CN117030820A - Measuring method of biosensor - Google Patents
Measuring method of biosensor Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000012491 analyte Substances 0.000 claims abstract description 16
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical group CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims description 108
- 238000011534 incubation Methods 0.000 claims description 63
- 229940109239 creatinine Drugs 0.000 claims description 54
- 238000000691 measurement method Methods 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 108010077078 Creatinase Proteins 0.000 claims description 6
- 102000003992 Peroxidases Human genes 0.000 claims description 5
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- 102000008118 Sarcosine oxidase Human genes 0.000 claims description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 5
- 238000005259 measurement Methods 0.000 abstract description 41
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention provides a measuring method of a biosensor, which comprises the following steps: a sample is added to the biosensor, a positive voltage is applied to the working electrode and the counter electrode, and then a negative voltage is applied, and the content of the analyte in the sample is calculated from the obtained electrical data. The invention can obviously improve the resolution of the measured current signal, thereby improving the detection precision and accuracy of the measurement, and being applicable to electrochemical detection products for detecting chronic complications and the like.
Description
Technical Field
The invention belongs to the field of electrochemical detection, and particularly relates to a measuring method of a biosensor.
Background
The basic structure of the biosensor comprises an insulating bottom plate, an electrode system positioned on the insulating bottom plate, a reaction reagent arranged on the electrode system, a working electrode and a counter electrode which are respectively connected with an analytical instrument pin through electrode leads, a sample channel arranged on the electrode, a sample to be detected (such as physiological liquid) contacted with the reaction reagent through the sample channel, and an analyte to be detected in the sample reacts with the reaction reagent to generate an electrical signal. The analysis instrument gives out a detection result according to the generated electrical signal.
Electrochemical methods are common methods for measuring the concentration of an analyte to be measured in a physiological fluid (e.g., blood or plasma by-products) using a biosensor. The analyte constituent to be measured is allowed to react with the specific reagent and produce an oxidizable (or reducible) substance proportional to the analyte concentration to be measured, which is ultimately converted into an electrical signal, such as a current signal or an impedance signal, that is readily measured by the instrument, and the analyte concentration to be measured is in a relationship to the signal magnitude. For example, in the electrochemical measurement of creatinine, creatinine is converted into a current signal value related to the concentration of creatinine by the action of a reactive enzyme such as creatinase, sarcosine oxidase, peroxidase, etc.
Improving the resolution of analyte measurement to be measured is an important consideration in biosensor design and analytical instrument measurement system design. For example, the accuracy of detection is affected by the current signal resolution, and thus how to improve the biosensor current signal resolution is an important research content in the field of biosensors.
One way to increase the resolution of the electrical signal of the biosensor is to adjust the formulation of the reagents in the biosensor. When the reagent formulation of the biosensor is determined, if it is desired to further change the magnitude of the current to increase the resolution of the electrical signal, it is generally adopted to change the electrode structure of the sensor or increase the sample channel to increase the reaction area of the sample and the reagent. All that is mentioned above is to redesign the biosensor, which necessarily increases the design cost and prolongs the product development period.
Disclosure of Invention
In order to solve the problems, the invention provides a novel measuring method applied to an electrochemical biosensor, and the measuring method can improve the resolution of a current signal measured by an electrochemical method, thereby improving the detection precision of the electrochemical biosensor.
Specifically, the invention provides a measurement method, which adopts an electrochemical biosensor for detection, wherein the electrochemical biosensor comprises a working electrode and a counter electrode, and the measurement process comprises the following steps:
step 1: adding a sample to the biosensor;
step 2: signal incubation for a duration of T1 seconds;
step 3: after the time T1 seconds has arrived, applying a voltage between the working electrode and the counter electrode of the biosensor for a time T2 seconds;
step 4: and (3) calculating the content of the analyte in the sample according to the electrical data obtained in the step (3).
Further, in step 2, a voltage is applied between the working electrode and the counter electrode of the biosensor such that the voltage difference between the working electrode and the counter electrode is positive, zero or negative.
Further, in step 3, a voltage is applied between the working electrode and the counter electrode of the biosensor, so that a voltage difference is generated between the working electrode and the counter electrode, and the voltage difference generated in step 3 is opposite to the voltage difference generated in step 2.
The voltage difference generated in step 3 is opposite to the voltage difference generated in step 2, which means that when the voltage difference generated in step 2 between the working electrode and the counter electrode is a positive voltage, the voltage difference generated in step 3 between the working electrode and the counter electrode is a negative voltage.
Further, in step 4, the content of the analyte in the sample is calculated based on the measured value at T2.
Further, step 1 further includes judging whether the sample addition amount is sufficient, after adding a sample to the biosensor, applying voltages to the working electrode and the counter electrode of the biosensor to make the voltage difference between the working electrode and the counter electrode be positive, and after detecting the current change, judging that the sample addition amount is sufficient to meet the requirement, and entering step 2.
More specifically, the invention provides a method for measuring creatinine, which adopts an electrochemical biosensor for detection, wherein the electrochemical biosensor comprises a working electrode and a counter electrode, and the measuring process comprises the following steps:
step 1: adding a sample to the biosensor;
step 2: applying a voltage to the working electrode and the counter electrode of the biosensor such that the voltage difference between the working electrode and the counter electrode is a positive voltage, or applying no voltage; duration T1 seconds;
step 3: after the time T1 seconds is reached, applying a voltage between the working electrode and the counter electrode of the biosensor, so that the voltage difference between the working electrode and the counter electrode is a negative voltage for a duration T2 seconds;
step 4: and (3) calculating the content of the analyte in the sample according to the electrical data obtained in the step (3).
Further, in step 2 of the creatinine measurement method, voltages are applied to the working electrode and the counter electrode of the biosensor such that the working electrode voltage is 0.4V higher than the counter electrode voltage.
More specifically, in step 2 of the creatinine measurement method, a voltage of 0.4V is applied to the working electrode, and a voltage of 0V is applied to the counter electrode.
Further, in step 3 of the creatinine measurement method, voltages are applied to the working electrode and the counter electrode of the biosensor such that the working electrode voltage is 0.4V lower than the counter electrode voltage.
More specifically, in step 3 of the creatinine measurement method, a voltage of 0.4V is applied to the working electrode, and a voltage of 0.8V is applied to the electrode.
Further, in step 4 of the creatinine measurement method, the content of the analyte in the sample is calculated based on the measured value at T2.
Further, in the measuring method of creatinine, a step of judging whether sample is injected is further included, specifically, in step 1 of the measuring method of creatinine, voltages are applied to the working electrode and the counter electrode of the biosensor, so that a voltage difference between the working electrode and the counter electrode is a positive voltage, specifically, when a current change is detected, it is indicated that the sample has entered the reaction area of the biosensor. Subsequently, step 2 is entered, and the analyzer starts the T1 timer.
Further, in the sample injection judgment step of the creatinine measurement method, the voltage is applied to the working electrode at 0.4V, and the voltage is applied to the counter electrode at 0V.
Further, in the creatinine measurement method, the T1 time is greater than or equal to 30S and the T2 time is greater than or equal to 3S.
Further, the biosensor for creatinine detection includes reagents for detection including creatininase, creatinase, sarcosine oxidase, peroxidase, and a reduced electron mediator.
The invention adds the step of signal incubation between the conventional sample adding step and the signal measuring and calculating step, so that the invention enters the conventional signal measuring and calculating step after waiting for a certain time (T1 s) after the sample adding step.
The signal incubation of the invention can adopt a mode of applying no voltage between the working electrode and the counter electrode, or after the voltage is applied, the voltage difference between the working electrode and the counter electrode is 0; alternatively, a voltage may be applied to the working electrode and the counter electrode, and the voltage difference generated between the working electrode and the counter electrode may be reversed from the voltage difference in the signal measurement stage.
The beneficial effects of the invention are as follows:
the signal incubation step is added in the measuring method, and the current signal resolution of the electrochemical method measurement is obviously improved under the condition of not changing the formula of the biosensor test paper and increasing a sample contact chamber (sample channel) by waiting for a certain time and combining a mode of increasing voltage, so that the measuring precision is improved. The method can be applied to electrochemical measurement of H2O2 generated by intermediate reactions of creatinine, blood sugar, uric acid, cholesterol, triglyceride and the like, and can realize high-resolution electrical signals and higher detection accuracy. Meanwhile, the research and development cost of the biosensor can be effectively reduced.
Drawings
FIG. 1 is a schematic diagram of a test circuit according to the present invention.
Fig. 2 is a waveform diagram of voltages applied across the working electrode and the counter electrode.
Fig. 3 is a schematic view of electrodes of a biosensor for creatinine detection covered with an insulating layer, but not covered with a gap layer and a cover layer.
Fig. 4 is an exploded schematic view of a biosensor for creatinine detection.
Fig. 5 is a cross-sectional view of a biosensor for creatinine detection.
Fig. 6 is a measurement flow chart of the present invention.
Fig. 7 is a graph of creatinine concentration versus current signal magnitude at various incubation voltages and times.
Fig. 8 shows the relationship between the concentration of creatinine and the magnitude of the current signal when the incubation voltages are the same and the incubation times are different.
Fig. 9 is a waveform diagram of the current signal over time during the signal test phase.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1 biosensor and analytical instrument for detecting creatinine content
The biosensor 100 for detecting creatinine content in a blood sample shown in fig. 3, 4 and 5 comprises an insulating substrate 103, a working electrode 101 and a counter electrode 102 arranged on the insulating substrate 103, an electrode lead 104 connected with the working electrode or the counter electrode, and an analytical instrument connected with the other end of the electrode lead. The insulating layer 105 is used as a reaction region forming layer to cover the electrodes, and an opening is formed in the insulating layer at a position corresponding to the signal reaction region 110 of the electrodes, and a reaction reagent is added in the opening. A sample channel 108 is formed between the insulating layer 105 and the cover layer 106 through a gap layer 107, and an air hole 109 is provided in the cover layer. Wherein fig. 3 is a schematic view of electrodes of a biosensor covered with an insulating layer, but not with a gap layer and a cover layer, showing the arrangement of the electrodes under the insulating layer. Signal reaction zone 110 contains a reagent that includes creatinase, sarcosine oxidase, peroxidase, enzyme mediators, surfactants, buffers, and the like.
The reaction principle of the biosensor for detecting creatinine is as follows: inserting the biosensor 100 for detecting creatinine content of a blood sample into an analysis instrument, and generating creatine (formula (1)) by creatinine in the blood sample under the action of creatinase and creatinase in the biosensor when the blood sample enters the biosensor through the sample channel 108; creatine, H2O and O2 react under the action of sarcosine oxidase to form formaldehyde, glycine and H2O2 (formula (2)); the reduced electron mediator reacts with H2O2 under the action of peroxidase and generates an oxidative electron mediator and H2O (formula (3)); after the measuring circuit applies the potential, the oxidizing electron mediator absorbs electrons to react to generate a reduced electron mediator (formula (4)). In equation (4), the number of electrons is positively correlated with the creatinine concentration, and the creatinine measurement analyzer can convert the detected current signal into a specific value of creatinine concentration via a solidified software system.
As shown in fig. 1, the test circuit of the creatinine measurement analyzer includes a metal dome 1 connected to a working electrode 101 of a biosensor 100 and a metal dome 2 connected to a counter electrode 102 of the biosensor. The metal spring plate 1 and the metal spring plate 2 are respectively connected with a current-voltage conversion circuit 3, and the current-voltage conversion circuit 3 is composed of a transimpedance amplifier. The metal spring may also be referred to as a pin. The current-voltage conversion circuit 3 is connected with the analog-digital conversion circuit 5, the analog-digital converter 5 is connected with the microcontroller 7, the microcontroller 7 is formed by taking a singlechip as a core and is used for controlling the 0.8V or 0V voltage switching circuit 6, collecting test signals of the analog-digital conversion circuit 5 and calculating test results, and a data storage unit 8 is arranged in the microcontroller. The test circuit further includes a 0.4V voltage generating circuit 4 and a 0.8V or 0V voltage switching circuit 6. The 0.4V voltage generating circuit 4 is divided by a reference source and is generated by following an output through an operational amplifier. The 0.8V or the 0.8V voltage in the 0V voltage switching circuit 6 is generated by dividing the reference source and following the output through the operational amplifier, and the 0V is directly connected with the system ground, and the switching between the two is realized by analog switch switching.
The voltage value of the voltage generation circuit 4 and the voltage value of the voltage switching circuit 6 may be set according to actual conditions, and are not necessarily 0.4V or 0.8V or the like.
Example 2 measurement of creatinine content in blood sample
The measuring method for measuring the creatinine content in the blood sample comprises the following steps.
Step 1: and (3) sample injection detection: the biosensor is inserted into an analytical instrument and a sample is added to the biosensor. The analytical instrument detects whether a sample enters the reaction area of the biosensor to judge whether sample is injected. After the instrument detects the sample injection, the instrument enters a signal incubation period.
Step 2: signal incubation period: during the incubation period, voltages are applied to the working and counter electrodes of the biosensor such that the voltage difference between the working and counter electrodes is a positive voltage (i.e., the working electrode voltage is higher than the counter electrode voltage) for a duration of T1 seconds, and then a signal measurement period is entered.
Step 3: signal measurement period: during the measurement period, a voltage is applied between the working electrode and the counter electrode of the biosensor, so that the voltage difference between the working electrode and the counter electrode is a negative voltage (i.e., the working electrode voltage is lower than the counter electrode voltage), at this time, a current signal is generated between the working electrode and the counter electrode, the current signal is converted into a voltage signal by a current-voltage conversion circuit, and the signal is sampled and recorded by an analog-digital conversion circuit for a duration of T2 seconds.
Step 4: and (3) calculating results: the measured value at the moment of the measurement period T2 seconds is taken and converted into the creatinine concentration through a calibration curve.
The specific durations of the durations T1 and T2 may be determined according to the experimental results, for example, as desired time for the biosensor to be selected after repeated laboratory tests.
As shown in fig. 1 to 6, the creatinine content in the blood sample was measured using the biosensor 100 of example 1 and the test circuit of the analytical instrument.
In the sample injection detection stage, a blood sample is added in a sample injection channel 108 of the biosensor, a test circuit of the analysis instrument applies a voltage of 0.4V on a working electrode 101 and a voltage of 0V on a counter electrode 102, at this time, an analog-to-digital conversion circuit 5 always works, when a controller 7 detects that a current change exists through the analog-to-digital conversion circuit 5, the sample is indicated to enter a reaction area 110, at this time, the controller starts T1 timing, and the signal incubation stage is measured.
In the signal incubation phase, when the addition of sample is detected, an incubation procedure is initiated. In this embodiment, a positive voltage incubation method is adopted, that is, after a voltage is applied, the voltage difference between the working electrode and the counter electrode is a positive voltage. Specifically, the voltage applied to the working electrode 101 is 0.4V, the voltage applied to the counter electrode 102 is 0V, the working electrode voltage is 0.4V with respect to the counter electrode voltage, the incubation voltage waveform is shown in a period T1 of fig. 2, and T1 represents that the incubation voltage of 0.4V is applied between the working electrode and the counter electrode for a duration T1 seconds. The analog-digital conversion circuit 5 does not need to work in the process, and when the controller 7 counts the time T1 seconds, incubation is finished, and the signal measurement stage is started. In this embodiment, the current signal generated during the incubation period does not participate in the calculation of the result. In a further embodiment the analog to digital conversion circuit 5 operates, but the current signal generated during the incubation period does not participate in the calculation of the result.
In the signal measurement phase, after the signal incubation is completed, a signal measurement procedure is initiated. In this embodiment, a negative voltage is applied to measure, i.e., the voltage difference between the working electrode and the counter electrode is a negative voltage. Specifically, a voltage of 0.4V is applied to the working electrode, the voltage applied to the electrode is controlled by the controller, the voltage is switched from 0V to 0.8V, the working electrode voltage at this time is-0.4V relative to the counter electrode voltage, the waveform is shown in a T2 period of fig. 2, T2 is a measured voltage of-0.4V applied between the working electrode and the counter electrode, and the duration is T2 seconds. The controller 7 continuously collects the voltage signal generated by the creatinine current signal flowing through the current-voltage conversion circuit 3 through the analog-to-digital conversion circuit 5 and stores the voltage signal in the memory unit 8 of the controller 7. The measurement is stopped after the T2 second time has arrived. The result calculation phase is entered.
In the result calculation phase, the controller 7 analyzes the data acquired in T2 seconds, takes the measured value at the time of the measurement period T2 seconds, and converts the measured value into the creatinine concentration by a calibration curve.
It is relatively speaking that the voltage difference is a positive voltage or that the voltage difference is a negative voltage.
When a voltage of the counter electrode is used as a reference, a voltage is applied to the working electrode and the counter electrode of the biosensor, and the working electrode voltage is higher than the counter electrode voltage, the voltage difference between the working electrode and the counter electrode is positive, for example, 0.4V is applied to the working electrode, and when a voltage of 0V is applied to the counter electrode, the voltage difference between the working electrode and the counter electrode is 0.4V, that is, the working electrode voltage is 0.4V to the counter electrode. When a voltage is applied to the working electrode and the counter electrode of the biosensor, the voltage difference between the working electrode and the counter electrode is negative, for example, when a voltage of 0.4V is applied to the working electrode and a voltage of 0.8V is applied to the counter electrode, the voltage difference between the working electrode and the counter electrode is-0.4V, that is, the voltage of the working electrode to the counter electrode is-0.4V.
Conversely, when the voltage of the working electrode is used as a reference, the voltages are applied to the working electrode and the counter electrode of the biosensor, so that the working electrode voltage is higher than the counter electrode voltage, the voltage difference between the working electrode and the counter electrode is negative, for example, when the voltage is 0V, the voltage difference between the working electrode and the counter electrode is-0.4V, that is, the working electrode voltage is-0.4V relative to the counter electrode voltage. When a voltage is applied to the working electrode and the counter electrode of the biosensor, the voltage difference between the working electrode and the counter electrode is a positive voltage, for example, when a voltage of 0.4V is applied to the working electrode and a voltage of 0.8V is applied to the counter electrode, the voltage difference between the working electrode and the counter electrode is 0.4V, that is, the voltage of the working electrode to the counter electrode is 0.4V.
Example 3 relation of creatinine concentration to Current Signal size at different incubation voltages, time
In order to compare the relationship between the creatinine concentration and the current signal in the sample under different incubation voltages and incubation times, three experimental schemes were used to develop comparative experiments in this example.
Experiment group 1
In this experimental group, the measurement step included the step 1 sample injection detection, the step 3 signal measurement and the step 4 result calculation in example 2. The experimental group did not contain the signal incubation in example 2. Specifically, after sample injection detection is completed according to the method of example 2, the signal measurement stage is directly entered, voltages are applied to the working electrode and the counter electrode of the biosensor, even if the working electrode voltage is-0.4V relative to the counter electrode voltage, the duration is T2 seconds, the result calculation stage is entered, and the measured value at the moment of T2 seconds is taken to calculate the creatinine concentration.
Experiment group 2
In this experimental set, the measurement step included step 1 sample detection, step 2 signal incubation, step 3 signal measurement and step 4 result calculation in example 2, except that the signal incubation was incubation without pressure. Specifically, after sample injection detection is completed according to the method of example 2, a signal incubation stage is performed, and in this stage, incubation is performed without applying pressure, for example, the voltage difference between the working electrode and the counter electrode is 0V. After the controller times T1 for 30s (namely, the biosensor is incubated for 30s without pressure), signal measurement is carried out, constant-0.4V voltage is applied to the working electrode and the counter electrode of the biosensor for a duration of T2 seconds, the controller enters a result calculation stage, and the measured value at the moment of T2 seconds is taken to calculate the creatinine concentration.
Experiment group 3
In this experimental set, the measurement step included step 1 sample detection, step 2 signal incubation, step 3 signal measurement and step 4 result calculation in example 2. Specifically, after sample injection detection is completed according to the method of example 2, a signal incubation stage is entered, voltages are applied to a working electrode and a counter electrode of the biosensor, so that the voltage of the working electrode is 0.4V relative to the voltage of the counter electrode, after the controller times T1 for 30 seconds, signal measurement is entered, in the signal measurement stage, constant-0.4V voltage is applied to the working electrode and the counter electrode of the biosensor for a duration of T2 seconds, a result calculation stage is entered, and measured values at the moment of T2 seconds are taken to calculate creatinine concentration.
The T2 time of the above three experimental groups was 3s.
The signal measurement stage of this embodiment uses a mode of applying a voltage of 0.4V to the working electrode and applying a voltage of 0.8V to the electrode. Of course, in other embodiments, a mode of applying 0.5V to the working electrode and 0.9V to the counter electrode may be adopted, as long as the voltage difference between the working electrode and the counter electrode is-0.4V. Similarly, the voltage applied during the incubation period can be selected according to the actual condition of the product design, for example, the voltage applied to the working electrode during the incubation period is 0.5V, and the voltage applied to the electrode is 0.1V.
In this example, a plurality of groups of samples with known creatinine concentrations were prepared using fresh blood, the specific creatinine concentrations in the samples are shown in table 1, and the test results are shown in table 1 and fig. 7. Fig. 7 is a graph showing the relationship between creatinine concentration and current signal magnitude at different incubation voltages and times, wherein 0V0S represents no incubation, 0V30S represents incubation for 30 seconds but no pressure is applied during the incubation period, and 0.4V30S represents the application of the incubation voltage of 0.4V during the incubation period.
Test was performed using test panel 1 (labeled 0V0S in fig. 7), i.e., the signal measurement applied with-0.4V was directly entered after the test sample injection. The test results showed that the slope of measurement of experimental group 1 was only 0.0002 uA/uM, with substantially no signal gradient.
Signal testing was performed using experimental group 2 (labeled 0V30S in fig. 7), i.e., incubation for 30S without pressure after detection sample injection, followed by-0.4V. The test result shows that the measurement slope of the experimental group 2 is 0.0013 uA/uM, which is more than 6 times of the slope of the experimental group 1 directly measured without incubation, and the gradient of the detection signal is greatly increased.
Test group 3 (indicated by 0.4V30S in FIG. 7) was used, i.e., incubation was performed for 30S with 0.4V applied after detection sample injection, and then signal testing was performed with-0.4V applied. The test result shows that the measured slope of the experimental group 3 is 0.0019 uA/uM, the slope of the experimental group 2 is increased by more than 40%, and the gradient of the detection signal is further increased.
Through the analysis, the gradient of the detection signal can be increased by adding an incubation step in the creatinine measurement process, and particularly, the gradient of the detection signal can be greatly increased after adding a pressurizing incubation step. This is probably because the incubation process contributes to electron accumulation during the reaction, especially incubation under pressure (e.g. incubation for 30s at 0.4V), can greatly increase the detection reaction current gradient.
TABLE 1
Example 4 analysis of the relationship between creatinine concentration and reaction current using different incubation times
The method of this example is essentially the same as experimental group 3 of example 3, except that the incubation times T1 are 10s, 30s, 40s, respectively. The test results are shown in Table 2 and FIG. 8. Fig. 8 is a graph showing the relationship between creatinine concentration and current signal at various incubation times, wherein 0.4V10S represents incubation for 10s with an applied 0.4V incubation voltage, 0.4V30s represents incubation for 30s with an applied 0.4V incubation voltage, and 0.4V40s with an applied 0.4V incubation voltage. The current signal slopes corresponding to the incubation times of 10S, 30S and 40S are respectively 0.002 uA/uM, 0.0026 uA/uM and 0.0029 uA/uM, and the current signals are increased with the increase of the incubation time. T1 may be chosen to be 30 seconds in view of test efficiency and signal detection gradient.
TABLE 2
Example 5 analysis of the waveform of the current signal over time during the measurement phase.
In this embodiment, the method of experiment set 3 in example 3 is adopted, the incubation voltage is 0.4V, the incubation time T1 is 30s, the concentration of creatinine in the sample to be tested is 227uM, the system does not measure the current value of the creatinine signal in 30s in the incubation period, the incubation is completed, and the signal measurement period is entered, at this time, the system monitors the creatinine current signal, the time-varying waveform chart of the electrical signal is shown in fig. 9, that is, fig. 9 is the time-varying waveform chart of the current signal value in the signal measurement period, specifically, the current signal is in a transition state in which the current signal is severely changed and has larger randomness in the period of time, so that the test result is not suitable for selecting the time period. When the signal measurement period is 2s later, the current signal is in a steady state, the signal changes slowly with time, and the signal acquisition is stable, so the test result should be selected in the state. In this embodiment, T2 selects a current signal test value at 3 seconds as a test value of the creatinine signal.
Example 6 stability test
This example uses the method of experimental group 3 of example 3 to test samples of different creatinine content in table 3, each sample being tested 5 times, and the test results are shown in table 3.
Table 3: five measurements with an applied incubation voltage of 0.4V30S
As can be seen from the data in Table 3, the method of the present invention, which is to apply a positive voltage of 0.4V after sample injection, incubate for 30s, apply a signal test of-0.4V, and obtain a current value at the time of T2, wherein CV values are less than 5% at 4 concentrations. Therefore, the measuring method can meet the measuring requirement and can be used for actual measurement.
Example 7 accuracy test
The creatinine analyzer prepared by the method of the invention tests test samples with different concentrations, each concentration sample is tested 5 times, and the test results are shown in Table 4:
table 4:
as can be seen from the data in Table 4, the creatinine analyzer manufactured by the method of the invention has high measurement accuracy and good accuracy.
Claims (13)
1. A method of measuring a biosensor, wherein the biosensor is configured to detect by an electrochemical biosensor, the electrochemical biosensor comprising a working electrode and a counter electrode, the measuring process comprising:
step 1: adding a sample to the biosensor;
step 2: signal incubation for a duration of T1 seconds;
step 3: after the time T1 seconds has arrived, applying a voltage between the working electrode and the counter electrode of the biosensor for a time T2 seconds;
step 4: and (3) calculating the content of the analyte in the sample according to the electrical data obtained in the step (3).
2. The measurement method according to claim 1, wherein in step 2, a voltage is applied between the working electrode and the counter electrode of the biosensor such that a voltage difference between the working electrode and the counter electrode is a positive voltage, zero or a negative voltage.
3. The measurement method according to claim 2, wherein in step 3, a voltage is applied between the working electrode and the counter electrode of the biosensor, such that a voltage difference is generated between the working electrode and the counter electrode, and the voltage difference generated in step 3 is opposite to the voltage difference generated in step 2.
4. The method according to claim 1, wherein in step 4, the content of the analyte in the sample is calculated from the measured value at T2.
5. The measurement method according to claim 1, wherein in step 1, after a sample is added to the biosensor, a voltage is applied to the working electrode and the counter electrode of the biosensor so that a voltage difference between the working electrode and the counter electrode is a positive voltage, and when a change in current is detected, the process proceeds to step 2.
6. The method of claim 1, wherein the analyte is creatinine and the measuring comprises:
step 1: adding a sample to the biosensor;
step 2: applying a voltage to the working electrode and the counter electrode of the biosensor such that the voltage difference between the working electrode and the counter electrode is a positive voltage, or applying no voltage; duration T1 seconds;
step 3: after the time T1 seconds is reached, applying a voltage between the working electrode and the counter electrode of the biosensor, so that the voltage difference between the working electrode and the counter electrode is a negative voltage for a duration T2 seconds;
step 4: and (3) calculating the content of the analyte in the sample according to the electrical data obtained in the step (3).
7. The method according to claim 6, wherein in step 2, voltages are applied to the working electrode and the counter electrode of the biosensor such that the working electrode voltage is 0.4V higher than the counter electrode voltage.
8. The method according to claim 7, wherein the voltage applied to the working electrode is 0.4V and the voltage applied to the counter electrode is 0V.
9. The method according to claim 6, wherein in step 3, voltages are applied to the working electrode and the counter electrode of the biosensor such that the working electrode voltage is lower than the counter electrode voltage by 0.4V.
10. The measurement method according to claim 9, wherein a voltage of 0.4V is applied to the working electrode, and a voltage of 0.8V is applied to the electrode.
11. The method according to claim 6, wherein in step 4, the content of the analyte in the sample is calculated based on the measured value at T2.
12. The method according to claim 6, wherein in step 1, after the sample is added to the biosensor, a voltage is applied to the working electrode and the counter electrode of the biosensor so that a voltage difference between the working electrode and the counter electrode is a positive voltage, and when a change in current is detected, the process proceeds to step 2.
13. The method of claim 6, wherein the reagent on the biosensor comprises creatinase, sarcosine oxidase, peroxidase, and a reduced electron mediator.
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