blood glucose concentrations after the second excitation wave applied to the first working electrode ends.

[0032] FIG7D is a graph showing the relationship between the decay potential at the first counter electrode and the analyte concentration.

[0033] FIG8 is a graph showing the output current of the second working electrode corresponding to the step wave at different hematocrits.

[0034] FIG9A is a graph showing the linear relationship between the current of the fourth step wave and the hematocrit at different blood glucose concentrations.

[0035] FIG9B is a graph showing the ratio of the current values ​​of the fourth step wave at different hematocrit levels to the current value of the fourth step wave at a hematocrit of 42%.

[0036] FIG9C is a graph showing the linear relationship between the current of the fifth step wave and the hematocrit at different blood glucose concentrations.

[0037] FIG9D is a graph showing the ratio of the current values ​​of the fifth step wave at different hematocrit values ​​to the current value of the fifth step wave at a hematocrit of 42%.

[0038] Figure 10A shows Δi at different blood glucose concentrations 43 Linear relationship graph with hematocrit.

[0039] Figure 10B shows Δi at different hematocrit levels. 43 Compared with Δi at 42% hematocrit 43 Relationship graph between the ratio of current values ​​and hematocrit.

[0040] Figure 10C shows Δi at different blood glucose concentrations. 54 Linear relationship graph with hematocrit.

[0041] Figure 10D shows Δi at different hematocrit levels. 54 Compared with Δi at 42% hematocrit 54 Relationship graph between the ratio of current values ​​and hematocrit.

[0042] Figure 10E shows Δi at different blood glucose concentrations 53 Linear relationship graph with hematocrit.

[0043] FIG10F shows Δi at different hematocrit levels. 53 Compared with Δi at 42% hematocrit 53 Relationship graph between the ratio of current values ​​and hematocrit.

[0044] FIG. 11 shows a diagram of the present invention ii 平均 An exemplary embodiment of a linear relationship plot versus glucose concentration.

[0045] FIG12 shows a comparison diagram of the deviation before and after calibration of Example 1;

[0046] FIG13 shows a comparison diagram of the deviation before and after calibration of Example 2;

[0047] FIG14 shows a comparison diagram of the deviation before and after calibration of Example 3;

[0048] FIG15 shows a comparison diagram of the deviations after calibration of Examples 1-3.

[0049] FIG16 is a schematic diagram of a detection circuit in an embodiment of the present application. DETAILED DESCRIPTION

[0050] The excitation described in the disclosure of the present invention is significant in that it applies an input signal to the electrodes in the sensor, wherein the applied signal includes a positive or negative DC constant voltage or a positive or negative DC constant current source, etc., so as to cause a complete or partial oxidation or reduction reaction to occur on the electrode surface. The system processing center of the sensor applies an excitation input signal to the corresponding electrode through the connection port of the instrument and the sensor strip. When the predetermined excitation time is over, the system will no longer apply a signal to the electrode, and the electrode will be in an open circuit state. The open circuit state is not zero input. For example, inputting a voltage of 0V (zero volts) to the electrode is not equal to an open circuit. Zero voltage is also a type of input voltage. When the electrode is in an open circuit state, the oxidation-reduction substances on and near the electrode surface will cause the residual signal to decay. The decay signal will give a state that reflects the electrode's past, thereby recording other information related to the sensor and the analyte.

[0051] Figure 1 shows a schematic diagram of a sensor, wherein D represents a first working electrode, which is coated with a first chemical agent corresponding to a predetermined analyte. C represents a first counterelectrode corresponding to the first working electrode, which is coated with at least a certain amount of a medium or the same chemical agent as the first working electrode. E represents the sample injection detection electrode. A represents a second working electrode in the sensor relative to the sandwich excitation method, which may be one of the electrode combinations used to measure impedance in the sensor. The surface may be blank, coated with an inert material, or coated with a second chemical agent different from the first chemical agent. B represents a second counterelectrode corresponding to the second working electrode.

[0052] The first chemical agent includes an oxidoreductase and a mediator, the oxidoreductase being selected from at least one of FAD dehydrogenase and PQQ dehydrogenase, and the mediator being selected from at least one of trichlorohexamine ruthenium, potassium ferrocyanide, a ferrocene derivative, phenazine methylsulfate, and a polyethersulfone resin. The inert substance is selected from at least one of carboxymethyl cellulose, hydroxymethyl cellulose, polyethylene glycol, and polyvinyl pyrrolidone.

[0053] One of the related aspects of this invention is the sandwich excitation method. Figure 2 is a schematic diagram of the main elements of the sandwich excitation method. A first excitation signal is applied to the first working electrode. After the circuit is opened, a sequence of excitation signals (i.e., the series of excitation signals described in the figure) is applied to the second working electrode. After the circuit is opened, a second excitation signal is applied to the first working electrode again.

[0054] FIG2 is a schematic diagram of the main excitation signals of the sandwich excitation method in this embodiment. The horizontal axis represents the sensor test time, and the vertical axis represents the excitation signal intensity, which can be the voltage applied to the electrode, or the constant current applied to the electrode, etc. There is a pre-test idle period in the figure, called the first idle period, during which the sensor does not receive any input signal. The time range of the first idle period is 0 to 2 seconds. 0s means that the sensor system starts the test program immediately after determining that the sample completely fills the sensor cavity. The idle time between 0 and 2 seconds will be used to incubate the enzyme catalytic reaction before applying the first excitation signal to the first working electrode of the sensor. In addition, the first idle period may also be used to measure the impedance of the sample by a specific electrode pair.

[0055] After the first idle period, a first excitation signal is applied to the first working electrode. The excitation voltage of the first excitation signal is 0.2-0.4 V, preferably 0.3 V, and the excitation time is 0.1-1 s, preferably 0.5 s.

[0056] After the first excitation signal is delivered, the system opens the circuit to the first working electrode, marking the first working electrode's second idle period. During this second idle period, the active area of ​​the first working electrode undergoes an enzyme-catalyzed reaction, incubating and accumulating the reduced mediator after the comb flow reaction, preparing for the electrooxidation reaction of the second excitation signal. Simultaneously, the sensor system can perform other tests, such as measuring the decay signal generated by the first excitation signal at the first working electrode, with a measurement time between 0.005 and 0.5 seconds.

[0057] The system then initiates a sequence of excitation signals for the second working electrode. Each excitation in the excitation signal sequence can last between 0.05 and 0.5 seconds and can be a continuous excitation sequence without intervals or a sequence of different excitation pulses with intervals. Preferably, the excitation signal sequence is a continuous excitation sequence without intervals. It is understood that this continuous excitation sequence can be a continuously increasing excitation sequence, or a sequence that first increases and then decreases, or first increases, then decreases, and then increases again. Preferably, the continuous excitation sequence is a continuously increasing excitation sequence. If the excitation signal sequence is a voltage, the voltage value is between 0.1 and 3V. The excitation signal sequence shown in the figure includes five excitations, and the excitation voltages of the five excitations can be, respectively, 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V, and the duration of each excitation can be 0.2 seconds. This excitation signal sequence can be referred to as a "staircase wave." After the excitation signal sequence for the second working electrode ends, the system places the second working electrode in an open circuit state and measures the decay signal of the second working electrode after the circuit is opened, with the measurement time ranging from 0.005 to 0.5 seconds.

[0058] Finally, the system applies a second excitation signal to the first working electrode. The excitation time is between 0.1 and 2 seconds, preferably 1.25 seconds, and the excitation voltage is 0.2-0.4 V, preferably 0.3 V. After the second excitation signal ends, the system places the first working electrode in an open circuit state and measures the potential decay signal of the corresponding electrode of the system. The measured value is between 0.005 and 0.5.

[0059] In summary, the test time and potential control in the test method are preferably as follows: (1) the pre-test idle period is 1.25 seconds; (2) a first excitation signal is applied to the first working electrode, a 0.3V potential difference is input to the first working electrode and the corresponding electrode for 0.5 seconds, and the output current signal is synchronously measured at intervals of 0.05 seconds; (3) after the first excitation signal ends, the first working electrode is set to an open circuit state, and the first potential decay signal of the first working electrode is measured at intervals of 0.005 seconds for 0.5 seconds; (4) while the first working electrode is kept in the open circuit state, an excitation signal sequence is input to the second working electrode and the corresponding electrode, and the excitation signal sequence includes 5 excitations, The excitation voltages are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V, respectively. Each excitation lasts for 0.2 seconds, and the output current signal is synchronously measured at intervals of 0.05 seconds (hereinafter referred to as step wave 1); (5) the second working electrode is set to an open circuit state; (6) after the first working electrode is open for 2.0 seconds, a second excitation signal is applied to the first working electrode at a voltage of 0.3V (volts) for 1.25 seconds, and the output current signal is synchronously measured at intervals of 0.05 seconds; (7) after the second excitation signal ends, the first working electrode is set to an open circuit state, and the potential decay signal of the corresponding electrode is measured at intervals of 0.005 seconds for 0.5 seconds. The total time for the above test steps is 1.25+0.5+2+1.25+0.5=5.5 seconds.

[0060] In addition to the excitation signal sequence implemented on the second working electrode as shown in FIG2 , other different excitation signal series, i.e., step waves, can also be implemented. FIG3 shows several different step wave examples in the sandwich excitation method: (1) Step wave 2 shown in FIG3A : the excitation voltage of the excitation signal increases continuously by three steps, then drops by one step and then increases continuously by four steps. The voltages of the excitation signal are 0.3V, 0.7V, 1.1V, 0.9V, 1.2V, 1.5V, 1.9V, and 2.3V in sequence. The voltage of each excitation signal lasts for 0.15 seconds. (2) Step wave 3 shown in FIG3B : the excitation voltage of the excitation signal increases continuously by three steps, then drops by one step, then increases continuously by three steps, then drops by one step, then increases continuously by three steps. The voltages of the excitation signal are 0.3V, 0.6V, 0.9V, 0.2V, ... 8V, 1.1V, 1.4V, 1.7V, 1.6V, 1.9V, 2.3V, 2.7V, and the voltage of each excitation signal lasts for 0.15 seconds; (3) Step wave 4 shown in Figure 3C: The growth rate of the first few excitation signals and the last few excitation signals in the excitation signal sequence is different. The voltage of the excitation signal is 0.3V, 0.5V, 0.7V, 0.9V, 1.2V, 1.6V, 2.0V, 2.4V in sequence, and each voltage lasts for 0.15 seconds; (4) Step wave 5 shown in Figure 3D: The excitation signal sequence is a discontinuous excitation signal. Each excitation pulse signal is separated by a short open circuit time, and the interval time does not exceed 50% of the pulse excitation time. The voltage of the excitation signal is 0.3V, 0.8V, 1.2V, 1.7V, 2.2V in sequence, and each excitation pulse lasts for 0.15 seconds. There is a 0.05 second open circuit interval between pulses.

[0061] Excitation signal sequence options 2, 3, and 4 can capture output signals at relatively low excitation intensities and relatively high excitation intensities. For example, when the input signal is a potential, a range of 0.3-1.2V is more suitable for measuring common chemicals in a sample, while an input signal between 1.2-3.0V will stimulate chemicals requiring high excitation, such as hematocrit. Finally, a series of discrete, spaced pulse excitation signals can produce relatively distinct output signals.

[0062] Figure 4 shows the output current signal of the second excitation signal at different blood glucose concentrations when the step wave between the first excitation signal and the second excitation signal is scheme two, three, and four. It can be seen from the figure that the excitation size and type of the step wave have no direct effect on the size of the output current signal of the second excitation signal.

[0063] As shown in Figure 4, continuing the working method of Figure 2, the excitation signals applied to the first working electrode can be two or more, and the step wave of the second working electrode is sandwiched between two to three of the excitation waves. Figures 5A, 5B, and 5C show other multi-excitation sandwich excitation methods. In Figure 5A, the excitation signal sequence (step wave) of the second working electrode is sandwiched between the first and second excitation waves of the first working electrode. The step wave signal can be the step wave shown in Figure 3. The first working electrode is then subjected to subsequent multi-pulse excitation, and each pulse of the excitation wave applied to the first working electrode is a constant value excitation. The application time of each excitation can be the same or different, and the duration of each excitation is between 0.2-2 seconds. The open circuit time between each excitation wave can be equal or different, and the open circuit time is between 0.2-5 seconds. In Figure 5B, the second working electrode excitation signal sequence is sandwiched between the second and third excitation waves of the first working electrode. Among them, the excitation signal sequence can be the step wave case in Figure 3. The first working electrode is then subjected to subsequent multi-pulse excitation waves. The excitation wave applied to the first working electrode is a constant excitation, and the application time of each excitation can be the same or different. The excitation duration is between 0.2-2 seconds. The open circuit time between each excitation wave can be equal or unequal, and the open circuit time is between 0.2-5 seconds. In Figure 5C, two excitation signal sequences (step waves) are applied to the second working electrode, the first step wave is sandwiched between the first and second excitation waves of the first working electrode, and the second step wave is sandwiched between the second and third excitation waves, and then subsequent multi-pulse excitation waves are applied to the first working electrode. The first step wave of the second working electrode is a low-intensity excitation, for example, the excitation potential can be 0.15V-1.2V; and the second step wave is a high-intensity excitation, for example, the excitation potential can be 1.2V-2.5V. The excitation wave applied to the first working electrode is a constant excitation, and the application time of each excitation can be the same or different. The excitation duration is between 0.2-2 seconds. The open circuit time between each excitation wave can be equal or unequal, and the open circuit time is between 0.2-5 seconds.

[0064] Figure 6A shows the output current when the sandwich excitation method includes two excitation signals and a staircase wave. 0, 53, 287, ..., 1190 in the figure are reference blood glucose concentrations (mg / dL). It can be seen that the output current of the staircase wave is independent of the blood glucose concentration. Figure 6B shows the linear relationship between the output current of the excitation signal to the first working electrode and the blood glucose concentration in the sandwich excitation method. Here, i_p1 is the output current of the first excitation wave, and i_p2 is the output current of the second excitation wave. The excitation and current acquisition for the first excitation wave were completed within 1.75 seconds. At this time, the sensor chemical was still in a metastable state, resulting in a relatively high current, but also a high background current, resulting in an intercept-to-slope ratio of ~47 mg / dL for the response regression line. The second excitation wave was initiated at 3.75 seconds and completed and acquired within 5 seconds. The slope was slightly lower than that of the response line for the first excitation wave, but the background current was significantly reduced, resulting in an intercept-to-slope ratio of ~12.1 mg / dL. When the blood glucose concentration reaches 1190 mg / dL, the current output by the first excitation wave and the current output by the second excitation wave are slightly lower than the linear relationship formed at low concentrations (0-800 mg / dL), proving that this method has a wide detection range.

[0065] Figure 6C shows the output current of the sandwich excitation method after six pulses of excitation at the first working electrode. The values ​​0, 45, 273, ..., and 1151 in the figure represent reference blood glucose concentrations (mg / dL). The upper limit of detection for blood glucose concentrations can approach 1200 mg / dL, almost double the upper limit of commonly used blood glucose meters (600 mg / dL). Each excitation wave is preceded by a short incubation period, namely the first and second idle periods in Figure 2, as well as the subsequent open-circuit and closed-circuit multi-pulse excitations. Compared to existing testing methods, which immediately apply voltage to the electrode at the start of the test, this method includes an idle period before each excitation signal. This idle period allows for the accumulation of oxidizable mediators during the incubation period, ensuring that the current generated by the first excitation wave is quite stable and can be used as a parameter for calculating analyte concentration. The second idle period is longer, allowing for the incubation and accumulation of more enzyme reactants, resulting in more stable and reproducible currents generated by the second excitation wave. The linear relationship between the output current and blood glucose concentration for other multi-pulse excitation waves is shown in Figure 6D. The linear relationship of the output current (i_p2, i_p3, i_p4, i_p5, i_p6) from the second excitation wave to the sixth excitation wave changes very little. As can be seen from Figure 6D, when the blood glucose concentration is 0-800 mg / dL, the output current from the second excitation wave to the sixth excitation wave has a good linear relationship with the blood glucose concentration. At 800-1150 mg / dL, the linear relationship is slightly worse, but still a linear relationship. Finally, Figure 6E shows the output current of the step wave, which is almost unchanged at different blood glucose concentrations. This feature forms an independent parameter that is unrelated to the output current of the first working electrode for testing other performance parameters related to blood glucose, wherein tp1 represents the output current of the first step wave, tp2 represents the output current of the second step wave, tp3 represents the output current of the third step wave, and tp4 represents the output current of the fourth step wave.

[0066] Table 1 summarizes the output current results for the six pulse excitation waves, including slope, intercept, and linear correlation coefficient. With the exception of the first excitation wave, the linear correlation coefficients for all other excitation waves are greater than 0.99. The linear constants for the second through fourth excitation waves are very similar. The fifth and sixth waves show a slight decrease, but remain quite close. The degree of similarity between the second through sixth excitation waves can be seen in the slope, intercept, correlation coefficient, and intercept / slope ratio, respectively. Multi-pulse output current provides more parameters related to blood glucose, improving the accuracy of test result calculations.

[0067] Table 1

[0068] Figure 7A shows the potential decay curves generated by the first working electrode at different analyte concentrations after the first excitation wave ends (i.e., the circuit is open). The values ​​51, 104, 282, 447, and 598 in the figure represent reference blood glucose concentrations (mg / dL). This set of decay curves demonstrates that the potential decay rate of the first working electrode after the first excitation wave ends is proportional to the analyte concentration. That is, the higher the analyte concentration, the faster the decay rate. Consequently, within a specific, short period of time after the circuit is opened, the higher the analyte concentration, the lower the output decay potential. This decay rate can be expressed as a relative decay coefficient, calculated as follows: subtract the potential at a certain point after decay from the initial potential (approximately 0.3 volts), and divide the result by the potential at that point. Figure 7B shows the relationship between the relative decay coefficient of the first working electrode's decay potential and the reference analyte concentration. In the figure, 1.75 / 1.754-1 = V1.75s / V1.754s-1. Similarly, 1.75 / 1.756-1 = V1.75s / V1.756s-1, and 1.75 / 1.758-1 = V1.75s / V1.758s-1. This figure shows that after the excitation of the first working electrode ends (open circuit), the relative attenuation coefficients can be used to indicate the analyte concentration. These three relative attenuation coefficients are nearly linearly related to the blood glucose concentration. Figure 7C shows the potential decay curve of the first corresponding electrode after the sensor system applies the second excitation wave to the first working electrode (open circuit). The values ​​51, 104, 282, 447, and 598 in the figure represent the reference blood glucose concentration (mg / dL). The potential difference between the first working electrode and the first corresponding electrode is based on the first corresponding electrode being zero, i.e., the instrument ground, while the first working electrode is input with the corresponding excitation potential. Therefore, the attenuation at the first corresponding electrode increases from zero, indicating a negative attenuation relationship. Figure 7D shows the relationship between the attenuation potential at the first corresponding electrode and the analyte concentration. In the figure, 5.008 represents the decay potential at 5.008 seconds, 5.01 represents the decay potential at 5.01 seconds, and 5.012 represents the decay potential at 5.012 seconds. This figure shows that the potential decay number at the first counter electrode is almost linearly proportional to the analyte concentration. As shown by the potential decays at the first working electrode and the first counter electrode, the decay potential number can be used as an auxiliary parameter for analyte concentration, helping to increase the accuracy and precision of analyte concentration measurements. For example, analyte concentration can be expressed using the multi-parameter regression equation: G = f(ip1,ip2,v1,1,v1,2,v1,3,v2,1,v2,2,v2,3…).

[0069] Figure 8 shows the output signals of the second working electrode under five step excitation waves in the first embodiment of the step wave method. Because the second working electrode is not covered by a chemical that reacts with the analyte, the current generated at the predetermined oxidation potential of 0.3V is almost zero. In other words, the second working electrode does not produce a response current related to blood glucose concentration. As the excitation potential increases, the current generated by the second working electrode also increases. In particular, when the excitation potential increases to 1.7V, the current generated by the second working electrode is quite high, reaching its peak at an excitation potential of 2.2V. However, as the hematocrit (%-HCT) value of the whole blood sample increases, the current generated decreases. This is because at high potentials, the current is generated by the complete oxidation of the oxidizable substances in the plasma (glucose cannot be directly oxidized). The HCT represents the number of red blood cells; higher HCT indicates the number of oxidizable substances, resulting in lower current. This intuitively demonstrates that the current generated by the second working electrode at relatively high excitation potentials can be used to represent the HCT value.

[0070] FIG9 is a graph showing the relationship between a set of current parameters extracted from the current generated by the second working electrode and the hematocrit value (%-HCT) in a whole blood sample. In the graph, 50, 250, and 400 represent blood glucose concentrations of 50 mg / dL, 250 mg / dL, and 400 mg / dL, respectively. FIG9A shows the linear relationship between the current of the fourth step wave and the hematocrit at different blood glucose concentrations. FIG9B shows the ratio of the current of the fourth step wave at different hematocrits to the current of the fourth step wave at 42% hematocrit on the ordinate, and the hematocrit on the abscissa. FIG9C shows the linear relationship between the current of the fifth step wave and the hematocrit at different blood glucose concentrations. FIG9D shows the ratio of the current of the fifth step wave at different hematocrits to the current of the fifth step wave at 42% hematocrit on the ordinate, and the hematocrit on the abscissa. As can be seen from FIG9B and FIG9D , the output current of the second working electrode exhibits a clear gradient in response to HCT. At higher excitation potentials (2.2 V versus 1.7 V), the gradient is more pronounced and the current is more independent of the analyte concentration (glucose concentration) of the blood sample.

[0071] Figure 10 shows the relationship between the current difference parameter set extracted from the current generated by the second working electrode and the hematocrit value (%-HCT) in the whole blood sample. In the figure, 50, 250, and 400 represent blood glucose concentrations of 50 mg / dL, 250 mg / dL, and 400 mg / dL, respectively. The current difference between the third and fourth step waves is Δi43 = i4 - i3, the current difference between the fourth and fifth step waves is Δi54 = i5 - i4, and the current difference between the third and fifth step waves is Δi53 = i5 - i3. 10A is a graph showing the linear relationship between Δi43 and hematocrit at different blood glucose concentrations. The ordinate of FIG. 10B represents the ratio of the current values ​​between Δi43 at different hematocrit values ​​and Δi43 at a hematocrit value of 42%, while the abscissa represents the hematocrit value. FIG. 10C is a graph showing the linear relationship between Δi54 and hematocrit at different blood glucose concentrations. The ordinate of FIG. 10D represents the ratio of the current values ​​between Δi54 at different hematocrit values ​​and Δi54 at a hematocrit value of 42%, while the abscissa represents the hematocrit value. FIG. 10E is a graph showing the linear relationship between Δi53 and hematocrit at different blood glucose concentrations. The ordinate of FIG. 10F represents the ratio of the current values ​​between Δi53 at different hematocrit values ​​and Δi53 at a hematocrit value of 42%, while the abscissa represents the hematocrit value. Compared to the gradients in Figures 9B and 9D, the gradients in Figures 10B, 10D, and 10F show a 10-15% improvement and are more independent of analyte concentration. Therefore, in practical applications, using direct current difference as a parameter for calculating HCT is more reliable and has greater resolution. This is why using a series of excitation signals is more useful and reliable than the current generated by a single high-potential excitation, and is one of the motivations for the present invention. Furthermore, the optimal regression equation for the %HCT of a whole blood sample can be found by using multiple parameters, namely, two or more current differences rather than a single high-potential current, i.e., %HCT = f(Δi43, Δi54, Δi53).

[0072] Example 1

[0073] The blood glucose concentration calculation method is applied to a biological sample analyte testing device, wherein the biological sample analyte testing device includes a first working electrode and a second working electrode, wherein the first working electrode is covered with a first chemical agent that can react with the analyte. Specifically, the first chemical agent includes a biological enzyme that can catalyze the analyte reaction, one or more mediators that can generate a combing effect between the enzyme reaction and the electrode reaction, and an inert substance that assists the chemical agent. The second working electrode is a blank electrode, covered with an inert substance that does not chemically react with the analyte, or covered with a second chemical agent different from the first chemical agent. A first excitation signal and a second excitation signal are input to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open circuit state.

[0074] Between the first excitation signal and the second excitation signal, an excitation signal sequence is input to the second working electrode, and after the excitation signal sequence is completed, the second working electrode is in an open circuit state; wherein the excitation signal sequence includes at least two continuous or non-continuous input signals, and each of the input signals is a constant value;

[0075] At regular intervals, the first excitation signal, the second excitation signal, and the output current of the current collection point of the excitation signal sequence are collected to obtain an output current group, and the blood glucose concentration is obtained. The blood glucose concentration G is calculated using Formula 1.

[0076] Formula 1 is The G in Equation 1 is calculated by Equation 2, and the f in Equation 1 is calculated by Equation 3, where f is a multi-parameter calibration factor.

[0077] Formula 2 is Where imean is the average value of the output currents of at least two current acquisition points in the second excitation signal, in nA; S is the slope of the linear equation between imean and glucose concentration. Exemplarily, S is obtained by linearly fitting the known historical imean and historical glucose concentrations. Alternatively, the historical imean and historical glucose concentrations are statistically correlated and their linear relationship is directly tabulated, and the slope S is obtained by table lookup.

[0078] Formula 3 is

[0079] Among them, in and ij are the output currents in the output current group, nA; i difference is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, nA; the value range of N is 10~53; the value range of J is 10~53; the value range of Xn is -500~500; the value range of Xj is -15~15; the value range of K is -30~0.

[0080] Alternatively, in and ij may be the output currents of consecutive sampling points in the output current group. For example, in may be i1, i2, i3, i4, i5, i6, i7, i8, i9, i10; and ij may be i6, i7, i8, i9, i10, i11, i12, i13, i14, i15. Alternatively, in and ij may be the output currents of non-consecutive sampling points in the output current group. For example, in may be i3, i5, i11, i13, i21; and ij may be i1, i2, i9, i17, i20, i29, i30. However, the present invention is not limited thereto, and those skilled in the art may select the values ​​of in and ij based on a statistical significance test method.

[0081] Optionally, the excitation time of the first excitation signal is 0.1-1s, and the excitation time of the second excitation signal is 0.1-2s; preferably, the excitation time of the first excitation signal is 0.5s, and the excitation time of the second excitation signal is 1.25s.

[0082] Optionally, i average is the average value of the output currents of at least three current collection points in the second excitation signal. For example, i average is the average value of the output currents of the last five current collection points in the second excitation signal.

[0083] Optionally, the voltage value of the first excitation signal is 0.2-0.4V, and the voltage value of the second excitation signal is 0.2-0.4V; preferably, the voltage value of the first excitation signal and the second excitation signal are both 0.3V.

[0084] Optionally, the total application time of the excitation signal sequence is at least 1 s, and the application time of each input signal in the excitation signal sequence is 0.1-0.3 s; preferably, the application time of each input signal in the excitation signal sequence is equal, both being 0.2 s.

[0085] Optionally, the voltage range of the excitation sequence is 0.1~3V, and the excitation signal sequence includes 4-8 consecutive input signals; preferably, the excitation signal sequence includes 5 consecutive input signals, and the voltage values ​​of the 5 consecutive input signals are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V respectively.

[0086] Optionally, the first excitation signal, the second excitation signal, and the output current of the current collection point of the excitation signal sequence are collected every 0.05 s.

[0087] Example 2

[0088] Based on Example 1, the main difference is that it further includes a step of applying an AC voltage to the second working electrode before inputting the first excitation signal to the working electrode to measure the impedance value of blood glucose, and obtaining the blood glucose concentration based on the output current group and the impedance value, wherein,

[0089] Where R is the impedance value, Ω; im is the output current in the output current group, nA; M ranges from 10 to 53; and Xm ranges from -120 to 120.

[0090] Compared with Example 1, Example 2 further considers the mutual influence between the impedance value and the output current.

[0091] Example 3

[0092] Based on Example 2, the method further includes the step of obtaining the measured ambient temperature, and obtaining the blood glucose concentration according to the output current group, the impedance value and the temperature, wherein:

[0093] Where T is the ambient temperature, in °C; iu is the output current in the output current group, in nA; U ranges from 10 to 53; and Xu ranges from -7 to 3.

[0094] Compared with Example 2, Example 3 further considers the mutual influence among the impedance value, ambient temperature and output current.

[0095] Experimental example

[0096] A sensor capable of executing the blood glucose concentration calculation method of the above-mentioned embodiments 1-3 is used to detect a blood sample to be tested. A calculation module is provided in the sensor, and the calculation module includes a calculation formula. The method and process of storing the calculation module are prior art and can be implemented by those skilled in the art, and will not be described in detail in this embodiment.

[0097] The test time and potential control in this test method are as follows: (1) a pre-test idle period of 1.25 seconds; during the test idle period, an AC voltage can be applied to the second working electrode to measure the impedance value of blood glucose; (2) a first excitation signal is applied to the first working electrode, a 0.3V potential difference is input to the first working electrode and the corresponding electrode for 0.5 seconds, and the output current is synchronously measured at intervals of 0.05 seconds; (3) after the first excitation signal ends, the first working electrode is set to an open circuit state, and the first potential decay signal of the first working electrode is measured at intervals of 0.005 seconds for 0.5 seconds; (4) while the first working electrode is in the open circuit state, an excitation signal is input to the second working electrode and the corresponding electrode. Signal sequence, the excitation signal sequence includes 5 excitations, the excitation voltages are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V, respectively, each excitation lasts for 0.2 seconds, and the output current is measured synchronously at intervals of 0.05 seconds; (5) the second working electrode is set to an open circuit state; (6) after the first working electrode is open for 2.0 seconds, a second excitation signal is applied to the first working electrode with a voltage of 0.3V for 1.25 seconds, and the output current is measured synchronously at intervals of 0.05 seconds; (7) after the second excitation signal ends, the first working electrode is set to an open circuit state, and the potential decay signal of the corresponding electrode is measured at intervals of 0.005 seconds for 0.5 seconds. The total time of the above test steps is 1.25+0.5+2+1.25+0.5=5.5 seconds.

[0098] Referring to Figure 11, Figure 11 shows a linear relationship between historical i average and historical glucose concentration, with a slope S of 22.181. In Examples 1-3, i average is the average of the output currents of the last five current collection points in the second excitation signal.

[0099] In Example 1, f=-18.78+1.068i3 / i average+3.46i5 / i average-5.58i6 / i average-5.14i9 / i average+6.51i10 / i average-11.95i12 / i average-8.05i18 / i average+3.355i21 / i average+4.292i23 / i average-6.792i24 / i average+0.991i26 / i average+0.3292i30 / i average+0.4228i31 / i average-2.718i32 / i average+1.779i33 / i average+0.790i37 / i average+18.22i52 / i average- 0.1262i difference i2 / i average -0.2950i difference i4 / i average +2.740i difference i8 / i average -2.310i difference i9 / i average +1.201i difference i11 / i average +1.653i difference i18 / i average -0.909i difference i22 / i average -1.059i difference i23 / i average +1.466i difference i24 / i average -0.01481i difference i27 / i average -0.04952i difference i29 / i average +0.3606i difference i32 / i average -0.3276i difference i34 / i average -0.298i difference i39 / i average +0.228i difference i48 / i average;

[0100] In Example 2, f=-27.81-33.79i12 / i average+11.87i19 / i average+1.321i23 / i average-6.96i26 / i average-0.2726i29 / i average+0.1939i31 / i average+1.046i33 / i average+28.46i52 / i average-0.0742i difference i2 / i average+0.2468i difference i8 / i average+1.290i difference i11 / i average-0.2111i difference i22 / i average-0.3908i difference i23 / i average+0.6853i difference i26 / i Average -0.02858i difference i30 / i average +0.0447i difference i31 / i average +0.3847i difference i33 / i average -0.675i difference i34 / i average +0.599i difference i37 / i average -0.580i difference i39 / i average +0.689Ri5 / i average -0.839Ri6 / i average +8.36Ri13 / i average -2.831Ri19 / i average +1.103Ri26 / i average -0.02016Ri27 / i average +0.1339Ri30 / i average -0.3000Ri32 / i average;

[0101] In Example 3, f=-18.27-51.0i11 / i average-3.232i24 / i average+4.20i27 / i average-4.77i30 / i average+12.12i31 / i average-13.90i32 / i average-440i51 / i average+476i52 / i average+2.690i differencei12 / i average+2.24i differencei14 / i average-0.541i Difference i23 / i average +0.675i Difference i24 / i average +0.0220i Difference i28 / i average -0.1763i Difference i29 / i average +0.1496i Difference i30 / i average +0.0723i Difference i31 / i average -0.2146i Difference i34 / i average +0.890i Difference i39 / i average -0.908i Difference i41 / i average +11.34R i11 / i average +0.659Ri23 / i average -1.100Ri27 / i average +1.258Ri30 / i average -3.069Ri31 / i average +3.511Ri32 / i average +112.4Ri51 / i average -116.8Ri52 / i average -0.0202TG calculated i differencei2 / i average +0.0574TG calculated i differencei3 / i Average -0.0318TG calculation i difference i4 / i average +2.381TG calculation i difference i11 / i average -6.085TG calculation i difference i12 / i average +0.0665TG calculation i difference i19 / i average -0.1297TG calculation i difference i22 / i average -0.1133TG calculation i difference i23 / i average +0.1401TG calculation i difference i26 / i average +0.03416TG calculation i difference i27 / i average +0.1098TG calculation i difference i29 / i average -0.1481TG calculation i difference i30 / i average -0.0645TG calculation i difference i32 / i average +0.0833TG calculation i difference i33 / i average -0.0434TG calculation i difference i36 / i average +0.02523TG calculation i difference i53 / i average;

[0102] In order to verify the accuracy of the detection method of this embodiment, the following verification is performed:

[0103] 1. Determine the reference value of blood glucose concentration. Measure the reference value of blood glucose concentration using a YSI 2300 blood glucose analyzer.

[0104] 2. Calculate the deviation. The deviation is the absolute deviation, which is calculated as follows: Deviation = blood glucose concentration test value - blood glucose concentration reference value.

[0105] Referring to Table 1, Table 2 shows the percentage of deviations of Examples 1-3 from the blood glucose concentration reference value within each range.

[0106] Table 2

[0107] As shown in Table 2, it can be seen that the blood glucose concentration calculated by the present invention, compared with the reference value of blood glucose concentration measured by the YSI2300 blood glucose analyzer, has a deviation of 66% within ±10%, 96% within ±15%, and 97% within ±20%.

[0108] Referring to Figures 12-14 , deviation comparisons of Examples 1-3 before and after calibration are shown, respectively. The figures before calibration show deviation comparisons of the calculated G value and the reference blood glucose concentration value, while the figures after calibration show deviation comparisons of the measured blood glucose concentration value G according to the present invention and the reference blood glucose concentration value. Figure 15 shows a deviation comparison of Examples 1-3 after calibration. It can be seen that the calibrated blood glucose concentration calculation method of the present invention comprehensively considers the interplay between multiple interfering factors related to blood glucose concentration, further improving the accuracy and precision of testing blood glucose concentration in biological samples.

[0109] Example 4

[0110] As shown in Figure 16, an embodiment of the present application provides a detection circuit for a blood glucose test device, which is built into the blood glucose test device. The blood glucose test device includes a test strip reaction unit 100 and a processing unit. The test strip reaction unit 100 is used to connect to a blood glucose test strip. The blood glucose test strip and the detection circuit can be independent and detachable structures, or they can be an integrated fixed structure.

[0111] As shown in Figure 1, a blood glucose test strip contains multiple electrodes, designated Electrode A through Electrode E. The upper ends of these electrodes are used to hold the blood glucose sample to be tested, while their lower ends connect to corresponding electrode interfaces (Interface A through Interface E) in the test strip's reaction unit 100. The electrodes in the blood glucose test strip correspond one-to-one with the electrode interfaces in the test strip's reaction unit 100. For ease of description, the following description assumes that the blood glucose test strip is connected to the test strip's reaction unit 100 unless otherwise specified.

[0112] Based on this, the multiple electrodes included in the test paper reaction unit 100 are combined. Depending on the different electrodes combined, corresponding electrode pairs can be formed, and depending on the different structures of the processing units connected to each electrode pair, it also has corresponding functions.

[0113] Specifically, the test paper reaction unit includes at least an impedance test electrode pair and a current test electrode pair. The impedance test electrode pair consists of a second working electrode A and a second pair of electrodes B, which are connected to the impedance detection module 101 in the processing unit. The current test electrode consists of a first working electrode C and a first pair of electrodes D, which are connected to the current detection module 102 and the potential tracking module 104 in the processing unit.

[0114] The impedance detection module 101 is used to test the impedance of the blood glucose sample, as shown in Figure 16, and includes a signal generator that can apply an AC signal or a DC signal to the impedance testing electrode pair based on demand.

[0115] The current detection module 102 can apply an excitation voltage to the current testing electrode pair to test the current of the blood glucose sample. After the excitation voltage is disconnected, the potential tracking module 104 can test the attenuation potential of the current testing electrode pair.

[0116] Compared to traditional methods that only measure the current to obtain the blood glucose value in the blood glucose sample, this method also measures the decay potential of the current test electrode pair. The decay potential can be used as a blood glucose calibration parameter to improve the accuracy of blood glucose testing.

[0117] In one embodiment, the impedance testing electrode pair includes a second working electrode (i.e., electrode A in FIG16 ) and a second pair of electrodes (i.e., electrode B in FIG16 ). In this case, the impedance detection module 101 further includes a first operational amplifier OP1, a signal generator is connected to the second working electrode, and the first operational amplifier OP1 is connected to the second pair of electrodes. The signal generator applies an AC signal or a DC signal to both ends of the impedance testing electrode pair based on demand, and impedance detection is performed through the operational amplifier OP1.

[0118] In one embodiment, the current testing electrode pair includes a first pair of electrodes (i.e., electrode C in FIG16 ) and a first working electrode (i.e., electrode D in FIG16 ). The current detection module 102 includes a second operational amplifier OP2, the first pair of electrodes is grounded via a switch, and the second operational amplifier OP2 is connected to the first working electrode via a switch. When blood in the blood glucose sample is absorbed into the test paper reaction unit, an excitation voltage V is applied between the current testing electrode pair, and the glucose enzyme undergoes a reduction reaction with the glucose in the blood glucose sample, thereby generating a weak current. The weak current in the current testing electrode pair is detected by the second operational amplifier OP2 and then converted into a blood glucose value.

[0119] Furthermore, the potential tracking module 104 includes a fourth operational amplifier OP4. The potential tracking module 104 is connected to the first working electrode and the first pair of electrodes via switches. Using a dual-pulse excitation method, when the excitation voltage across the current testing electrode pair is disconnected, the potential tracking module 104 can detect the decay potentials of the first working electrode and the first pair of electrodes, thereby enhancing blood glucose detection accuracy.

[0120] In one embodiment, the test paper reaction unit 100 further includes a sample injection detection electrode (ie, electrode E in FIG. 1 and FIG. 16 ), and the processing unit further includes a sample injection detection module 103 . The sample injection detection module 103 includes a third operational amplifier OP3 .

[0121] The sampling detection module 103 is connected to the sampling detection electrode through a switch. The sampling detection module 103 is based on the third operational amplifier OP3. It detects whether the blood glucose sample covers the test paper reaction unit 100 through the sampling detection electrode, and then determines whether the test paper reaction unit is full of blood. Only when it is full, will the corresponding blood glucose value be detected.

[0122] In one embodiment, one of the impedance testing electrode pairs is electrically connected to one of the current testing electrode pairs. As shown in FIG1 , electrode A and electrode C may be electrically connected, and electrode B and electrode D may be electrically connected.

[0123] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method for testing an analyte in a biological sample, characterized in that, Comprising the following steps: Bringing a working electrode into contact with a biological sample, wherein the working electrode includes a first working electrode and a second working electrode, the first working electrode is covered with a first chemical agent capable of reacting with an analyte, and the second working electrode is a blank electrode, covered with an inert substance that does not chemically react with the analyte, or covered with a second chemical agent different from the first chemical agent; Inputting at least two excitation signals to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open-circuit state; Between at least two of the excitation signals, inputting an excitation signal sequence to the second working electrode, and after the excitation signal sequence is completed, the second working electrode is in an open-circuit state; wherein the excitation signal sequence includes at least two consecutive or non-consecutive input signals, and each input signal is a constant value; Measuring at least one output signal in response to the excitation signal to obtain an output signal group, and at least one output signal sequence in response to the excitation signal sequence; Obtaining the concentration of the analyte in the biological sample according to the output signal group and the output signal sequence; 2. The method according to claim 1, wherein Further comprising: Measuring the attenuation signals after at least two excitation signals are completed to obtain an attenuation signal group; Obtaining the concentration of the analyte in the biological sample according to the output signal group, the attenuation signal group, and the output sequence signal; Preferably, after the excitation signal is completed, the attenuation signal is measured within no more than one second after the first working electrode is opened.

3. The method according to claim 1, wherein The at least two excitation signals include a first excitation signal and a second excitation signal, measuring at least one first output signal in response to the first excitation signal and at least one second output signal in response to the second excitation signal; Obtaining the concentration of the analyte in the biological sample according to the first output signal, the second output signal, and the output signal sequence; 4. The method according to claim 3, characterized in that, Measuring a first attenuation signal after the first excitation signal is completed and a second attenuation signal after the second excitation signal is completed; Obtaining the concentration of the analyte in the biological sample according to the first output signal, the second output signal, the first attenuation signal, the second attenuation signal, and the output signal sequence; 5. The method according to claim 4, characterized in that Further comprising measuring a third attenuation signal after the excitation signal sequence is completed; Obtaining the concentration of the analyte in the biological sample according to the first output signal, the second output signal, the first attenuation signal, the second attenuation signal, the third attenuation signal, and the output signal sequence; Preferably, after the excitation signal sequence is completed, the third attenuation signal is measured within no more than one second after the second working electrode is opened.

6. The method according to claim 3, characterized in that, The voltage values of the first excitation signal and the second excitation signal are equal, and the application time of the first excitation signal is less than the application time of the second excitation signal; Preferably, the voltage value of the first excitation signal is 0.2 - 0.4V, and the application time is at least 0.1 - 1s; the voltage value of the second excitation signal is 0.2 - 0.4V, and the application time is 0.1 - 2s; More preferably, the voltage values of the first excitation signal and the second excitation signal are both 0.3V, the application time of the first excitation signal is 0.5s, and the application time of the second excitation signal is 1.25s.

7. The method according to claim 1, wherein The excitation signal sequence includes at least two continuously applied input signals, and the output voltage of the excitation signal sequence increases or decreases continuously, with a voltage range of 0.1 - 3V; Preferably, the excitation signal sequence includes 4 - 8 consecutive input signals; More preferably, the excitation signal sequence includes 5 consecutive input signals, and the voltage values of the 5 consecutive input signals are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V respectively.

8. The method according to claim 1, wherein The excitation signal sequence includes at least two non - continuously applied input signals, and the output voltage of the excitation signal sequence increases or decreases discontinuously, with a voltage range of 0.1 - 3V; Preferably, the excitation signal sequence includes 4 - 8 consecutive input signals; More preferably, the excitation signal sequence includes 8 consecutive input signals, and the voltage values of the 6 consecutive input signals are 0.3V, 0.7V, 1.1V, 0.9V, 1.2V, 1.5V, 1.9V, and 2.3V respectively.

9. The method according to claim 1, wherein The excitation signal sequence includes 4 - 6 non - consecutive input signals; Preferably, the excitation signal sequence includes 5 non - consecutive input signals, and the voltage values of the 5 non - consecutive input signals are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V respectively.

10. The method according to claim 1, characterized in that, The total application time of the excitation signal sequence is at least 1s; preferably, the application time of each input signal in the excitation signal sequence is 0.1 - 0.3s; More preferably, the application time of each input signal in the excitation signal sequence is equal, all being 0.2s.

11. The method according to claim 1, characterized in that, Before inputting at least two excitation signals to the first working electrode, it further includes the step of applying an alternating voltage to the second working electrode to measure the impedance value of the biological sample; According to the output signal group, output signal sequence, and impedance value, the concentration of the analyte in the biological sample is obtained.

12. The method according to claim 1, wherein The first chemical agent includes redox enzyme and mediator, the redox enzyme is selected from at least one of FAD dehydrogenase and PQQ dehydrogenase, and the mediator is selected from one of ruthenium hexamine trichloride, potassium ferricyanide, ferrocene derivatives, methylene blue sulfate, and polyethersulfone resin.

13. The method according to claim 1, characterized in that, The inert substance is selected from at least one of carboxymethyl cellulose, hydroxymethyl cellulose, polyethylene glycol, and polyvinylpyrrolidone.

14. A sensor for testing an analyte in a biological sample, characterized in that, Comprising: A biosensor, the biosensor includes a first working electrode, wherein the first working electrode is covered with a first chemical agent capable of reacting with the analyte, the first working electrode can receive at least two excitation signals, and after each excitation signal is completed, the first working electrode is in an open - circuit state; An electrochemical sensor, the electrochemical sensor includes a second working electrode, wherein the second working electrode is a blank electrode, covered with an inert substance that does not chemically react with the analyte, or covered with a second chemical agent different from the first chemical agent. Between at least two of the excitation signals, the second working electrode can receive an excitation signal sequence, the excitation signal sequence includes at least two input signals, and each of the input signals is a constant value. Measure at least one output signal in response to the excitation signal to obtain a set of output signals, and at least one output signal sequence in response to the excitation signal sequence to determine the concentration of the target analyte in the biological sample.

15. The sensor according to claim 14, wherein After each excitation signal is received by the first working electrode, measure the attenuation signal of each excitation signal to obtain a set of attenuation signals, and determine the concentration of the analyte in the biological sample based on the set of output signals, the set of attenuation signals, and the output sequence signals.

16. A system for testing an analyte in a biological sample, characterized in that, Comprising: A biosensor, the biosensor includes a first working electrode and a corresponding first pair of electrodes, and a first chemical agent capable of reacting with the target analyte is covered on the first working electrode; An electrochemical sensor, the electrochemical sensor includes a second working electrode and a corresponding second pair of electrodes, and the second working electrode is a blank electrode or covered with an inert substance that does not chemically react with the analyte; A signal collection device, the signal collection device is capable of inputting at least two excitation signals to the first working electrode, and between at least two excitation signals, is capable of inputting an excitation signal sequence to the second working electrode, the excitation signal sequence includes at least two input signals, and each of the input signals is a constant value.

17. The system according to claim 16, wherein, The signal collection device includes: an operational amplifier, a signal generation module, a data acquisition module, and a data storage module.

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