Method, sensor and system for testing analyte in biological sample

Abstract

The invention discloses 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. The method comprises the following steps: contacting a first working electrode and a second working electrode with a biological sample, and inputting a first excitation signal and a second excitation signal to the first working electrode; inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal; 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 sequence of output signals responsive to the sequence of excitation signals on the first working electrode; and 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. 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 influence of interferents can be eliminated, and the detection accuracy of the test method is improved.

Description

Technical Field

[0001] 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

[0002] When detecting and analyzing an analyte in a biological sample, an input signal is applied to a working electrode of a sensor by a measuring device, and the working electrode transmits the input signal into the biological sample. The analyte in the biological sample undergoes an oxidation-reduction reaction to generate an output signal in response to the input signal, so as to determine the concentration of the analyte in the biological sample according to the output signal. The analyte includes substances such as blood glucose, blood ketone, blood lactic acid, cholesterol, uric acid, triglyceride, coagulation factor, anticoagulation factor, etc. 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.

[0003] Traditional biosensors achieve test selectivity through biomolecular catalysis of a single reaction, which is based on two chain reactions. Among them, the reduced state of the mediator in the second reaction (shuttle reaction) diffuses to the electrode and is then oxidized to generate a corresponding current signal. The catalytic process of the analyte can be described by the following three steps:

[0004] (1) Analyte + Enzyme 氧化态 = Biochemical reaction product + Enzyme 还原态

[0005] (2) Mediator 氧化态 + Enzyme 还原态 = Mediator 还原态 + Enzyme 氧化态

[0006] (3) Mediator 还原态 (electrode)= Mediator 氧化态 + e -

[0007] The enzyme biomolecule of the glucose detection sensor system solely catalyzes the oxidation of glucose molecules in blood. The added mediator reacts with the enzyme to convert the enzyme from the reduced state back to the oxidized state, thus playing a role in shuttling electrons. The glucose enzyme can be an oxidase or a dehydrogenase. Since oxidase is oxidized by dissolved oxygen in the blood sample while dehydrogenase is not, there is no "dissolved oxygen effect". Dehydrogenase has been the preferred enzyme in biosensors in the past two decades. The catalytic enzymes for other analytes may be oxidases or dehydrogenases, and the principles of their biosensors are basically the same. However, during the testing process, although the enzymatic catalytic reaction is basically single, there may be other oxidizable interfering substances 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 in the shuttling reaction, such as ferricyanide (Fe(CN)6 -3 ), the more likely it is to generate interference signals because the higher the electrode oxidation potential, the more interfering substances will be oxidized.

[0008] In addition to the above chemical interfering substances, there are other testing interferences in biosensors. For example, the percentage of hematocrit (%-HCT) is a relatively obvious interfering factor. This interference is due to the fact that during the diffusion of the mediator after the shuttling reaction to the electrode, its diffusion is hindered by blood cells, resulting in the diffusion coefficient changing with %-HCT. And the current in the electrochemical reaction will be affected by different %-HCT. Therefore, the factory batch calibration of the sensor test strip usually centers around 42% HCT. If the user's %-HCT is higher than 42%, the test will show a negative deviation. If the user's %-HCT is lower than 42%, the test will show a positive deviation. In addition, the aging effect generated during the storage time of the test strip will also interfere with the test results, that is, as the storage time prolongs, the sensitivity of the sensor test relatively declines, or more oxidizable substances that can provide background signals will be generated. Additionally, interfering factors include the test temperature.

[0009] Traditional biosensors generally apply a voltage to the electrode in contact with the analyte sample to oxidize the reduced mediator generated after the shuttling reaction, thereby generating a current signal responsive to the analyte. Through corresponding calculation methods, the current signal is converted into the analyte concentration. For the interfering factors during the testing process, existing methods mostly use different single parameters to exclude or weaken the interference compensation, so as to achieve the purpose of improving the test accuracy and precision. However, these methods of using single parameters for interference compensation often ignore the mutual influence relationships among multiple interfering factors. For example, the mutual influence between temperature and HCT, the mutual influence between temperature and test strip aging, the mutual influence between HCT and test strip aging, and so on.

[0010] For a long time, traditional biosensors have generated output signals with a single excitation and determined the analyte concentration from this single output signal. The later-developed multi-pulse excitation method, such as the method disclosed in CN109690304A, provides multiple parameters to improve the accuracy and precision of the sensor, overcome the influence of chemical interferents, and provide discrimination between whole blood samples and quality control solutions, thus significantly improving the performance of the biosensor. However, this method requires alternating input of pulse signals between two working electrodes, which is prone to test vibrations that affect the continuity of subsequent input signals, resulting in error signals that are difficult to identify, leading to a decrease in the accuracy of test results due to excessive uncertain factors.

[0011] Therefore, in order to avoid the generation of interference signals, there is still much room for improvement in the test methods for analytes in biological samples in terms of multi-pulse excitation and multi-parameter compensation, etc., to further improve the accuracy and precision of testing analytes in biological samples. Summary of the Invention

[0012] To solve the above problems, the inventor discloses an apparatus, system, and method for testing an analyte in a biological sample. The disclosed apparatus, system, and method are related to measuring the concentration of one or more analytes in a biological sample. The method involves applying at least two excitation signals to a first working electrode and, between the at least two excitation signals applied to the first working electrode, applying a sequence of excitation signals to a second working electrode. The input signals to the first and second working electrodes do not overlap in time. That is, after the excitation signal for the first working electrode is completed, the system controls the first working electrode in an open-circuit state, and then starts to apply the sequence of excitation signals to the second working electrode. After the sequence of excitation signals for the second working electrode is completed, the system controls the second working electrode in an open-circuit state, and then starts to apply another excitation signal to the first working electrode. The excitation signals applied to the first and second working electrodes are defined as the "sandwiched pulsing excitation". Among them, the first working electrode is covered with a first chemical agent that can chemically react with a predetermined analyte. The first chemical agent includes a biological enzyme that can catalyze the reaction of the analyte, one or more mediators that can generate a combing effect between the enzyme reaction and the electrode reaction, and an inert substance that aids the chemical agent. The chemical agent covered on the first pair of electrodes corresponding to the first working electrode includes at least a mediator, or has the same composition as the first chemical agent. The second working electrode is a blank electrode or is covered with an inert substance that does not chemically react with the analyte. Corresponding to the above-mentioned sandwiched pulsing excitation, the sensor system will measure the output signals generated by each input excitation signal, that is, the output signals generated by the first and second excitation signals applied to the first electrode, and the series of output signals generated by the series of excitation signals applied to the second electrode. At the same time, the measured signals also include the residual decay signal generated by the corresponding electrode conjugate to the first electrode after the first excitation signal of the first electrode stops, and the residual decay signal generated by the first electrode after the second excitation signal of the first electrode stops, and so on.

[0013] One aspect of the disclosed content of the sandwiched pulsing excitation will be used to determine one or more analytes in a biological sample. The analyte concentration is determined from one or more signal values in the first and second output signals of the first electrode, one or more signal values in the series of output signals of the second electrode, and the residual decay signal values of the first and second input signals to the first electrode.

[0014] Another aspect of the sandwich excitation method, particularly the values of the output signal series generated by the series of input excitation signals in the sandwich, will be used to measure other parameters in biological samples, including the hematocrit value (% - HCT) of whole blood samples, and to give appropriate compensation and correction to the analyte measurement through parameters related to the hematocrit value, excluding or reducing the influence of blood cells on the analyte measurement, thereby improving the accuracy and precision of the analyte measurement. Description of the Drawings

[0015] Figure 1 Schematic diagram of the sensor for testing analytes in biological samples of the present invention.

[0016] Figure 2 Schematic diagram of the main excitation signal of the sandwich excitation method in this embodiment.

[0017] Figure 3 Schematic diagram of different times of the staircase wave in the sandwich excitation method.

[0018] Figure 4 Output current signals of different staircase waves at different blood glucose concentrations.

[0019] Figure 5A Schematic diagram of another implementation manner of the multi - pulse sandwich excitation method.

[0020] Figure 5B Schematic diagram of yet another implementation manner of the multi - pulse sandwich excitation method.

[0021] Figure 5C Schematic diagram of yet another implementation manner of the multi - pulse sandwich excitation method.

[0022] Figure 6A Output current graph of the sandwich excitation method at different blood glucose concentrations.

[0023] Figure 6B Linear relationship graph between the output current of the excitation signal for the first working electrode in the sandwich excitation method and the blood glucose concentration.

[0024] Figure 6C Output current graph of the multi - pulse sandwich excitation method at different blood glucose concentrations.

[0025] Figure 6D Linear relationship graph between the output current of the excitation signal for the first working electrode of the multi - pulse sandwich excitation method and the blood glucose concentration.

[0026] Figure 6E Output current graph of different staircase waves at different blood glucose concentrations.

[0027] Figure 7A Potential decay curve at different blood glucose concentrations after the end of the first excitation wave applied to the first working electrode.

[0028] Figure 7B It is a graph showing the relationship between the relative attenuation coefficient of the decay potential of the first working electrode and the reference concentration of the analyte.

[0029] Figure 7C It is the potential decay curve at different blood glucose concentrations after the end of the second excitation wave applied to the first working electrode.

[0030] Figure 7D It is a graph showing the relationship between the decay potential on the first corresponding electrode and the analyte concentration.

[0031] Figure 8 It is a graph of the output current corresponding to the staircase wave of the second working electrode at different hematocrits.

[0032] Figure 9A It is a graph showing the linear relationship between the current of the fourth staircase wave and the hematocrit at different blood glucose concentrations.

[0033] Figure 9B It is a graph showing the ratio of the current value of the fourth staircase wave at different hematocrits to the current value of the fourth staircase wave at 42% hematocrit.

[0034] Figure 9C It is a graph showing the linear relationship between the current of the fifth staircase wave and the hematocrit at different blood glucose concentrations.

[0035] Figure 9D It is a graph showing the ratio of the current value of the fifth staircase wave at different hematocrits to the current value of the fifth staircase wave at 42% hematocrit.

[0036] Figure 10A It is for Δi at different blood glucose concentrations 43 and the graph of the linear relationship with the hematocrit.

[0037] Figure 10B It is for Δi at different hematocrits 43 and the graph of the relationship between the ratio of the current value of Δi at 42% hematocrit 43 and the hematocrit.

[0038] Figure 10C It is for Δi at different blood glucose concentrations 54 and the graph of the linear relationship with the hematocrit.

[0039] Figure 10D It is for Δi at different hematocrits 54 and the graph of the relationship between the ratio of the current value of Δi at 42% hematocrit 54 and the hematocrit.

[0040] Figure 10E It is for Δi at different blood glucose concentrations 53Graph of the linear relationship with hematocrit.

[0041] Figure 10F Δi at different hematocrits 53 and Δi at 42% hematocrit 53 Graph of the relationship between the ratio of current values and hematocrit.

[0042] Figure 11 Shows an exemplary embodiment of the linear relationship graph of the present invention i i平均 with glucose concentration.

[0043] Figure 12 Shows the deviation comparison graph before and after calibration in Example 1;

[0044] Figure 13 Shows the deviation comparison graph before and after calibration in Example 2;

[0045] Figure 14 Shows the deviation comparison graph before and after calibration in Example 3;

[0046] Figure 15 Shows the deviation comparison graph after calibration in Examples 1 - 3.

[0047] Figure 16 Schematic diagram of the detection circuit in the embodiments of the present application. Detailed implementation manners

[0048] The excitation described in the disclosure of the present invention is to apply an input signal to the electrodes in the sensor. The applied signals include positive and negative DC constant voltages or positive and negative DC constant current sources, etc., so that a complete or partial oxidation or reduction reaction occurs 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 test strip. When the predetermined excitation time ends, 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, applying a voltage of 0V (zero volts) to the electrode is not equal to an open circuit. Zero voltage is also a kind of input voltage. After the electrode is in an open circuit state, the oxidation-reduction substances on the electrode surface and nearby will cause the attenuation process of the remaining signal. This attenuation signal will give a reflection of the state that the electrode has experienced, so as to record other information related to the sensor and the analyte.

[0049] Figure 1The figure shows a schematic diagram of a sensor, where D represents the first working electrode, which is covered with a first chemical agent corresponding to a predetermined analyte. C represents the first pair of electrodes corresponding to the first working electrode, which is covered with at least a certain amount of mediator or the same chemical agent as the first working electrode. E represents the injection detection electrode, A represents the second working electrode in the sensor corresponding to the sandwich excitation method, which may be one of the electrode combinations for measuring impedance in the sensor, and its surface may be blank, or covered with an inert substance or a second chemical agent different from the first chemical agent, and B is the second pair of electrodes corresponding to the second working electrode.

[0050] The first chemical agent includes a redox enzyme and a 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. The inert substance is selected from at least one of carboxymethyl cellulose, hydroxymethyl cellulose, polyethylene glycol, and polyvinylpyrrolidone.

[0051] One of the related contents disclosed in the present invention is the sandwich excitation method. Figure 2 It is a schematic diagram of the main elements of the sandwich excitation method. First, a first excitation signal is applied to the first working electrode. After opening the circuit, an excitation signal sequence (i.e., the excitation series signal described in the figure) is continuously applied to the second working electrode. After opening the circuit, a second excitation signal is applied to the first working electrode again.

[0052] Figure 2 It is a schematic diagram of the main excitation signals of the sandwich excitation method in this embodiment. Where the horizontal axis is 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 from 0 to 2 seconds. 0s means that after the sensor system determines that the sample completely fills the sensor cavity, the test program is immediately started, and the idle time between 0 and 2 seconds will be used for the enzyme-catalyzed reaction incubation 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 pair of electrodes.

[0053] 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 - 0.4V, preferably 0.3V, and the excitation time is 0.1 - 1s, preferably 0.5s.

[0054] After the first excitation signal is completed, the system opens the first working electrode, which is the second neutral gear of the first working electrode. During the second neutral gear, the active area of the first working electrode will undergo an enzymatic catalysis reaction to incubate and accumulate the reduced mediator after the comb current reaction, preparing for the electro-oxidation reaction of the second excitation signal. At the same time, the sensor system can perform other tests, such as measuring the attenuation signal generated after the first excitation signal on the first working electrode, and the measurement time is between 0.005 and 0.5 seconds.

[0055] Then, the system will start the excitation signal sequence for the second working electrode. The time of each excitation in the excitation signal sequence can be between 0.05 and 0.5 seconds. It can be a continuous excitation sequence without intervals or a sequence composed of different excitation pulses with intervals. Preferably, the excitation signal sequence is a continuous excitation sequence without intervals. It can be understood that this continuous excitation sequence can be a continuously increasing excitation sequence, or an excitation sequence that first increases and then decreases, first increases and then decreases and then increases, etc. Preferably, this continuous excitation sequence is a continuously increasing excitation sequence. If the excitation series signal is a voltage, the voltage value is between 0.1 and 3V. The excitation series signal shown in the figure includes 5 excitations, and the excitation voltages of the 5 excitations can be 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V in sequence, and the time of each excitation can be 0.2 seconds. This excitation series signal can be called a "ramp wave". After the excitation signal sequence of the second working electrode ends, the system places the second working electrode in an open circuit state and measures the attenuation signal of the second working electrode after opening the circuit, and the measurement time is between 0.005 and 0.5 seconds.

[0056] Finally, the system applies a second excitation signal to the first working electrode, the excitation time is between 0.1 and 2 seconds, preferably 1.25s, and the excitation voltage is 0.2 - 0.4V, preferably 0.3V. After the second excitation signal ends, the system places the first working electrode in an open circuit state and measures the potential attenuation signal of the corresponding electrode of the system, and the measurement is between 0.005 and 0.5.

[0057] As described above, preferably, in this test method, the test time and potential control are as follows: (1) The pre-test neutral gear is 1.25 seconds; (2) Apply a first excitation signal to the first working electrode, input a potential difference of 0.3V to the first working electrode and the corresponding electrode, and last for 0.5 seconds, and synchronously measure the output current signal at intervals of 0.05 seconds; (3) After the first excitation signal ends, set the first working electrode to an open circuit state, and measure the first potential decay signal of the first working electrode at intervals of 0.005 seconds, lasting for 0.5 seconds; (4) While the first working electrode is continuously in the open circuit state, input an excitation signal sequence to the second working electrode and the corresponding electrode. The excitation signal sequence includes 5 excitations, and the excitation voltages are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V in sequence. The duration of each excitation is 0.2 seconds, and the output current signal (hereinafter referred to as step wave one) is synchronously measured at intervals of 0.05 seconds; (5) Set the second working electrode to an open circuit state; (6) After the first working electrode is open for 2.0 seconds, apply a second excitation signal to the first working electrode, with a voltage of 0.3V (volt), lasting for 1.25 seconds, and synchronously measure the output current signal at intervals of 0.05 seconds; (7) After the second excitation signal ends, set the first working electrode to an open circuit state, and measure the potential decay signal of the corresponding electrode at intervals of 0.005 seconds, lasting 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.

[0058] In addition to Figure 2 the excitation signal sequence applied to the second working electrode shown in Figure 3 several different step wave cases in the sandwich excitation method are as follows: (1) Figure 3 Step wave two shown in A: After the excitation voltage of the excitation signal continuously increases by three steps, it drops by one step and then continuously increases 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) Figure 3 Step wave three shown in B: After the excitation voltage of the excitation signal continuously increases by three steps, it drops by one step, then continuously increases by three steps and drops by one step, and then continuously increases by three steps. The voltages of the excitation signal are 0.3V, 0.6V, 0.9V, 0.8V, 1.1V, 1.4V, 1.7V, 1.6V, 1.9V, 2.3V, and 2.7V in sequence. The voltage of each excitation signal lasts for 0.15 seconds; (3) Figure 3Step wave four as shown in C: The growth amplitudes of the first few and the last few excitation signals in the excitation signal sequence are different. The voltages of the excitation signals are 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) Figure 3 Step wave five as shown in D: The excitation signal sequence is a discontinuous excitation signal, and 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 voltages of the excitation signals are 0.3V, 0.8V, 1.2V, 1.7V, 2.2V in sequence, each excitation pulse lasts for 0.15 seconds, and there is an open - circuit interval of 0.05 seconds between pulses.

[0059] Schemes two, three, and four of the excitation signal sequence can collect output signals at relatively low excitation intensities and output signals at relatively high excitation intensities. For example, when the input signal is a potential, the range of 0.3 - 1.2V is more suitable for measuring common chemicals in the sample, while when the input signal is between 1.2 - 3.0V, it will excite chemicals that require high - intensity excitation, such as hematocrit values. Finally, the discontinuous interval pulse excitation series signals will generate relatively different output signals.

[0060] Figure 4 The output current signals 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 of scheme two, three, or four. It can be seen from the figure that the magnitude and type of the step - wave excitation have no direct influence on the magnitude of the output current signal of the second excitation signal.

[0061] Such as Figure 4 shown, continuing Figure 2 the working method, the excitation signal applied to the first working electrode can be two or multiple, and the step wave of the second working electrode is sandwiched between two to three excitation waves among them. Figure 5A , 5B, 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 as shown in Figure 3 . Then, subsequent multi - pulse excitation is performed on the first working electrode. Each pulse of the excitation wave applied to the first working electrode is a constant - value excitation, and the application time of each excitation can be the same or different. 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 excitation signal sequence of the second working electrode is sandwiched between the second and third excitation waves of the first working electrode. Among them, the excitation signal sequence can be Figure 3The case of the staircase wave. Then, a subsequent multi-pulse excitation wave is applied to the first working electrode. The excitation wave applied to the first working electrode is a constant-value excitation, and the application time of each excitation can be the same or different. The excitation duration is between 0.2 and 2 seconds. The open-circuit time between each excitation wave can be equal or unequal, and the open-circuit time is between 0.2 and 5 seconds. In Figure 5C , two excitation signal sequences (staircase waves) are applied to the second working electrode. The first staircase wave is sandwiched between the first and second excitation waves of the first working electrode, and the second staircase wave is sandwiched between the second and third excitation waves. Then, a subsequent multi-pulse excitation wave is applied to the first working electrode. The first staircase wave of the second working electrode is a low-intensity excitation. For example, the excitation potential can be 0.15V - 1.2V; while the second staircase 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-value excitation, and the application time of each excitation can be the same or different. The excitation duration is between 0.2 and 2 seconds. The open-circuit time between each excitation wave can be equal or unequal, and the open-circuit time is between 0.2 and 5 seconds.

[0062] Figure 6A is the output current when the sandwich excitation method includes two excitation signals and a staircase wave. 0, 53, 287,..., 1190 in the figure are the blood glucose reference concentrations (mg / dL) respectively. It can be seen that the output current of the staircase wave has nothing to do with the blood glucose concentration. Figure 6B is a linear relationship diagram of the output current of the excitation signal for the first working electrode in the sandwich excitation method and the blood glucose concentration. Among them, 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 of the first excitation wave are completed within 1.75 seconds. At this time, the sensor chemical agent is still in a metastable state, the current is quite high, but the background current is also very high, so that the ratio of the intercept to the slope of the response regression line is at ~47 mg / dL. The second excitation wave starts at 3.75 seconds and completes and acquires the current at 5 seconds. The slope is slightly lower than that of the first excitation wave response line, but the background current drops significantly, so that the ratio of the intercept to the slope reaches ~12.1 mg / dL. After 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 by the low concentration (0 - 800 mg / dL). It is proved that this method has a wide detection range.

[0063] Figure 6C is the output current of six-pulse excitation for the first working electrode in the sandwich excitation method. 0, 45, 273,..., 1151 in the figure are the blood glucose reference concentrations (mg / dL) respectively. The upper limit of the detection of the blood glucose concentration can be close to 1200 mg / dL, which is almost twice the upper limit of the commonly used blood glucose meters (600 mg / dL) on the market. There is a short incubation period before each excitation wave, that isFigure 2 The first and second neutral gears and subsequent open-circuit and closed-circuit multi-pulse excitations. Compared with the existing test methods, that is, applying voltage to the electrode immediately at the start of the test, there is a neutral gear before each excitation signal in this test method. The accumulation of oxidizable mediators brought about by the neutral gear as an incubation period ensures that the current generated by the first excitation wave is quite stable and can be used as one of the parameters for calculating the analyte concentration. The second neutral gear has a longer time, incubating and accumulating more enzyme reactants, so that the current generated by the second excitation wave is more stable and has better repeatability. The linear relationship between the output currents of other multi-pulse excitation waves and the blood glucose concentration is shown by Figure 6D It can be seen. The change in the linear relationship of the output currents (i_p2, i_p3, i_p4, i_p5, i_p6) from the second excitation wave to the sixth excitation wave is very small. From Figure 6D It can be seen that when the blood glucose concentration is 0 - 800 mg / dL, the linear relationship between the output currents from the second excitation wave to the sixth excitation wave and the blood glucose concentration is good. When it is 800 - 1150 mg / dL, the linear relationship is slightly worse, but still linear. Finally, Figure 6E shows the output current of the staircase wave, which is almost unchanged at different blood glucose concentrations. This feature exactly forms an independent parameter unrelated to the output current of the first working electrode for testing other performance parameters related to blood glucose. Among them, tp1 represents the output current of the first staircase wave, tp2 represents the output current of the second staircase wave, tp3 represents the output current of the third staircase wave, and tp4 represents the output current of the fourth staircase wave.

[0064] Table 1 summarizes the results of the output currents of six pulse excitation waves, including slope, intercept, and linear correlation coefficient. Except for the first excitation wave, the linear correlation coefficients of other excitation waves are all greater than 0.99. Among them, the linear constants of the second to fourth excitation waves are very close. The fifth and sixth ones decrease, but are also quite close. The closeness of the second to sixth excitation waves can be seen from the slope, intercept, correlation coefficient, and intercept / slope ratio respectively. The multi-pulse output current will provide more parameters related to blood glucose and can improve the calculation accuracy of the test results.

[0065] Table 1

[0066]

[0067] Figure 7AIt is the potential decay curve generated by the first working electrode at different analyte concentrations after the end of the first excitation wave applied to the first working electrode (i.e., open circuit). 51, 104, 282, 447, and 598 in the figure are the blood glucose reference concentrations (mg / dL) respectively. This set of decay curves shows that after the first working electrode is excited by the first excitation wave and ends, the potential decay rate is proportional to the concentration of the analyte. That is, the higher the analyte concentration, the faster the decay rate. Thus, within a short specific time after open circuit, the higher the analyte concentration, the lower the output decay potential. And this decay rate can be expressed by the relative decay coefficient and can be obtained by the following calculation: subtract the potential at a certain time point after decay from the initial potential (nearly 0.3 volts), and then divide by the potential at that time point. Figure 7B It is a graph showing the relationship between the relative decay coefficient of the decay potential of the first working electrode and the analyte reference 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 graph shows that after the first working electrode is excited and ends (open circuit), the relative decay coefficient can be used to indicate the analyte concentration. These three relative decay coefficients have an approximately linear relationship with the blood glucose concentration. Figure 7C It is the potential decay curve of the first corresponding electrode after the sensor system applies the second excitation wave to the first working electrode and ends (open circuit). 51, 104, 282, 447, and 598 in the figure are the blood glucose reference concentrations (mg / dL) respectively. The potential difference relationship between the first working electrode and the first corresponding electrode is that the first corresponding electrode is zero, that is, the instrument ground wire, and the first working electrode is input with the corresponding excitation potential. Therefore, the decay on the first corresponding electrode starts from zero and rises, belonging to a negative decay relationship. Figure 7D It is a graph showing the relationship between the decay potential on the first corresponding electrode and the analyte concentration. 5.008 in the figure represents the decay potential at the 5.008th second, 5.01 represents the decay potential at the 5.01st second, and 5.012 represents the decay potential at the 5.012th second. This graph shows that the potential decay number of the first corresponding electrode has an almost linear proportional relationship with the analyte concentration. From the potential decays of the above-mentioned first working electrode and the first corresponding electrode, it can be seen that the decay potential number can be used as an auxiliary parameter for the analyte concentration, which is beneficial to increasing the accuracy and precision of measuring the analyte concentration. For example, the analyte concentration can be expressed by a multi-parameter regression equation: G = f(ip1, ip2, v1,1, v1,2, v1,3, v2,1, v2,2, v2,3...).

[0068] Figure 8In one embodiment of the staircase wave, it is the output signal of the second working electrode under five staircase excitation waves. Since the second working electrode is not covered with a chemical agent that can react with the analyte, at a predetermined oxidation potential of 0.3 V, the generated current is almost close to zero. That is to say, the second working electrode does not generate a response current related to the blood glucose concentration. As the excitation potential increases, the current generated by the second working electrode also increases. Especially when the excitation potential increases to 1.7 V, the current generated by the second working electrode is quite high and reaches the maximum at an excitation potential of 2.2 V. However, as the hematocrit value (% - HCT) of the whole blood sample increases, the generated current decreases. This is because at high potentials, all the oxidizable substances (glucose cannot be directly oxidized) in the plasma part of the blood are oxidized to obtain current, and HCT represents the number of red blood cells. The higher the HCT, the fewer the oxidizable substances, and the lower the obtained current. This intuitively proves that when the second working electrode is at a relatively high excitation potential, the current it generates can be used to represent the HCT value.

[0069] Figure 9 is a graph showing the relationship between the relevant current parameter group extracted from the current generated by the second working electrode and the hematocrit value (% - HCT) in the whole blood sample. The 50, 250, and 400 in the figure represent blood glucose concentrations of 50 mg / dL, 250 mg / dL, and 400 mg / dL respectively. Among them, Figure 9A is a linear relationship graph of the current of the fourth staircase wave and the hematocrit under different blood glucose concentrations, Figure 9B the ordinate of which is the ratio of the current value of the fourth staircase wave at different hematocrits to the current value of the fourth staircase wave at 42% hematocrit, and the abscissa is the hematocrit, Figure 9C is a linear relationship graph of the current of the fifth staircase wave and the hematocrit under different blood glucose concentrations, Figure 9D the ordinate of which is the ratio of the current value of the fifth staircase wave at different hematocrits to the current value of the fifth staircase wave at 42% hematocrit, and the abscissa is the hematocrit. From Figure 9B and 9D it can be seen that the output current of the second working electrode has an obvious gradient in response to HCT. The higher the excitation potential (2.2 V compared to 1.7 V), the more obvious the gradient, and the current is more independent of the analyte concentration (blood glucose concentration) of the blood sample.

[0070] Figure 10 is a graph showing the relationship between the relevant current difference parameter group extracted from the current generated by the second working electrode and the hematocrit value (% - HCT) in the whole blood sample. The 50, 250, and 400 in the figure represent blood glucose concentrations of 50 mg / dL, 250 mg / dL, and 400 mg / dL respectively. The current difference between the third and fourth staircase waves is Δi43 = i4 - i3, the current difference between the fourth and fifth staircase waves is Δi54 = i5 - i4, and the current difference between the third and fifth staircase waves is Δi53 = i5 - i3. Among them,Figure 10A It is a linear relationship graph of Δi43 and hematocrit at different blood glucose concentrations. Figure 10B The ordinate of is the ratio of the current values between Δi43 at different hematocrits and Δi43 at 42% hematocrit, and the abscissa is the hematocrit. Figure 10C It is a linear relationship graph of Δi54 and hematocrit at different blood glucose concentrations. Figure 10D The ordinate of is the ratio of the current values between Δi54 at different hematocrits and Δi54 at 42% hematocrit, and the abscissa is the hematocrit. Figure 10E It is a linear relationship graph of Δi53 and hematocrit at different blood glucose concentrations. Figure 10F The ordinate of is the ratio of the current values between Δi53 at different hematocrits and Δi53 at 42% hematocrit, and the abscissa is the hematocrit. Compared with Figure 9B and 9D the gradients in, Figure 10B , 10D , the gradients in 10F have a 10 - 15% improvement and are more independent of the influence of analyte concentration. Therefore, in practical applications, it is more reliable and has a higher resolution to use the direct current difference as the parameter for calculating HCT. This is where using the excitation series signal is more useful and reliable than the current generated by a single high potential excitation, which is also one of the motivations of the present invention. At the same time, the %HCT of the whole blood sample can also find the optimal regression equation number, that is, %HCT = f(Δi43, Δi54, Δi53), through multiple parameters, namely the current differences generated by more than two current differences rather than a single high potential.

[0071] Example 1

[0072] The blood glucose concentration calculation method is applied to a biological sample analyte testing device, which includes a first working electrode and a second working electrode. 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 current effect between the enzyme reaction and the electrode reaction, and an inert substance that aids 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.

[0073] An excitation signal sequence is input to the second working electrode between the first excitation signal and the second excitation signal. 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 discontinuous input signals, and each input signal is a constant value.

[0074] The output current of the current acquisition points of the first excitation signal, the second excitation signal, and the excitation signal sequence is collected at regular intervals to obtain an output current group, and the concentration of the blood glucose is obtained. The calculation method of the blood glucose concentration G is obtained through Equation 1.

[0075] Equation 1 is And the calculation of G in Equation 1 is obtained through Equation 2, and f in Equation 1 is obtained through Equation 3, where f is a multi-parameter calibration factor.

[0076] Equation 2 is Wherein, i_avg 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 of i_avg and the glucose concentration. Exemplarily, S is obtained by linearly fitting the known historical i_avg and historical glucose concentration. Alternatively, the linear relationship between the historical i_avg and historical glucose concentration is obtained through statistical laws and then directly listed, and the slope S value is obtained by looking up the table.

[0077] Equation 3 is

[0078] Wherein, in and ij are both output currents in the output current group, in nA; i_diff is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, in nA; the value range of N is 10 to 53; the value range of J is 10 to 53; the value range of Xn is -500 to 500; the value range of Xj is -15 to 15; the value range of K is -30 to 0.

[0079] Optionally, in and ij can be the output currents of consecutive acquisition points in the output current group. For example, in can be i1, i2, i3, i4, i5, i6, i7, i8, i9, i10; ij can be i6, i7, i8, i9, i10, i11, i12, i13, i14, i15. Optionally, in and ij can be the output currents of non-consecutive acquisition points in the output current group. For example, in can be i3, i5, i11, i13, i21; ij can be i1, i2, i9, i17, i20, i29, i30. However, the present invention is not limited thereto, and those skilled in the art can select the values of in and ij according to the statistical significance test method.

[0080] Optionally, the excitation time of the first excitation signal is 0.1 - 1 s, and the excitation time of the second excitation signal is 0.1 - 2 s; preferably, the excitation time of the first excitation signal is 0.5 s, and the excitation time of the second excitation signal is 1.25 s.

[0081] Optionally, i_avg is the average value of the output currents at at least three current acquisition points in the second excitation signal. For example, i_avg is the average value of the output currents at the last 5 current acquisition points in the second excitation signal.

[0082] Optionally, the voltage value of the first excitation signal is 0.2 - 0.4 V, and the voltage value of the second excitation signal is 0.2 - 0.4 V; preferably, the voltage values of the first excitation signal and the second excitation signal are both 0.3 V.

[0083] 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.

[0084] Optionally, the voltage range of the excitation sequence is 0.1 - 3 V, 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 0.3 V, 0.8 V, 1.2 V, 1.7 V, and 2.2 V respectively.

[0085] Optionally, the output current at the current acquisition point of the first excitation signal, the second excitation signal, and the excitation signal sequence is collected every 0.05 s.

[0086] Embodiment 2

[0087] Based on Embodiment 1, the main difference is that it further includes the step of applying an alternating voltage to the second working electrode before applying the first excitation signal to the working electrode to measure the impedance value of blood glucose, and obtaining the concentration of the blood glucose according to the output current group and the impedance value, where

[0088]

[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; Xm ranges from - 120 to 120.

[0090] Compared with Embodiment 1, in Embodiment 2, the mutual influence between the impedance value and the output current is further considered.

[0091] Embodiment 3

[0092] Based on Embodiment 2, it further includes the step of obtaining the measured ambient temperature, and obtaining the concentration of the blood glucose according to the output current group, the impedance value, and the temperature, where,

[0093]

[0094] where, T is the ambient temperature, °C; iu is the output current in the output current group, nA; U has a value range of 10 to 53; Xu has a value range of -7 to 3.

[0095] Compared with Embodiment 2, in Embodiment 3, the mutual influence among the impedance value, the ambient temperature, and the output current is further considered.

[0096] Experimental Example

[0097] The sensor capable of executing the blood glucose concentration calculation method of the above Embodiments 1-3 is used to detect the blood sample to be tested. A calculation module is provided in the sensor. The calculation module includes a calculation formula. The method and process of storing the calculation module are prior arts and can be implemented by those skilled in the art, which will not be elaborated in this embodiment.

[0098] In this test method, the test time and potential control are as follows: (1) The pre-test neutral gear is 1.25 seconds; during the test neutral gear, an alternating voltage can be applied to the second working electrode to measure the impedance value of the blood glucose; (2) Apply a first excitation signal to the first working electrode, input a potential difference of 0.3V to the first working electrode and the corresponding electrode, and last for 0.5 seconds, and synchronously measure the output current at intervals of 0.05 seconds; (3) After the first excitation signal ends, set the first working electrode to the open circuit state, and measure the first potential decay signal of the first working electrode at intervals of 0.005 seconds, lasting for 0.5 seconds; (4) While the first working electrode is in the continuous open circuit state, input an excitation signal sequence to the second working electrode and the corresponding electrode. The excitation signal sequence includes 5 excitations, and the excitation voltages are 0.3V, 0.8V, 1.2V, 1.7V, and 2.2V in sequence. The duration of each excitation is 0.2 seconds, and the output current is synchronously measured at intervals of 0.05 seconds; (5) Set the second working electrode to the open circuit state; (6) After the first working electrode is open for 2.0 seconds, apply a second excitation signal to the first working electrode, with a voltage of 0.3V, lasting for 1.25 seconds, and synchronously measure the output current at intervals of 0.05 seconds; (7) After the second excitation signal ends, set the first working electrode to the open circuit state, and measure the potential decay signal of the corresponding electrode at intervals of 0.005 seconds, lasting 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.

[0099] Reference Figure 11 as shown Figure 11A graph showing a linear relationship between the historical i average and the historical glucose concentration is presented, with a slope S of 22.181. In Examples 1-3, the i average is the average of the output currents of the last 5 current acquisition points in the second excitation signal.

[0100] 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;

[0101] 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;

[0102] 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 difference i12 / i average + 2.24i difference i14 / 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 + 11.34Ri11 / 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 calculation i difference i2 / i average + 0.0574TG calculation i difference i3 / 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;

[0103] To verify the accuracy of the detection method in this example, the following verification was carried out:

[0104] 1. Determine the reference value of blood glucose concentration. The reference value of blood glucose concentration was measured by a YSI 2300 blood glucose analyzer.

[0105] 2. Calculate the deviation. The deviation is the absolute deviation, and the deviation = the measured value of blood glucose concentration - the reference value of blood glucose concentration.

[0106] As shown in Table 1 for reference, Table 2 shows the proportion of the deviations of Examples 1 - 3 from the reference value of blood glucose concentration within each range.

[0107] Table 2

[0108]

[0109] As can be seen from the reference Table 2, the deviation of the blood glucose concentration calculated by the present invention is within ±10% for 66%, within ±15% for 96%, and within ±20% for 97% compared with the reference value of the blood glucose concentration measured by the YSI 2300 blood glucose analyzer.

[0110] Reference Figures 12 - 14 shown, respectively show the deviation comparison diagrams before and after calibration of Examples 1-3. Among them, the diagram before calibration in the figure represents the deviation comparison diagram between G calculation and the reference value of blood glucose concentration, and the diagram after calibration in the figure represents the deviation comparison diagram between the detected value G of the blood glucose concentration of the present invention and the reference value of blood glucose concentration. Figure 15 The deviation comparison diagrams after calibration of Examples 1-3 are shown. It can be seen that the blood glucose concentration calculation method after calibration of the present invention comprehensively considers the mutual influence relationship between various interference factors related to blood glucose concentration, and further improves the accuracy and precision of measuring blood glucose concentration in biological samples.

[0111] Example 4

[0112] As Figure 16 shown, an embodiment of the present application provides a detection circuit for a blood glucose testing device, and this detection circuit is built in the blood glucose testing device. The blood glucose testing device includes a test strip reaction unit 100 and a processing unit. The test strip reaction unit 100 is used to connect with a blood glucose test strip, and the blood glucose test strip and the detection circuit can be a mutually independent detachable structure or an integrated fixed structure.

[0113] As Figure 1 shown, the blood glucose test strip contains multiple electrodes, namely electrode A to electrode E. The upper ends of these multiple electrodes are used to place the blood glucose sample to be detected, and the lower ends are connected to the corresponding electrode interfaces (respectively interface A to interface E) in the test strip reaction unit 100. The electrodes in the blood glucose test strip and the electrode interfaces in the test strip reaction unit 100 are in one-to-one correspondence. For the convenience of description, when described below, if there is no additional explanation, it is default that the blood glucose test strip has been connected to the test strip reaction unit 100.

[0114] Based on this, a plurality of electrodes included in the test strip reaction unit 100 are combined. According to the different combinations of electrodes, corresponding electrode pairs can be formed, and according to the different structures of the processing units connected to each electrode pair, they also have corresponding functions.

[0115] Specifically, the test strip reaction unit at least includes an impedance test electrode pair and a current test electrode pair. Among them, the impedance test electrode pair is composed of a second working electrode A and a second counter electrode B, which is connected to the impedance detection module 101 in the processing unit. The current test electrode is composed of a first working electrode C and a first counter electrode D, which is connected to the current detection module 102 and the potential tracking module 104 in the processing unit.

[0116] The impedance detection module 101 is used to test the impedance of the blood glucose sample. As Figure 16 shown, it contains a signal generator. Based on requirements, the signal generator can apply an AC signal or a DC signal to the impedance test electrode pair.

[0117] The current detection module 102 can apply an excitation voltage to the current test 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 decay potential of the current test electrode pair.

[0118] Compared with the traditional solution where only the current is detected to obtain the blood glucose value in the blood glucose sample, the decay potential of the current test electrode pair can also be measured. And the decay potential can be used as one of the blood glucose calibration parameters, which can improve the accuracy of blood glucose detection.

[0119] In one embodiment, the impedance test electrode pair includes a second working electrode (i.e., Figure 16 electrode A in Figure 16 ), a second counter electrode (i.e.,

[0120] electrode B in Figure 16 ). At this time, the impedance detection module 101 further includes a first operational amplifier OP1. The signal generator is connected to the second working electrode, and the first operational amplifier OP1 is connected to the second counter electrode. The signal generator applies an AC signal or a DC signal to both ends of the impedance test electrode pair based on requirements, and realizes impedance detection through the operational amplifier OP1. Figure 16 In one embodiment, the current test electrode pair includes a first counter electrode (i.e.,

[0121] electrode C in Figure 16 ), a first working electrode (i.e., Figure 16 electrode D in Figure 16 ). The current detection module 102 includes a second operational amplifier OP2. The first counter electrode is grounded through a switch, and the second operational amplifier OP2 is connected to the first working electrode through a switch. When the blood in the blood glucose sample is inhaled into the test strip reaction unit, an excitation voltage V is applied between the current test electrode pair. The glucose enzyme reacts with glucose in the blood glucose sample to produce a weak current. The weak current in the current test electrode pair is detected by the second operational amplifier OP2, and then converted into a blood glucose value.Further, 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 respectively through switches. By adopting a double-pulse excitation mode, when the excitation voltage across the current test electrode pair is disconnected, the potential tracking module 104 can detect the decay potential of the first working electrode and the first pair of electrodes, so as to use it for the detection of blood glucose value, thereby increasing the detection accuracy.

[0122] In one embodiment, the test strip reaction unit 100 further includes a sample injection detection electrode (i.e., Figure 1 and Figure 16 the electrode E in), and the processing unit further includes a sample injection detection module 103, and the sample injection detection module 103 includes a third operational amplifier OP3.

[0123] The sample injection detection module 103 is connected to the sample injection detection electrode through a switch. Based on the third operational amplifier OP3, the sample injection detection module 103 detects whether the blood glucose sample covers the test strip reaction unit 100 through the sample injection detection electrode, and then judges whether the blood in the test strip reaction unit is full. Only after it is full, the corresponding blood glucose value will be detected.

[0124] In one embodiment, one of the impedance test electrode pairs is electrically connected to one of the current test electrode pairs. As Figure 1 shown, it can be that electrode A and electrode C are electrically connected, and electrode B and electrode D are electrically connected.

[0125] As described above, only the embodiments of the present application are given. The protection scope 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, various changes and modifications can be made to the present application. Any modifications, equivalent replacements, improvements, etc. made within the technical idea and principle of the present application shall be included in the protection scope 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 It further includes: 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, wherein Measuring the first attenuation signal after the first excitation signal is completed and the 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, wherein It further includes 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, characterized in that, The excitation signal sequence includes at least two continuously applied input signals, and the output voltage of the excitation signal sequence increases continuously 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 discontinuously 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 applying 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; Based on 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 a redox enzyme and a 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 by 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 including 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, and between at least two of the excitation signals, the second working electrode is capable of receiving an excitation signal sequence, the excitation signal sequence including at least two input signals, each of the input signals being 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, characterized in that, 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 according to 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 by comprising: A biosensor, the biosensor including a first working electrode and a corresponding first pair of electrodes, the first working electrode being covered with a first chemical agent capable of reacting with the target analyte; An electrochemical sensor, the electrochemical sensor including a second working electrode and a corresponding second pair of electrodes, the second working electrode being 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 being capable of inputting at least two excitation signals to the first working electrode, and between at least two excitation signals, being capable of inputting an excitation signal sequence to the second working electrode, the excitation signal sequence including at least two input signals, each of the input signals being 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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