TWI493183B - Method of measuring absolute concentration of analyte - Google Patents

Description

Method for measuring the absolute concentration of a test object

The present invention relates to a method of measuring concentration, and more particularly to a method of measuring absolute concentration using electrochemistry.

A variety of methods have been developed to measure the concentration of a test substance in a solution, such as an electrochemical method, a fluorescence method, a chemical fluorescence method, and the like. In the prior art, when concentration measurement is performed by an electrochemical method, a current-to-concentration calibration curve is usually established by using a sample of a known concentration in a buffer. When a sample concentration of an unknown concentration is desired, it is desirable to substitute the current obtained by measuring the sample of the unknown concentration into the previously established calibration curve to obtain the concentration of the analyte. However, in fact, whenever the measurement is repeated, the difference in the position of the sensor, the difference in the sensitivity of the sensor, or the difference in the environmental disturbance, even if the concentration of the target in the sample is zero, the measurement is performed. The background current value will be different. Therefore, when the sample of unknown concentration is repeatedly measured, only the amount of change in the concentration of the analyte can be obtained due to the difference in the background current value at each measurement, and the absolute concentration of the analyte cannot be known.

The invention provides a method for measuring the absolute concentration of a test object, which can detect the absolute concentration of the test object by avoiding the influence of the background current.

The present invention proposes a method for measuring the absolute concentration of a test object, which includes the following steps. Step (a) placing the working electrode and the reference electrode in the first solution, and obtaining a current value I ₁ from the working electrode by electrochemical amperometry, wherein the first solution contains the analyte of unknown concentration. Step (b) adding a buffer solution to the first solution to form a second solution, and obtaining an electric current value I ₂ from the working electrode by electrochemical amperometry, wherein the buffer solution contains no analyte. Step (c) adding a standard solution to the second solution to form a third solution, and obtaining an electric current value I ₃ from the working electrode by electrochemical amperometry, wherein the standard solution contains the analyte having a concentration of Y. Step (d) calculates X at least according to Equation 1, Equation 2 and Equation 3: I ₁ = a × (X / V ₁ ) + b Equation 1, I ₂ = a × (X / (V ₁ + V ₂ )) +b Formula 2, I ₃ = a × ((X + V ₃ ) / (V ₁ + V ₂ )) + b Formula 3, where V ₁ represents the volume of the first solution, and V ₂ represents the volume of the buffer solution, V ₃ represents the volume of the standard solution, and X, a, and b are variables to be solved. Step (e) calculates the unknown concentration of the analyte based on X.

In an embodiment of the invention, the analyte to be tested comprises hydrogen peroxide, and the unknown concentration ranges from 5 μM to 150 μM.

In an embodiment of the invention, the analyte to be tested comprises perglutamic acid, and the unknown concentration ranges from 10 μM to 80 μM.

In an embodiment of the invention, before the step (a) above, the glutamic acid oxidase is further adhered to the working electrode.

In an embodiment of the present invention, in the above step (c), the method further comprises adding the standard solution to the third solution successively to obtain the fourth, fifth, ... Nth solution, and after each standard solution is added. Obtaining current values I ₄ , I ₅ ... I _N from the working electrode by electrochemical amperometry, and in the above step (d), further comprising calculating X:I ₄ = according to Equation 4, Equation 5... a × ((X + V ₃ + V ₄ ) / (V ₁ + V ₂ )) + b Equation 4, I ₅ = a × ((X + V ₃ + V ₄ + V ₅ ) / (V ₁ + V ₂ )) +b Equation 5, I _N = a × ((X + V ₃ + V ₄ + V ₅ + ... + V _N ) / (V ₁ + V ₂ )) + b of the formula N, wherein V ₁ represents the volume of the first solution, and V ₂ represents The volume of the buffer solution, V ₃ represents the volume of the standard solution, V ₄ , V ₅ ... V _N represents the volume of the standard solution added successively, X, a, b are the variables to be solved, and N is a positive integer greater than or equal to 4.

In an embodiment of the invention, the manner of performing step (d) above comprises the following steps. Calculate a according to Equation 3 to Formula N, and calculate X according to a and Equations 1 and 2.

In an embodiment of the invention, the manner of performing step (e) above comprises calculating the unknown concentration of the object to be tested according to formula a: the unknown concentration of the object to be tested = (X×Y)/V ₁ Wherein V ₁ represents the volume of the first solution, and Y represents the concentration of the analyte in the standard solution.

The present invention further provides a method for measuring the absolute concentration of a test object, which includes the following steps. Step (a) placing the working electrode and the reference electrode in the first solution, and obtaining a current value I ₁ from the working electrode by electrochemical amperometry, wherein the first solution contains the analyte of unknown concentration. Step (b) adding a standard solution to the first solution to form a second solution, and obtaining an electric current value I ₂ from the working electrode by electrochemical amperometry, wherein the standard solution contains the analyte having a concentration of Y. Step (c) adding a buffer solution to the second solution to form a third solution, and obtaining an electric current value I ₃ from the working electrode by electrochemical amperometry, wherein the buffer solution contains no analyte. Step (d) calculates X at least according to Equation 1, Equation 2 and Equation 3: I ₁ = a × (X / V ₁ ) + b Equation 1, I ₂ = a × ((X + V ₂ ) / (V ₁ )) + b Formula 2, I ₃ = a × ((X + V ₂ ) / (V ₁ + V ₃ )) + b Formula 3, where V ₁ represents the volume of the first solution, and V ₂ represents the volume of the standard solution V ₃ represents the volume of the buffer solution, and X, a, and b are variables to be solved. Step (e) calculates the unknown concentration of the analyte based on X.

Based on the above, the method for measuring the absolute concentration of the analyte to be tested according to the present invention can directly determine the unknown concentration of the analyte by the operations of Equation 1, Equation 2, and Equation 3. Therefore, by measuring the unknown concentration of the analyte by this measurement method, the absolute concentration of the analyte can be obtained in a simple and convenient step under the influence of the background current value.

The above described features and advantages of the invention will be apparent from the following description.

S _i ~S _vi , S _i' ~S _vi' ‧‧‧ Section

1 is a graph showing the relationship between time and current of a hydrogen peroxide solution measured by a constant potential amperometric method in Experimental Example 1, wherein the concentration of the hydrogen peroxide solution was 35.3 μM.

Fig. 2 is a graph showing the relationship between the prepared concentration of hydrogen peroxide in Experimental Example 1 and the absolute concentration obtained after the calculation.

Fig. 3 is a graph showing the relationship between time and current for measuring a glutamic acid solution by a constant potential amperometric method in Experimental Example 2, wherein the concentration of the glutamic acid solution was 10 μM.

Fig. 4 is a graph showing the relationship between the concentration of glutamic acid prepared in Experimental Example 2 and the absolute concentration obtained after the calculation.

Hereinafter, a method of measuring the absolute concentration of the analyte will be described in detail with reference to the embodiments.

An embodiment of the present invention provides a method for measuring an absolute concentration of a test object, comprising the steps of: step (a): placing a working electrode and a reference electrode in a first solution, and electrochemically amperaging from the working electrode Obtaining a current value I ₁ , wherein the first solution contains an unknown concentration of the analyte; and step (b): adding a buffer solution to the first solution to form a second solution, and obtaining an electric current from the working electrode by electrochemical amperometry a value I ₂ , wherein the buffer solution contains no analyte; step (c): adding a standard solution to the second solution to form a third solution, and obtaining a current value I ₃ from the working electrode by electrochemical amperometry, wherein The standard solution contains the analyte of concentration Y; step (d): at least X is calculated according to the following formula 1, formula 2 and formula 3: I ₁ = a × (X / V ₁ ) + b Formula 1, I ₂ = a × (X / (V ₁ + V ₂ )) + b Formula 2, I ₃ = a × ((X + V ₃ ) / (V ₁ + V ₂ )) + b Formula 3, where V ₁ represents the first The volume of the solution, V ₂ represents the volume of the buffer solution, V ₃ represents the volume of the standard solution, X, a, b are the variables to be solved; and step (e): the unknown concentration of the analyte is calculated according to X.

First, step (a) will be described.

In the present embodiment, the current value I1 is obtained by performing an electrochemical amperometric method using a three-pole battery system, wherein the three-pole battery system includes an auxiliary electrode in addition to the aforementioned working electrode and reference electrode. The working electrode is, for example, a platinum electrode, a glassy carbon electrode, a pyrolytic graphite electrode or a titania electrode. The reference electrode is, for example, a silver/silver chloride electrode, copper/copper sulfate. The auxiliary electrode is, for example, a platinum electrode or a gold electrode. In the present embodiment, although the electrochemical amperage method is performed using a three-electrode battery system, the present invention is not limited thereto. In other embodiments, the electrochemical amperometric method can also be performed through a two-pole battery system.

Specifically, in step (a), by maintaining a certain potential between the working electrode and the reference electrode, the object to be tested in the first solution can be oxidized at the surface of the working electrode to generate a current, and the current can be stabilized after the current is stabilized. The working electrode obtains a current value I ₁ . The current value I ₁ will be in a linear relationship with the concentration of the analyte, that is, the higher the current value obtained, the higher the concentration of the analyte. In one embodiment, when the working electrode is a platinum electrode, the reference electrode is a silver/silver chloride electrode, and the auxiliary electrode is a platinum electrode, the constant potential used is 0.4V to 0.7V, preferably 0.7V.

In the present embodiment, the analyte is, for example, hydrogen peroxide or perglutamic acid. In one embodiment, when the analyte is hydrogen peroxide, the unknown concentration range of the analyte of the first solution is, for example, between 4.41 μM and 150 μM. In another embodiment, when the analyte is perglutamic acid, the unknown concentration range of the analyte of the first solution is, for example, between 10 μM and 80 μM. That is, the measuring method of the present invention has a wide range of linear concentrations.

It is worth noting that when the analyte is glutamic acid, it is further included on the working electrode to adhere to glutamate oxidase before step (a). In detail, in the case where the glutamic acid oxidase is attached to the working electrode, in the step (a), the glutamate oxidase can catalyze the oxidation reaction of the glutamic acid (the analyte) in the first solution. Hydrogen peroxide is produced. As a result, the generated hydrogen peroxide is oxidized on the surface of the working electrode to generate electrons, and the current value I ₁ can be obtained from the working electrode.

Next, the step (b) will be described.

The buffer solution is added to the first solution in the step (a) to form a second solution, and the current value I _{2 is} obtained from the working electrode by electrochemical amperometry. In the step (b), the electrochemical amperometric method can be carried out in the same manner and conditions as the electrochemical ampere method described in the step (a), and thus will not be described again.

In the present embodiment, the buffer solution is, for example, a sodium phosphate buffer solution. (SPB, pH = 7.4), carbonate buffer (CAB, pH = 7.4) or acetate buffer (Acetate Buffer Solution, pH = 3.5). It is worth noting that since the buffer solution does not contain the analyte, in step (d), when the simultaneous linear equation is solved for X, a linear independent equation can be provided (the related description will be described below).

Next, the step (c) will be described.

The standard solution is added to the second solution in the step (b) to form a third solution, and the current value I _{3 is} obtained from the working electrode by electrochemical amperometry. In the step (c), the electrochemical amperometric method can also be carried out in the same manner and conditions as the electrochemical ampere method described in the step (a), and thus will not be described again. In the present embodiment, the volume added by the standard solution is much smaller than the volume of the first solution, and the volume added by the standard solution is much smaller than the volume to which the buffer solution is added.

Next, the step (d) will be described. In the step (d), the current values I ₁ in the foregoing steps (a), (b) and (c) are represented by three mathematical expressions (i.e., the above formula 1, formula 2, and formula 3), A linear relationship between the current value I ₂ and the current value I ₃ and the converted concentration. As used herein, the term "conversion concentration" is defined as: the solution volume of the analyte containing the known concentration Y / the sum of the volume of the solution containing the analyte and the solution containing no analyte, and the variable X to be solved is It is defined as the volume of the unknown concentration of the analyte in the first solution converted to the known concentration Y of the analyte in the standard solution. Further, in general, the variable a to be solved represents the sensitivity, and the variable b to be solved represents the background current value.

It should be noted that, in the foregoing step (b), since the buffer solution added to the first solution does not contain any analyte, the denominator of the conversion concentration in Formula 1 is not the same as the denominator of the conversion concentration in Formula 2. . In the foregoing step (c), since the volume added by the standard solution is much smaller than the volume of the first solution, and the volume added by the standard solution is much smaller than the volume added by the buffer solution, in Equation 3, the denominator of the conversion concentration can be The volume V _{3 of the} standard solution is omitted. In this way, the three equations (ie, Equation 1, Formula 2, and Formula 3) obtained through the foregoing steps (a), (b), and (c) are linearly independent of each other.

In view of this, a can be calculated by subtracting the above-described equations 2 and 3 which are linearly independent from each other. After a is obtained, a is substituted into the equation a obtained by subtracting the above-described linearly independent equations 1 and 2 to obtain X: I _{1 -} I ₂ = a × ((X/V ₁ ) - (X) /(V ₁ +V ₂ )) Formula a, where V ₁ represents the volume of the first solution, V ₂ represents the volume of the buffer solution, and X, a are the variables to be solved.

Next, the step (e) will be described in detail. Since the variable X to be solved is defined as the volume of the analyte of unknown concentration converted to the known concentration Y, after X is obtained in step (d), X is substituted into the following formula b to obtain the analyte. Unknown concentration: unknown concentration of the analyte = (X × Y) / V ₁ Formula b, where V ₁ represents the volume of the first solution, and Y represents the known concentration of the analyte in the standard solution.

Further, in the present embodiment, although the standard solution is added after the buffer solution is added, the present invention is not limited thereto. In other embodiments, the measuring method of the present invention may also be followed by adding a standard solution and then adding a buffer solution.

Based on the above, the measurement of the absolute concentration of the analyte in the present invention is known. In the method, by using the above Equation 1, Equation 2, Equation 3, and Equation b, the unknown concentration of the analyte can be directly obtained. Therefore, by measuring the unknown concentration of the analyte by this measurement method, the absolute concentration of the analyte can be obtained without being affected by the background current value.

Although the absolute concentration of the analyte in the solution can be obtained by the above steps (a) to (e), in order to improve the accuracy and precision of the measurement, the method for measuring the absolute concentration of the analyte of the present invention can also use the following method. Come on.

In the foregoing step (c), the method further comprises adding the standard solution to the third solution successively to obtain the fourth, fifth, ... Nth solution, and after each time the standard solution is added, also working from the electrochemical amperometric method. The electrodes obtain current values I ₄ , I ₅ ... I _N , where N is a positive integer greater than or equal to four. That is, in the step (c), the standard solution may be added in portions to a solution containing an unknown concentration of the analyte, or may be added only once. Additionally, the invention does not limit the number of times a standard solution is added in portions. Further, the same as the condition in which the standard solution was added to the second solution, the volume of the standard solution was added successively much smaller than the volume of the first solution, and much smaller than the volume to which the buffer solution was added. Further, in the step (c), the volume of the standard solution may be the same or different from each other.

Further, in the step (d), in addition to the formula 1, the formula 2 and the formula 3, it is further calculated that X: I ₄ = a × ((X + V ₃ + V _{4 ) is} calculated according to the following formula 4, formula 5, ... ) / (V ₁ + V ₂ )) + b Equation 4, I ₅ = a × ((X + V ₃ + V ₄ + V ₅ ) / (V ₁ + V ₂ )) + b Equation 5, I _N = a × ((X + V ₃ + V ₄ + V ₅ + ... + V _N ) / (V ₁ + V ₂ )) + b Formula N, where V ₁ represents the volume of the first solution, and V ₂ represents The volume of the buffer solution, V ₃ represents the volume of the standard solution, V ₄ , V ₅ ... V _N represents the volume of the standard solution added successively, X, a, b are the variables to be solved, and N is a positive integer greater than or equal to 4.

As described above, Formula 4, Formula 5, Formula N, respectively, represent a linear relationship between the current values I ₄ , I ₅ ... I _N and the conversion concentration obtained after sequentially adding the standard solution in the step (c). In addition, since the volume of the standard solution added is much smaller than the volume of the first solution, and much smaller than the volume of the buffer solution, in the formula N, the formula 5, the denominator of the conversion concentration can omit the standard solution. Volume V ₄ , V ₅ ... V _N . In this way, Equations 4 and 5 are linearly independent of Equations 1 and 2, respectively.

In detail, the manner in which X is calculated according to at least Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, Formula N includes the following steps. First, the converted value after shifting X/(V ₁ +V ₂ ) to the left is plotted using the current values in Equations 3 to N to calculate the average value of a. In general, the slope of the graph after translation (ie a) does not change. Next, after the average value of a is obtained, X is obtained by substituting it into the above formula a in the same manner.

"experiment"

Features of the present invention will be described more specifically below with reference to experimental examples. Although the following experiments are described, the materials used, the amounts and ratios thereof, the processing details, the processing flow, and the like can be appropriately changed without departing from the scope of the invention. Therefore, the invention should not be construed restrictively by the experiments described below.

Experimental example 1 Concentration measurement of hydrogen peroxide

The instruments used are as follows: Instrument: Potentiostat (Model: VSP-300, Manufacturer: Bio-Logic); Working electrode: Platinum electrode, which is obtained by soldering white gold wire and silver plating wire with tin, and the length is about 1.5 cm. The diameter is 0.002 inches and the exposed area is 4 mm ² ; Reference electrode: silver/silver chloride water reference electrode

Auxiliary electrode: platinum corresponding electrode (VC-3 cell 5cm Pt)

The experimental method is as follows.

First, a hydrogen peroxide solution (manufacturer: Panreac) with a concentration of 8.824M was diluted 1000 times with deionized water to prepare a hydrogen peroxide solution with a concentration of 8.824 mM. This solution was added to 10 mL of buffer at different ratios. A set of hydrogen peroxide solutions of different concentrations were prepared, and the final concentrations were 4.41 μM, 8.8 μM, 17.6 μM, 35.3 μM, 70.6 μM, and 132.36 μM, respectively. Next, the hydrogen peroxide solutions having different concentrations are separately subjected to the following experimental steps to measure the concentration of hydrogen peroxide (test substance) in each hydrogen peroxide solution: first, the platinum electrode, silver/chlorination The silver water reference electrode and the platinum counter electrode were simultaneously placed in a volume of 10 mL of the hydrogen peroxide solution to be measured, and 0.7V (vs. silver/silver chloride) was applied by a potentiostat by a constant potential amperometric method. The constant potential of the water reference electrode). Next, 5 mL of a sodium phosphate buffer solution (pH = 7.4) was added. Thereafter, a volume of 20 μL of a standard hydrogen peroxide solution having a concentration of 8.824 mM was added, and this step was repeated three times. After the current is stabilized, observe the measured amount of current change, as shown in Figure 1, where the segment S _i is the steady current of the hydrogen peroxide solution to be measured, and the segment S _ii is stable after adding the sodium phosphate buffer solution. The current, the section S _iii is the steady current after the first addition of the standard hydrogen peroxide solution, the section S _iv is the steady current after the second addition of the standard hydrogen peroxide solution, and the section S _v is the third addition standard The steady current after the hydrogen peroxide solution, the section S _vi is the steady current after the fourth addition of the standard hydrogen peroxide solution.

Thereafter, each of the segments S _i to S _vi is taken out for each of the average current values I _i to I _vi calculated for the current value of 10 seconds, the volume of the hydrogen peroxide solution to be measured is 10 mL, and the volume of the sodium phosphate buffer solution is 5 mL. The volume of the standard hydrogen peroxide solution is 20 μL, 40 μL, 60 μL, 80 μL, and the concentration of the standard hydrogen peroxide solution of 8.824 mM is substituted into the above formulas 1 to 6, formula a and formula b, and the following formula i to formula vi, formula c and Formula d: I _i = a × (X/10mL) + b Formula i, I _ii = a × (X / (10mL + 5mL)) + b Formula ii, I _iii = a × ((X + 20μL) / ( 10 mL + 5 mL)) + b Formula iii, I _iv = a × ((X + 40 μL) / (10 mL + 5 mL)) + b Formula iv, I _v = a × ((X + 60 μL) / (10 mL + 5 mL) ) + b Formula v, I _vi = a × ((X + 80 μL) / (10mL + 5mL)) + b Formula vi, I _i -I _ii = a × ((X/10mL) - (X / (10mL + 5 mL)) Formula c, concentration of hydrogen peroxide to be measured = (X x 8.824 mM) / 10 mL Formula d.

Next, according to the formulas iii to vi, the current values I _iii to I _vi are plotted against the converted concentration after translation (as shown in Table 1), and after a is calculated, a is substituted into the formula c to obtain X. After X is obtained, the concentration of hydrogen peroxide to be measured can be obtained by substituting X into the formula d.

Experimental results:

2 is a graph showing the relationship between the prepared concentration of the hydrogen peroxide solution of Experimental Example 1 and the absolute concentration obtained after the calculation. As can be seen from Fig. 2, the data points coincide very much with the 45 degree line on the graph, which means that the absolute concentration calculated by the measuring method of the present invention is very close to the pre-formulated concentration. Therefore, the measuring method of the present invention can measure the absolute concentration of hydrogen peroxide in a simple and convenient step.

Experimental example 2 Concentration measurement of glutamic acid

The instruments used are as follows:

Instrument: potentiostat (model: VSP-300 manufacturer: BioLogic); working electrode: platinum electrode, its making method is as follows; reference electrode: silver / silver chloride water reference electrode

Auxiliary electrode: platinum corresponding electrode (VC-3 cell 5cm Pt)

Production of working electrodes:

First, cut a piece of platinum wire and use fire to remove the Teflon covered on the outer head and tail to expose the platinum wire. The head is about 5 mm long. Degree, the tail is exposed to a length of 1 mm. Next, cut a silver-plated wire and solder the silver-plated wire and the white gold wire with tin, and then protect the solder joint with AB glue. After the AB gel is cured, place the bare part of the platinum wire into the isopropanol and shake it in an ultrasonic oscillator for 3 minutes, then rinse it again with DI-Water. Next, the cleaned platinum wire was placed on a potentiostat, and a 20 μM poly-pyrrole solution was added for cyclic voltammetry (the number of turns was 25 laps, and the potential interval was -10 V to 10 V, reference) The electrode is a silver/silver chloride water-based reference electrode). Subsequently, the platinum wire deposited with polypyrrole was immersed in a perfluorosulfonic acid resin (Nafion) solution, and then baked in an oven at 180 ° C for 3 minutes and repeated three times to form another barrier layer. Finally, the glutamate oxidase enzyme was applied to the platinum wire (approx. 100 layers) under a microscope by hand coating to complete the preparation of the platinum electrode.

The concentration of glutamic acid in Experimental Example 2 was measured in the same manner as in Experimental Example 1, except that the test substance of Experimental Example 2 was glutamic acid, and the concentration of glutamic acid prepared by deionized water was 5 μM, A set of glutamic acid solutions of 10 μM, 20 μM, 40 μM, 80 μM, 150 μM. In detail, the method for preparing the glutamic acid solution comprises uniformly mixing 22.05 mg of L-Glutamic acid (molecular weight: 147.13 g/mol, manufacturer: sigma) with 15 mL of deionized water to A glutamic acid solution having a concentration of 10 mM was obtained. Next, 10 mM glutamic acid solution was added to 10 mL of buffer solution at different ratios to prepare a group of glutamic acid solutions at different concentrations, and the final concentrations were 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, and 150 μM. Amino acid solution.

In addition, in Experimental Example 2, the measured amount of current change is as shown in FIG. 3, in which the segment S _{i '} is the steady current of the glutamic acid solution to be measured, and the segment S _{ii '} is the sodium phosphate buffer solution. After the steady current, the segment S _{iii '} is the steady current after the first addition of the standard glutamic acid solution, the segment S _{iv '} is the steady current after the second addition of the standard glutamic acid solution, the segment S _{v '} The steady current after the third addition of the standard glutamic acid solution, the section S _vi' is the steady current after the fourth addition of the standard glutamic acid solution.

Experimental results:

Fig. 4 is a graph showing the relationship between the preparation concentration of the glutamic acid solution of Experimental Example 2 and the absolute concentration obtained after the calculation. As can be seen from Fig. 4, in the range of 10 μM to 80 μM glutamic acid concentration, the data points coincide very much with the 45-degree line on the graph, which means that the absolute concentration calculated by the measuring method of the present invention is very close to the pre-formulated concentration. Therefore, the measuring method of the present invention can measure the absolute concentration of glutamic acid in a simple and convenient step.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and any one of ordinary skill in the art can make some changes and refinements without departing from the spirit and scope of the present invention. The scope of the invention is defined by the scope of the appended claims.

Claims (10)

A method for measuring an absolute concentration of a test object, comprising the steps of: (a) placing a working electrode and a reference electrode in a first solution, and obtaining a current value from the working electrode by electrochemical amperometry; I ₁ , wherein the first solution contains an analyte of unknown concentration; step (b) adding a buffer solution to the first solution to form a second solution, and electrochemically amperometrically from the working electrode Obtaining a current value I ₂ , wherein the buffer solution does not contain the analyte; step (c) adding a standard solution to the second solution to form a third solution, and working from the electrochemical amperometric method The electrode obtains a current value I ₃ , wherein the standard solution contains the analyte of concentration Y; and step (d) calculates X at least according to Equation 1, Equation 2 and Equation 3: I ₁ = a × (X/V ₁ +b Equation 1, I ₂ = a × (X / (V ₁ + V ₂ )) + b Equation 2, I ₃ = a × ((X + V ₃ ) / (V ₁ + V ₂ )) + b Formula 3, wherein V ₁ represents the volume of the first solution, V ₂ represents the volume of the buffer solution, V ₃ represents the volume of the standard solution, X, a, b are variables to be solved; and step (e) is based on X Calculating the untested object Know the concentration. A method for measuring an absolute concentration of a test object as described in claim 1, wherein the test object comprises hydrogen peroxide. A method of measuring an absolute concentration of a test object as described in claim 2, wherein the unknown concentration ranges from 4.41 μM to 150 μM. A method for measuring an absolute concentration of a test object as described in claim 1, wherein the analyte comprises perglutamic acid. The method for measuring the absolute concentration of the analyte according to item 4 of the patent application, before step (a), further comprises adhering glutamic acid oxidase to the working electrode. A method of measuring an absolute concentration of a test object as described in claim 4, wherein the unknown concentration ranges from 10 μM to 80 μM. The method for measuring the absolute concentration of the analyte according to the first aspect of the patent application, in the step (c), further comprising adding the standard solution to the third solution successively to obtain the fourth, fifth, ... N solution, and after each addition of the standard solution, current values I ₄ , I ₅ ... I _{N are} obtained from the working electrode by electrochemical amperometry, and in step (d), further including according to formula 4 5... Equation N calculates X: I ₄ = a × ((X + V ₃ + V ₄ ) / (V ₁ + V ₂ )) + b Equation 4, I ₅ = a × ((X + V ₃ + V ₄ + V ₅ ) / (V ₁ + V ₂ )) + b Equation 5, I _N = a × ((X + V ₃ + V ₄ + V ₅ + ... + V _N ) / (V ₁ + V ₂ ) +b formula N, wherein V ₁ represents the volume of the first solution, V ₂ represents the volume of the buffer solution, V ₃ represents the volume of the standard solution, and V ₄ , V ₅ ... V _N represents the sequential addition of the standard solution. The volume, X, a, b are the variables to be solved, and N is a positive integer greater than or equal to 4. The method for measuring the absolute concentration of the analyte according to claim 7, wherein the manner of performing step (d) comprises: calculating a according to formula 3 to formula N; and calculating according to a and formula 1 and formula 2 Out X. A method for measuring an absolute concentration of a test object according to claim 1, wherein the manner of performing step (e) comprises calculating the unknown concentration of the test object according to formula b: the unknown concentration of the test object = (X × Y) / V ₁ Formula b, wherein V ₁ represents the volume of the first solution, and Y represents the concentration of the analyte in the standard solution. A method for measuring an absolute concentration of a test object, comprising the steps of: (a) placing a working electrode and a reference electrode in a first solution, and obtaining a current value from the working electrode by electrochemical amperometry; I ₁ , wherein the first solution contains an analyte of unknown concentration; step (b) adding a standard solution to the first solution to form a second solution, and electrochemically amperometrically from the working electrode Obtaining a current value I ₂ , wherein the standard solution contains the analyte of concentration Y; and step (c) adding a buffer solution to the second solution to form a third solution, and electrochemical amperometric method Obtaining a current value I ₃ from the working electrode, wherein the buffer solution does not contain the analyte; and step (d) calculates X according to Equation 1, Equation 2 and Equation 3: X ₁ = a × (X/V ₁ ) + b Equation 1, I ₂ = a × ((X + V ₂ ) / (V ₁ )) + b Equation 2, I ₃ = a × ((X + V ₂ ) / (V ₁ + V ₃ )) +b Equation 3, where V ₁ represents the volume of the first solution, V ₂ represents the volume of the standard solution, V ₃ represents the volume of the buffer solution, X, a, b are variables to be solved; and step (e) Calculating the object to be tested according to X Known concentration.