Method and sensor for testing analyte concentration in biological sample based on multiple excitation modes

Abstract

The invention discloses a method and a sensor for testing the concentration of an analyte in a biological sample based on a multi-excitation mode, and belongs to the technical field of instant detection of the concentration of the analyte in the biological sample. The method comprises the following steps: inputting a first excitation signal and a second excitation signal to a first working electrode; inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal; acquiring the first excitation signal, the second excitation signal and the output current of the current acquisition point of the excitation signal sequence at regular intervals to obtain an output current group; and acquiring output voltages of voltage acquisition points of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal at regular intervals to obtain an output voltage group. According to the method, the mutual influence relationship among various interference factors related to the analyte is eliminated or weakened, and the accuracy, precision and aging resistance of concentration detection of the analyte in the biological sample are improved.

Description

Technical Field

[0001] The present application relates to a method and a sensor for testing the concentration of an analyte in a biological sample based on a multi-excitation mode, and belongs to the technical field of analyte concentration detection. Background Art

[0002] Traditional biosensors achieve test selectivity by catalyzing a single reaction through biomolecules, which is based on two chain reactions. The reduced state of the electron mediator in the second reaction (comb flow 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:

[0003] (1) Analyte + enzyme oxidation state → biochemical reaction product + enzyme reduction state;

[0004] (2) Electron mediator oxidized state + enzyme reduced state → electron mediator reduced state + enzyme oxidized state;

[0005] (3) Electron mediator reduced state (working electrode) → electron mediator oxidized state +e-.

[0006] The enzyme biomolecules of the glucose detection sensor system catalyze the oxidation of glucose molecules in the blood. The added electron mediator reacts with the enzyme, converting the enzyme from the reduced state back to the oxidized state, thus playing a combing role.

[0007] Glucose enzymes can be either oxidases or dehydrogenases. Since oxidases can be oxidized by dissolved oxygen in blood samples, while dehydrogenases cannot, there is no "dissolved oxygen effect"; dehydrogenases have been the enzyme of choice in biosensors for nearly two decades. The catalytic enzymes of other analytes may be oxidases or dehydrogenases, and the principles of their biosensors 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 and thus generate interference signals. Generally speaking, the higher the standard potential of the electron mediator of the comb flow reaction, such as ferric cyanide (Fe(CN)6 -3 ), it is easier to generate interference signals, because higher electrode oxidation potential will oxidize more interference substances.

[0008] In addition to the above-mentioned chemical interferences, there are other test interferences in glucose detection sensors. The hematocrit percentage (%-HCT) is one of the more obvious interference factors. This interference is due to the diffusion of the electron mediator after the comb flow reaction to the electrode. Its diffusion is hindered by blood cells, so that the diffusion coefficient changes due to %-HCT, and the current in the electrochemical reaction will be affected by different %-HCT. Therefore, the factory batch calibration of the sensor test strip is usually centered on 42% HCT. If the user's %HCT is higher than 42%, the test current or reading will have a negative deviation; if the user's %HCT is lower than 42%, the test current or reading will have a positive deviation. In addition, the aging effect produced by the test strip during the storage time will also interfere with the blood glucose test results, that is, as the storage time increases, the sensitivity of the sensor test is relatively declining, and more oxidizable substances are generated for background signals, and the interference factors also include the test environment temperature.

[0009] Traditional blood glucose detection sensors generally apply voltage to the electrodes in contact with the analyte sample to oxidize the reduced electron mediators generated from the comb flow reaction, thereby generating a current signal in response to the analyte, and converting the current signal into the analyte concentration through a corresponding calculation method. For interference factors in the test process, most existing methods use different single parameters to eliminate or reduce interference compensation to achieve the purpose of improving test accuracy and precision. However, these single parameter interference compensation methods often ignore the mutual influence between multiple interference factors, such as 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, the mutual influence between temperature, current and HCT, etc.

[0010] Therefore, in order to avoid the generation of interference signals, there is still much room for improvement in the testing methods of blood glucose concentration and other analyte concentrations in biological samples in terms of multi-pulse excitation, attenuation signals and multi-parameter compensation, so as to further improve the detection accuracy, precision and anti-aging properties of blood glucose concentration and other analyte concentrations in biological samples. Summary of the invention

[0011] In order to solve the above problems, a method for testing the concentration of analytes in biological samples based on multi-excitation modes is provided. This method inputs excitation signals to the first working electrode and the second working electrode, and obtains the concentration of the target analyte by fitting the input signal and the output signal, which can improve the accuracy, precision and anti-aging performance of analyte concentration detection.

[0012] According to one aspect of the present application, a method for testing the concentration of an analyte in a biological sample based on a multi-excitation mode is provided, which is applied to a biological sample analyte testing device, wherein the biological sample analyte testing device comprises 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, 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;

[0013] Inputting a first excitation signal and a second excitation signal to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open circuit state, collecting the first working electrode attenuation signal after the first excitation signal is completed, and collecting the counter electrode attenuation signal after the second excitation signal is completed;

[0014] Inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal, wherein the second working electrode is in an open circuit state after the excitation signal sequence is completed; collecting an attenuation signal of the second working electrode, wherein the excitation signal sequence includes at least two continuous or discontinuous input signals, and each of the input signals is a constant value;

[0015] The output currents of the current collection points of the first excitation signal, the second excitation signal and the excitation signal sequence are collected at regular intervals to obtain an output current group; the output voltages of the voltage collection points of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal are collected at regular intervals to obtain an output voltage group, and the analyte concentration G1 is calculated according to the following formula:

[0016]

[0017]

[0018]

[0019] Among them, i 平均 is the average value of the output current of at least two current collection points in the second excitation signal, nA; S is i 平均 The slope of the linear equation with respect to analyte concentration; i n and i j are the output currents in the output current group, nA; i 差 is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, nA; v o is the output voltage in the output voltage group, V; V is the excitation voltage of the first excitation signal or the second excitation signal, V; the value range of N is 10 to 53; the value range of J is 10 to 53; X n The value range of X is -500 to 500;j The value range of is -15 to 15; the value range of K is -30 to 0, the value range of O is 1 to 21, and the value range of X is o The value range is -15 to 15.

[0020] The method is implemented by inputting a first excitation signal and a second excitation signal to a first working electrode, and inputting an excitation signal sequence to a second working electrode between the first excitation signal and the second excitation signal, wherein the input signals of the first working electrode and the second working electrode do not overlap in time, that is, after the excitation signal to the first working electrode is completed, the control of the first working electrode by a testing device is in an open circuit state, the potential decay signal of the first working electrode is tracked and collected, and then the input excitation signal sequence to the second working electrode is started; after the excitation signal sequence to the second working electrode is completed, the control of the second working electrode by the testing device is in an open circuit state, the potential decay signal of the second working electrode is tracked and collected, and then the second excitation signal to the first working electrode is started again, and the potential decay signal of the electrode is tracked and collected.

[0021] The excitation signal input to the first working electrode and the second working electrode in this application is defined as a "sandwich excitation method". The output signal of the first working electrode and the second working electrode can be obtained by using this excitation method. Combining the output signals and using the above formula for calculation can eliminate the multi-parameter interference in the test, thereby improving the accuracy and precision of the method and the anti-aging property of the test strip.

[0022] Optionally, the method further comprises the step of applying an alternating voltage to the second working electrode before inputting the first excitation signal to the working electrode, so as to measure the impedance value of the biological sample, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the impedance value, wherein,

[0023]

[0024] Where R is the impedance value, Ω; i m is the output current in the output current group, nA; M ranges from 10 to 53; X m The value range is -120 to 120.

[0025] The above calculation formula takes into account the influence between the impedance value and the output current, so as to further improve the accuracy of the multi-parameter calibration factor f, thereby improving the accuracy and precision of the analyte concentration detection result.

[0026] Optionally, the method further comprises the step of obtaining the measured ambient temperature, and obtaining the analyte concentration G1 according to the output current group, the output voltage group, the impedance value and the temperature, wherein:

[0027]

[0028] Where T is the ambient temperature, °C; i u is the output current in the output current group, nA; U ranges from 10 to 53; X u The value range is -7 to 3.

[0029] Through the above test and calculation formula, the mutual influence between the ambient temperature and the output current can be further eliminated or weakened, and the accuracy and precision of the method can be further improved.

[0030] Optionally, it further includes calculating HCT using parameters, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the HCT value, wherein,

[0031]

[0032] Where HCT is the hematocrit value, Ω; i f 、i p is the output current of the output current group, nA; the value range of F is 1-5, the value range of P is 1-5; X f , X p The value range is -60 to 60.

[0033] In the above method, by calculating HCT first through parameters, HCT can be optimized according to HCT-related parameters, preventing misidentification of HCT caused by changes in a single variable impedance, thereby further improving the accuracy and precision of detection.

[0034] In the above calculation method, i n 、i j 、i m 、i u 、i f 、i p All are output currents in the output current group. In the calculation, the above four currents can be the same, different, or partially the same. They are preferably different. The reason is that selecting different current values ​​increases the number of parameter items in the calculation formula, which can reduce the large fluctuation interference on the overall result caused by the fluctuation of individual parameter items, and improve the accuracy and repeatability of the results.

[0035] 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;

[0036] 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.

[0037] Optionally, i 平均is the average value of the output current of the last five current collection points in the second excitation signal.

[0038] 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;

[0039] Preferably, the voltage values ​​of the first excitation signal and the second excitation signal are both 0.3V.

[0040] 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.

[0041] Optionally, the voltage range of the excitation signal sequence is 0.1-3V, and the excitation signal sequence includes 4-8 consecutive input signals;

[0042] 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; the application time of each input signal in the excitation signal sequence is equal, which is 0.2s.

[0043] 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.

[0044] Optionally, the output attenuation voltage of the voltage collection point of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal sequence is collected every 0.05 s.

[0045] Optionally, the voltage range of the attenuation signal of the first working electrode is 0.3 to 0 V, the voltage range of the attenuation signal of the counter electrode is 0 to 0.3 V, and the voltage range of the attenuation signal of the second working electrode is 2.2 to 0 V;

[0046] The first working electrode attenuation signal, the counter electrode attenuation signal, and the second working electrode attenuation signal are 4-7 continuous output signals respectively;

[0047] Preferably, the first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are all 5 continuous output signals.

[0048] According to another aspect of the present application, a sensor for testing the concentration of an analyte in a biological sample based on multiple excitation modes is provided, wherein the sensor can execute any of the methods for testing the concentration of an analyte in a biological sample based on multiple excitation modes as described above.

[0049] The beneficial effects of this application include but are not limited to:

[0050] 1. The method of testing the concentration of analytes in biological samples based on multi-excitation methods in the present application can solve the technical problem that the method of using a single parameter for interference compensation in the prior art often ignores the mutual influence relationship between multiple interference factors, resulting in low accuracy and precision of blood glucose concentration testing.

[0051] 2. In the method of testing the concentration of analytes in biological samples based on multiple excitation modes of the present application, the output signal series values ​​generated by the sandwich input excitation signal series will be used to measure other parameters in the biological sample, including the hematocrit value (%-HCT) of the whole blood sample, and appropriate compensation and correction will be given to the analyte measurement through parameters related to the hematocrit value, thereby eliminating or reducing the influence of blood cells on the analyte measurement, thereby improving the accuracy and precision of the analyte measurement.

[0052] 3. The method of testing the analyte concentration in a biological sample based on a multi-excitation mode of the present application introduces a new attenuation electrical signal, which eliminates or weakens the mutual influence of various interference factors related to the blood glucose concentration or other analyte concentrations, thereby improving the accuracy and precision of testing the blood glucose concentration or other analyte concentrations in the biological sample, and improving the anti-aging property of the test strip, thereby extending the use time of the test strip.

[0053] 4. The method of testing the concentration of analytes in biological samples based on multiple excitation modes of the present application eliminates or weakens the mutual influence between various interference factors related to blood glucose concentration, thus making it possible to realize the barcode-free test strips when leaving the factory. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0055] Figure 1 An exemplary embodiment of a schematic diagram of the main excitation signal of the sandwich excitation method of the present application is shown;

[0056] Figure 2 This application shows 平均 An exemplary embodiment of a linear relationship plot versus glucose concentration.

[0057] Figure 3 A comparison diagram of the deviations before and after calibration of Example 1 is shown;

[0058] Figure 4 A comparison diagram of the deviations before and after calibration of Example 2 is shown;

[0059] Figure 5 A comparison diagram of the deviations before and after calibration of Example 3 is shown;

[0060] Figure 6 A comparison diagram of the deviations before and after calibration of Example 4 is shown;

[0061] Figure 7 A comparison diagram of the deviation after calibration of Examples 1-4 is shown;

[0062] Figure 8 A comparison diagram of the deviations before and after calibration of Example 5 is shown. DETAILED DESCRIPTION

[0063] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0064] Unless otherwise specified, the raw materials in the examples and comparative examples of the present application were purchased through commercial channels.

[0065] Unless otherwise specified, the methods used in the examples and comparative examples of the present application are conventional methods in the prior art.

[0066] The excitation described in the disclosure of the present invention is to apply an input signal to the electrode in the biological testing device (such as a sensor), wherein the applied signal includes a positive or negative DC constant voltage or a positive or negative DC constant current source, etc., so that a complete or partial oxidation or reduction reaction occurs on the electrode surface. The system processing center of the biological testing device applies an excitation input signal to the corresponding electrode through the connection port of the instrument and the test strip. After the predetermined excitation time is over, the system will no longer apply a signal to the electrode, and the electrode is in an open circuit state. The open circuit state is not zero input. For example, inputting 0V (zero volt) voltage 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 state substances on the electrode surface and nearby will cause the attenuation process of the residual signal. The attenuation signal will give a state that reflects the electrode has occurred, thereby recording other information related to the biological testing device and the analyte concentration.

[0067] Example 1

[0068] This embodiment relates to a method for testing blood glucose concentration in a biological sample based on a multi-excitation mode, which is applied to a biological sample analyte testing device, wherein the biological sample analyte testing device comprises 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 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;

[0069] Inputting a first excitation signal and a second excitation signal to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open circuit state, collecting the first working electrode attenuation signal after the first excitation signal is completed, and collecting the counter electrode attenuation signal after the second excitation signal is completed;

[0070] Inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal, wherein the second working electrode is in an open circuit state after the excitation signal sequence is completed; collecting an attenuation signal of the second working electrode, wherein the excitation signal sequence includes at least two continuous or discontinuous input signals, and each of the input signals is a constant value;

[0071] The output currents of the current collection points of the first excitation signal, the second excitation signal and the excitation signal sequence are collected at regular intervals to obtain an output current group; the output voltages of the voltage collection points of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal are collected at regular intervals to obtain an output voltage group, and the blood glucose concentration G1 is calculated according to Formula 1:

[0072] G in Formula 1 计算 It is obtained by formula 2, and f in formula 1 is obtained by formula 3, where f is the multi-parameter calibration factor;

[0073] Where S is i 平均 The slope of the linear equation with the analyte concentration, S, is obtained by linear fitting of the known historical i average and historical glucose concentrations; or, the historical i average and historical glucose concentrations of the multiple batches of products are directly tabulated after obtaining their linear relationship through statistical laws, and the slope S value is obtained by looking up the table;

[0074]

[0075] Among them, i 平均 is the average value of the output current of at least two current collection points in the second excitation signal, nA; i n and i j are the output currents in the output current group, nA; i 差 is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, nA; v o is the output voltage in the output voltage group, V; V is the excitation voltage of the first excitation signal or the second excitation signal, V; the value range of N is 10 to 53; the value range of J is 10 to 53; X n The value range of X is -500 to 500; j The value range of is -15 to 15; the value range of K is -30 to 0, the value range of O is 1 to 21, and the value range of X iso The value range is -15 to 15.

[0076] Specifically, the first chemical agent includes a biological enzyme that can catalyze the reaction of the analyte, one or more electron mediators that can generate a combing effect between the enzyme reaction and the electrode reaction, and an inert substance that assists the chemical agent.

[0077] Optionally, i n and i j It can be the output current of the continuous sampling points in the output current group. For example, i n It can be i1, i2, i3, i4, i5, i6, i7, i8, i9, i 10 ;i j It can be i6, i7, i8, i9, i 10 、i 11 、i 12 、i 13 、i 14 、i 15 .

[0078] Optionally, i n and i j It can be the output current of non-continuous sampling points in the output current group. For example, i n Can be i3, i5, i 11 、i 13 、i 21 ;i j Can be i1, i2, i9, i 17 、i 20 、i 29 、i 30 .

[0079] Optionally, v o It can be the output voltage of the continuous sampling points in the output voltage group. For example, v o Can be v1, v2, v3, v4, v5, v6, v7, v8, v9, v 10…… v 21 .

[0080] Optionally, v o The output current of the non-continuous sampling points in the output voltage group can be obtained. For example, v o Can be v1, v3, v6, v 11…… v 21 .

[0081] However, the present invention is not limited thereto, and those skilled in the art can select i according to a statistical significance test method. n 、i j and v o value.

[0082] 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.

[0083] Optionally, i 平均 is the average value of the output current of the last five current collection points in the second excitation signal.

[0084] 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 values ​​of the first excitation signal and the second excitation signal are both 0.3V.

[0085] 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, that is, 0.2 s.

[0086] Optionally, the voltage range of the excitation signal 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.

[0087] 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.

[0088] Optionally, the output attenuation voltage of the voltage collection point of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal sequence is collected every 0.01 s.

[0089] Optionally, the voltage range of the attenuation signal of the first working electrode is 0.3 to 0 V, the voltage range of the attenuation signal of the counter electrode is 0 to 0.3 V, and the voltage range of the attenuation signal of the second working electrode is 2.2 to 0 V;

[0090] The first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 4-7 continuous output signals. Preferably, the first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 5 continuous output signals.

[0091] Example 2

[0092] Based on Example 1, this embodiment further includes the 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 the biological sample, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the impedance value, wherein:

[0093]

[0094] Where R is the impedance value, Ω; i m is the output current in the output current group, nA; M ranges from 10 to 53; X m The value range is -120 to 120.

[0095] Example 3

[0096] Based on Example 2, this embodiment further includes the step of obtaining the measured ambient temperature, and obtaining the analyte concentration G1 according to the output current group, the output voltage group, the impedance value and the temperature, wherein:

[0097]

[0098] Where T is the ambient temperature, °C; i u is the output current in the output current group, nA; U ranges from 10 to 53; X u The value range is -7 to 3.

[0099] Example 4

[0100] Based on Example 2, this embodiment further includes using relevant parameters such as impedance to preferentially synthesize HCT, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the HCT value, wherein:

[0101]

[0102]

[0103] Where HCT is the hematocrit value, Ω; i f 、i p is the output current of the output current group, nA; the value range of F is 1-5, the value range of P is 1-5; X f , X p The value range is -60 to 60.

[0104] Comparative Example 1

[0105] Compared with Example 1, Comparative Example 1 does not collect the voltages of the first working electrode attenuation signal, the second working electrode attenuation signal, and the counter electrode attenuation signal, and obtains the analyte concentration G1 according to the output current group, wherein:

[0106]

[0107] Comparative Example 2

[0108] The test temperature T, the blood glucose test current value and the impedance value R are obtained by comparison, and the temperature compensation impedance value and the temperature compensation current value of the blood glucose are obtained by processing. The temperature compensation impedance value is fitted according to the HCT fitting equation to obtain the HCT value, and then the HCT value is converted into i according to the HCT compensation equation. 初始 , and then combine the current temperature compensation equation (to obtain i) and the current-reading equation to obtain the final blood glucose reading.

[0109] Experimental example

[0110] A sensor capable of executing the above-mentioned embodiment and comparative blood glucose concentration calculation method 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 arts and can be implemented by those skilled in the art, and will not be described in detail in this embodiment.

[0111] The test time and potential control in this test method are:

[0112] (1) A pre-test idle period of 1.25 seconds, during which an AC voltage can be applied to the second working electrode to measure the impedance value of blood glucose;

[0113] (2) applying a first excitation signal to the first working electrode, inputting a 0.3 V potential difference between the first working electrode and the corresponding electrode for 0.5 seconds, and synchronously measuring the output current at intervals of every 0.05 seconds;

[0114] (3) after the first excitation signal ends, the first working electrode is set to an open circuit state, and a first potential decay signal of the first working electrode is measured at intervals of 0.05 seconds for 0.25 seconds;

[0115] (4) while the first working electrode is continuously in an open circuit state, an excitation signal sequence is input to the second working electrode and the corresponding electrode, the excitation signal sequence includes five excitations, the excitation voltages are 0.3 V, 0.8 V, 1.2 V, 1.7 V, and 2.2 V, respectively, each excitation lasts for 0.2 seconds, and the output current is synchronously measured at intervals of 0.05 seconds;

[0116] (5) setting the second working electrode to an open circuit state, and measuring the second potential decay signal of the second working electrode at intervals of 0.05 seconds for 0.25 seconds;

[0117] (6) after the first working electrode is open circuited for 2.0 seconds, a second excitation signal is applied to the first working electrode at a voltage of 0.3 V for 1.25 seconds, and the output current is synchronously measured at intervals of 0.05 seconds;

[0118] (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 0.05 seconds for 0.5 seconds.

[0119] The total time of the above test steps is 1.25+0.5+1.5+1.25+0.5=5 seconds.

[0120] refer to Figure 1 As shown, Figure 1 The main excitation signal diagram of the sandwich excitation method is shown. Figure 1 The horizontal axis represents the sensor test time, and the vertical axis represents the excitation signal intensity, which is the voltage applied to the electrode.

[0121] refer to Figure 2 As shown, Figure 2 Shows the history 平均 The graph is linearly related to the historical glucose concentration, and the slope S is 22.181. 平均 is the average value of the output current of the last five current collection points in the second excitation signal.

[0122] In Example 1, f = -227.6-2.779i2 / i 平均 +16.52i4 / i 平均 -18.64i5 / i 平均 +115.8i8 / i 平均 +114.5i 10 / i 平均 +0.000043i 差 *i1 / i 平均 -0.000238i 差 *i3 / i 平均 +0.000467i 差 *i 10 / i 平均 +9.65v2 / V-8.765v3 / V.

[0123] In Example 2, f = 54.6-0.399i1 / i 平均 -53.7i9 / i 平均 -0.000024i 差 *i2 / i 平均 -0.000279i 差 *i7 / i 平均 +0.000431i 差*i9 / i 平均 +4.005v2 / V - 3.406v3 / V + 0.000037R*i1 / i 平均 -0.000190R*i6 / i 平均 。

[0124] In Example 3, f = 44.1 + 0.6512i1 / i 平均 -44.8i9 / i 平均 +0.832v2 / v - 0.000063R*i1 / i 平均 -0.000000T*G meter*i 差 *i6 / i 平均 +0.000000T*G 计 *i 差 *i8 / i 平均 。

[0125] In Example 4, HCT = 0.3208 + 3.685(i5 - i3) - 0.01616(i4 - i3*T + 0.005405(i5 - i3)*T - 0.00636(i4 - i3)*i1 / i 平均 -3.136(i4 - i3)*i8 / i 平均 -2.040(i5 - i4)*i9 / i 平均 -1.974(i5 - i4)*i 10 / i 平均 。

[0126] f = 2.845 - 0.7258i1 / i 平均 -1.130i2 / i 平均 +0.000038i 差 *i1 / i 平

[0127] 均 +6.507v2 / V - 5.922v3 / V + 1.064HCT*i1 / i 平均 -3.442HCT*i7 / i 平均 。

[0128] In Comparative Example 1, f = -80.10 + 8.91i4 / i 平均 +70.54i 10 / i 平均 -0.000082i 差 *i2 / i 平均 -0.000361i 差 *i4 / i 平均 -0.002330i 差 *i7 / i 平均+0.00699i 差 *i8 / i 平均 -0.004043i 差 *i9 / i 平均 .

[0129] The HCT fitting equation in Comparative Example 2: HCT = -2.5*R^2+30.04*R-78.6;

[0130] HCT compensation equation: 初始 =8.85*HCT^2-10.23*HCT+3.56;

[0131] Current temperature compensation equation: i = -0.0325*i 初始 ^2+4.89*i 初始 -120.75;

[0132] Current-reading equation: reading = -0.564*i^2+5.889*i-4.716.

[0133] In order to verify the accuracy of the detection method of this embodiment, the following verification is performed:

[0134] 1. Determine the reference value of blood glucose concentration: Determine the reference value of blood glucose concentration using the YSI2300 blood glucose analyzer.

[0135] 2. Calculate the deviation: The deviation is a relative deviation, deviation = [(blood glucose concentration test value - blood glucose concentration reference value) / blood glucose concentration reference value] × 100%.

[0136] 3. The proportion of the three relative deviations within ±10%, ±15% and ±20% is calculated respectively. The proportion calculation formula is: the number of deviations within ±10% (or ±15% or ±20%) / the total number of tests.

[0137] The test results are shown in Table 1, which shows the percentage of deviations between Examples 1-4 and Comparative Examples 1-2 and the blood glucose concentration reference value in each range.

[0138] Table 1

[0139]

[0140]

[0141] As shown in Table 1, 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 67.6% within ±10%, 84.8% within ±15%, and 95.2% within ±20%, which proves that the present invention can effectively improve the accuracy and precision of blood glucose concentration in testing biological samples.

[0142] refer to Figure 3-6 As shown, the deviation comparison diagrams of embodiments 1-4 before and after calibration are respectively shown, wherein the deviation before calibration in the figure indicates G 计算 A deviation comparison diagram with a blood glucose concentration reference value, wherein the figure shows a deviation comparison diagram between the blood glucose concentration detection value G of the present invention after calibration and the blood glucose concentration reference value.

[0143] Figure 7 A comparison diagram of the deviations after calibration of Examples 1-4 is shown. It can be seen that the method for calculating blood glucose concentration 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 testing blood glucose concentration in biological samples.

[0144] Example 5

[0145] The present embodiment relates to a method for testing the concentration of uric acid in a biological sample, which is applied to a biological sample analyte testing device, wherein the biological sample analyte testing device comprises 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, 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;

[0146] Inputting a first excitation signal and a second excitation signal to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open circuit state, collecting the first working electrode attenuation signal after the first excitation signal is completed, and collecting the counter electrode attenuation signal after the second excitation signal is completed;

[0147] Inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal, wherein the second working electrode is in an open circuit state after the excitation signal sequence is completed; collecting an attenuation signal of the second working electrode, wherein the excitation signal sequence includes at least two continuous or discontinuous input signals, and each of the input signals is a constant value;

[0148] The output currents of the current collection points of the first excitation signal, the second excitation signal and the excitation signal sequence are collected at regular intervals to obtain an output current group; the output voltages of the voltage collection points of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal are collected at regular intervals to obtain an output voltage group, and the blood glucose concentration G1 is calculated according to Formula 1:

[0149] G in Formula 1 计算 It is obtained by formula 2, and f in formula 1 is obtained by formula 3, where f is the multi-parameter calibration factor.

[0150] Among them, i平均 is the average value of the output current of at least two current collection points in the excitation signal, nA; S is i 平均 The slope of the linear equation with respect to the analyte concentration. Where S is determined by the known history i 平均 and historical analyte concentrations; or, historical i of multiple batches of products 平均 After obtaining the linear relationship between the historical analyte concentrations through statistical laws, they are directly tabulated and the slope S value is obtained by looking up the table.

[0151]

[0152] Among them, i 平均 is the average value of the output current of at least two current collection points in the second excitation signal, nA; i n and i j are the output currents in the output current group, nA; i 差 is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, nA; v o is the output voltage in the output voltage group, V; V is the excitation voltage of the first excitation signal or the second excitation signal, V; the value range of N is 10 to 53; the value range of J is 10 to 53; X n The value range of X is -500 to 500; j The value range of is -15 to 15; the value range of K is -30 to 0, the value range of O is 1 to 21, and the value range of X is o The value range is -15 to 15.

[0153] Specifically, the first chemical agent includes a biological enzyme that can catalyze the reaction of the analyte, one or more electron mediators that can generate a combing effect between the enzyme reaction and the electrode reaction, and an inert substance that assists the chemical agent.

[0154] Optionally, i n and i j It can be the output current of the continuous sampling points in the output current group. For example, i n It can be i1, i2, i3, i4, i5, i6, i7, i8, i9, i 10 ;i j It can be i6, i7, i8, i9, i 10 、i 11 、i 12 、i 13 、i 14 、i 15 .

[0155] Optionally, i n and i jIt can be the output current of non-continuous sampling points in the output current group. For example, i n Can be i3, i5, i 11 、i 13 、i 21 ;i j Can be i1, i2, i9, i 17 、i 20 、i 29 、i 30 .

[0156] Optionally, v o It can be the output voltage of the continuous sampling points in the output voltage group. For example, v o Can be v1, v2, v3, v4, v5, v6, v7, v8, v9, v 10…… v 21 .

[0157] Optionally, v o The output current of the non-continuous sampling points in the output voltage group can be obtained. For example, v o Can be v1, v3, v6, v 11…… v 21 .

[0158] However, the present invention is not limited thereto, and those skilled in the art can select i according to the significance test method in mathematical statistics. n 、i j and v o value.

[0159] 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.

[0160] Optionally, i 平均 is the average value of the output current of the last five current collection points in the second excitation signal.

[0161] 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 values ​​of the first excitation signal and the second excitation signal are both 0.3V.

[0162] 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, that is, 0.2 s.

[0163] Optionally, the voltage range of the excitation signal 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.

[0164] 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.

[0165] Optionally, the output attenuation voltage of the voltage collection point of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal sequence is collected every 0.01 s.

[0166] Optionally, the voltage range of the attenuation signal of the first working electrode is 0.3 to 0 V, the voltage range of the attenuation signal of the counter electrode is 0 to 0.3 V, and the voltage range of the attenuation signal of the second working electrode is 2.2 to 0 V;

[0167] The first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 4-7 continuous output signals. Preferably, the first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 5 continuous output signals.

[0168] In Example 5, f = 13.63 -0.557i1 / i 平均 +0.767i2 / i 平均 -1.558i3 / i 平均 -5.91i5 / i 平均 +11.99i7 / i 平均 -19.20i 10 / i 平均 -0.000247i 差 *i2 / i 平均 +0.000386i 差 *i3 / i 平均 +0.1210v2 / V+0.381v4 / V.

[0169] The test results are shown in Table 2, which shows the percentage of deviations of Example 5 from the reference value of uric acid concentration in each range. Figure 8 As shown, the deviation comparison diagrams before and after calibration of Example 5 are respectively shown, wherein the deviation before calibration in the figure indicates G 计算 A deviation comparison diagram of the uric acid concentration reference value is shown in the figure, which shows the deviation comparison diagram of the uric acid concentration detection value G of the present invention after calibration and the uric acid concentration reference value.

[0170] Table 2

[0171]

[0172] The above is only the embodiment of the present application, and 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, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for testing the concentration of an analyte in a biological sample based on a multi-stimulation method, characterized in that: Applicable to a biological sample analyte testing device, the biological sample analyte testing device comprises 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, 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 a first excitation signal and a second excitation signal to the first working electrode, and after each excitation signal is completed, the first working electrode is in an open circuit state, collecting the first working electrode attenuation signal after the first excitation signal is completed, and collecting the counter electrode attenuation signal after the second excitation signal is completed; Inputting an excitation signal sequence to the second working electrode between the first excitation signal and the second excitation signal, wherein the second working electrode is in an open circuit state after the excitation signal sequence is completed; collecting an attenuation signal of the second working electrode, wherein the excitation signal sequence includes at least two continuous or discontinuous input signals, and each of the input signals is a constant value; Collecting the output current of the first excitation signal, the second excitation signal and the current collection point of the excitation signal sequence at regular intervals to obtain an output current group; The output voltages of the voltage collection points of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal are collected at regular intervals to obtain an output voltage group, and the analyte concentration G1 is calculated according to the following formula: Among them, i 平均 is the average value of the output current of at least two current collection points in the second excitation signal, nA; S is i 平均 The slope of the linear equation with respect to analyte concentration; i n and i j are the output currents in the output current group, nA; i 差 is the difference between the maximum output current of the excitation signal sequence and the output current of the last acquisition point, nA; v o is the output voltage in the output voltage group, V; V is the excitation voltage of the first excitation signal or the second excitation signal, V; the value range of N is 10 to 53; the value range of J is 10 to 53; X n The value range of X is -500 to 500; j The value range of is -15 to 15; the value range of K is -30 to 0, the value range of O is 1 to 21, and the value range of X is o The value range is -15 to 15.

2. The method according to claim 1, characterized in that The method further includes applying an alternating voltage to the second working electrode before inputting the first excitation signal to the working electrode to measure the impedance value of the biological sample, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the impedance value, wherein: Where R is the impedance value, Ω; i m is the output current in the output current group, nA; M ranges from 10 to 53; X m The value range is -120 to 120.

3. The method according to claim 2, characterized in that The method further includes the step of obtaining the measured ambient temperature, and obtaining the analyte concentration G1 according to the output current group, the output voltage group, the impedance value and the temperature, wherein: Where T is the ambient temperature, °C; i u is the output current in the output current group, nA; U ranges from 10 to 53; X u The value range is -7 to 3; or It also includes first calculating HCT using parameters, and obtaining the analyte concentration G1 according to the output current group, the output voltage group and the HCT value, wherein, Where HCT is the hematocrit value, Ω; i f 、i p is the output current of the output current group, nA; the value range of F is 1-5, the value range of P is 1-5; X f , X p The value range is -60 to 60.

4. The method according to any one of claims 1 to 3, characterized in that: 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 0.5 s, and the excitation time of the second excitation signal is 1.25 s.

5. The method according to claim 4, characterized in that i 平均 is the average value of the output current of the last five current collection points in the second excitation signal.

6. The method according to any one of claims 1 to 3, characterized in that: 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 values ​​of the first excitation signal and the second excitation signal are both 0.3V.

7. The method according to any one of claims 1 to 3, characterized in that: 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; The voltage range of the excitation signal sequence is 0.1 to 3 V, and the excitation signal sequence includes 4 to 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; the application time of each input signal in the excitation signal sequence is equal, which is 0.2s.

8. The method according to any one of claims 1 to 3, characterized in that: Collecting the output current of the first excitation signal, the second excitation signal and the current collection point of the excitation signal sequence every 0.05 s; and / or The output attenuation voltage of the voltage collection point of the first working electrode attenuation signal, the second working electrode attenuation signal and the counter electrode attenuation signal sequence is collected every 0.05 s.

9. The method according to any one of claims 1 to 3, characterized in that The voltage range of the attenuation signal of the first working electrode is 0.3 to 0 V, the voltage range of the attenuation signal of the counter electrode is 0 to 0.3 V, and the voltage range of the attenuation signal of the second working electrode is 2.2 to 0 V; The first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 4-7 continuous output signals. Preferably, the first working electrode attenuation signal, the counter electrode attenuation signal and the second working electrode attenuation signal are respectively 5 continuous output signals.

10. A sensor for testing the concentration of an analyte in a biological sample based on a multi-stimulation method, characterized in that: The sensor can execute the method for testing the concentration of an analyte in a biological sample based on multiple excitation modes as described in any one of claims 1 to 9.

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