A method for constructing a photoelectrochemical enzyme sensor based on signal polarity inversion

Abstract

The application provides a construction method of a photoelectrochemical enzyme sensor based on signal polarity flip, and steps are as follows: step 1, preparation of BiOBr nanosheet; step 2, preparation of CuMOF nanosheet; step 3, preparation of BiOBr / CuMOF nanocomposite; and step 4, construction of a signal flip type photoelectrochemical sensor. The prepared BiOBr / CuMOF is used as a working electrode to create a sensitive photoelectrochemical sensing platform. The polarity of a photoelectric current signal is flipped by using a target, and the detection system plays a role in eliminating background signals and enhancing anti-interference capability. When the target ALP exists, ascorbic acid-2-phosphate magnesium (AAP) in the solution is decomposed under the catalysis of ALP to generate ascorbic acid, and the Bi 3+ -O bond in BiOBr is broken to generate an oxygen vacancy, so that the photoelectric current signal is flipped, the relationship between the photoelectric current response value and the ALP concentration is established, and the purpose of rapid, sensitive and selective detection of the ALP content in human serum is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of electrochemical detection, and relates to a method for constructing a photoelectrochemical enzyme sensor based on signal polarity inversion, in particular to a method for constructing a photoelectrochemical sensor for detecting alkaline phosphatase in serum and use thereof. BACKGROUND

[0002] Alkaline phosphatase (ALP) is an important biomarker in human tissues and organs. Abnormal expression of ALP in serum is closely related to many diseases such as breast cancer, prostate cancer, bone disease, liver dysfunction and diabetes, and plays an important role in the diagnosis of many diseases. Currently, the continuous monitoring method of disodium nitrophenyl phosphate is commonly used in clinical colorimetric detection of ALP. This method usually requires the cooperation of large biochemical analysis instruments for single detection, which is costly and not conducive to application in instant and on-site detection scenarios. Therefore, it is of great significance to develop a simple, rapid and sensitive method for the detection of alkaline phosphatase.

[0003] Photoelectrochemical (PEC) sensor is a new emerging analytical detection technology, which has been widely used in many fields due to its high sensitivity, good selectivity and easy operation. PEC sensor combines electrochemical detection and photoexcitation, and relies on the photoelectric conversion characteristics of photoactive substances to quantitatively or qualitatively detect the content of the target substance. Traditional PEC sensors are constructed based on one-way signal weakening and signal enhancement. Traditional PEC sensors inevitably have high background signals and are affected by other substances during detection. Therefore, the anti-interference ability and sensitivity of PEC biosensors are inevitably limited, especially for trace analysis in complex systems. In order to improve the detection performance and realize sensitive detection in complex systems, signal inversion type PEC sensors have been developed. When target recognition occurs, a photoelectric current in the opposite direction is generated, which can effectively eliminate the influence of high background signals and interfering substances on detection accuracy. PEC sensors based on signal inversion have lower detection limit and stronger anti-interference ability, which can effectively expand the application range of PEC sensors, and are a very potential analytical detection strategy.

[0004] The application prepares a bismuth oxybromide (BiOBr) modified copper-based metal organic framework (CuMOF) nanocomposite as a photoelectric active material, realizes the introduction of oxygen vacancies in the semiconductor material through the combined action of the target and light, thereby converting the cathode photocurrent into an anode photocurrent, successfully establishes a signal flipping PEC sensing platform, is used for PEC detection of ALP in human serum, constructs a rapid and sensitive PEC sensor for detecting ALP, establishes the corresponding relationship between the ALP concentration and the photocurrent response value, and realizes the purpose of simple, sensitive and rapid detection of ALP. Therefore, the prepared sensor can be used for detecting the ALP content in human serum. Currently, no related reports have been found. SUMMARY

[0005] The application aims to provide a PEC sensor with the advantages of high sensitivity, high selectivity and wide measurement range. The sensor has a simple preparation process, low cost and realizes the purpose of rapid quantitative detection of ALP.

[0006] The adopted scheme is summarized as follows:

[0007] The prepared BiOBr / CuMOF nanocomposite is used as a photoelectric active material to create a super-sensitive photoelectrochemical sensing platform. The polarity of the photocurrent signal is flipped by the combined action of the target and light, which plays a role in eliminating the background signal and enhancing the anti-interference ability of the detection system. When the target ALP is added to the solution, ascorbic acid-2-phosphate magnesium (AAP) in the electrolyte solution is decomposed under the enzyme catalysis of ALP, and ascorbic acid is produced to reduce BiOBr. Under the synergistic action of light, BiOBr is reduced to BiOBr, and the heterojunction structure is changed, so that the photocurrent response signal is flipped, and the relationship between the photocurrent response value and the ALP concentration is established, so as to achieve the purpose of rapid, sensitive and selective detection of the ALP content in human serum. 3+ The breaking of the -O bond can effectively produce oxygen vacancies, so that the heterojunction structure is changed, the photocurrent response signal is flipped, the relationship between the photocurrent response value and the ALP concentration is established, and the purpose of rapid, sensitive and selective detection of the ALP content in human serum is achieved.

[0008] The application is realized through the following specific technical schemes:

[0009] The construction method of the photoelectrochemical enzyme sensor based on signal polarity flipping is as follows:

[0010] Step 1, preparation of BiOBr nanosheet:

[0011] Bismuth nitrate pentahydrate is dissolved in dilute nitric acid, and then the solution is added to a cetyltrimethylammonium bromide (CTAB) solution. After stirring uniformly, the solution is transferred to a water bath and heated under constant pressure and sealed conditions. After the reaction is completed, the BiOBr nanosheet is obtained by washing and drying.

[0012] Step 2, preparation of CuMOF nanosheet:

[0013] Copper nitrate trihydrate and polyvinylpyrrolidone (PVP) were dissolved in a mixed solvent of N,N-dimethylformamide (DMF) and ethanol, then a certain amount of trifluoroacetic acid (TFA) was added to the above solution to obtain solution A;

[0014] The ligand meso-tetrakis(4-carboxyphenyl) porphyrin (TCPP) was added to a mixed solvent of DMF and ethanol, and ultrasonically dispersed to obtain solution B;

[0015] Then solution B was added to solution A and ultrasonically mixed to form a clear and transparent solution C; solution C was transferred to a sealed glass bottle for solvothermal reaction, after the reaction was completed, the product was washed, centrifuged and freeze-dried to obtain the CuMOF nanosheet;

[0016] Step 3, preparation of BiOBr / CuMOF nanocomposite:

[0017] The BiOBr nanosheet obtained in step 1 was dispersed in ethanol and stirred uniformly, then the CuMOF nanosheet in step 2 was added and ultrasonically mixed to obtain a mixed solution D; the mixed solution D was transferred to a three-necked flask for heating and refluxing reaction, after the reaction was completed, the product was washed and vacuum dried to obtain the BiOBr / CuMOF nanocomposite;

[0018] Step 4, construction of signal inversion detection ALP photoelectrochemical sensor:

[0019] The BiOBr / CuMOF nanocomposite was dispersed in anhydrous ethanol to obtain a BiOBr / CuMOF dispersion liquid, an appropriate amount of BiOBr / CuMOF dispersion liquid was dropped on the FTO electrode and placed under an infrared lamp for drying to obtain a BiOBr / CuMOF / FTO electrode, which constituted a BiOBr / CuMOF / FTO photoelectrochemical sensor for detecting ALP.

[0020] In step 1, the preparation of BiOBr nanosheet: 0.13 g of bismuth nitrate pentahydrate was dissolved in 50 mL of HNO3 (2 wt%). Then, the bismuth nitrate solution was added dropwise to 50 mL of CTAB solution (8×10 -3 M) and stirred and heated in a water bath at 80°C for 3 h. After the reaction was completed, the product was washed with ethanol and water several times, then transferred to an oven and dried at 60°C for 24 h to obtain BiOBr nanosheet.

[0021] In step 2,

[0022] In solution A, the amount ratio of copper nitrate trihydrate, PVP, mixed solvent of DMF and ethanol, trifluoroacetic acid TFA was 3.6 mg: 10 mg: 12 mL: 10 μL; wherein the concentration of TFA was 1.0 M.

[0023] The volume ratio of TCPP, DMF and ethanol in solution B is 4mg:4mL.

[0024] The volume ratio of solution A and solution B is 3:1.

[0025] The volume ratio of DMF and ethanol in the mixed solvent of DMF and ethanol is 3:1.

[0026] The temperature of the solvothermal reaction is 80℃, and the time is 24h.

[0027] In step 3, the ratio of the amount of BiOBr nanosheets and CuMOF nanosheets is 100mg:1-9mg; the temperature of the reflux reaction is 60℃, and the time is 12-24h.

[0028] In step 4, the concentration of the BiOBr / CuMOF dispersion is 2mg / mL, and the drop coating amount is 20-30μL.

[0029] The use of the photoelectrochemical enzyme sensor based on signal polarity inversion prepared by the application for detecting alkaline phosphatase ALP, the specific detection steps are:

[0030] (1) BiOBr / CuMOF / FTO electrode as the working electrode, saturated calomel electrode as the reference electrode, platinum wire as the counter electrode, PBS solution containing AAP as the electrolyte solution, to form a signal inversion type photoelectrochemical sensor for detecting alkaline phosphatase, under the irradiation of xenon lamp light source and nitrogen atmosphere, the photoelectric current value is used to reflect the change of ALP concentration through the three-electrode system of the electrochemical workstation;

[0031] (2) A series of ALP solutions with different concentrations are dropped on the surface of the BiOBr / CuMOF / FTO sensor, incubated at 37℃ for 60-120min, placed in the above-mentioned three-electrode system, and the photoelectric current value is detected, the results show that the ALP concentration and the photoelectric current value have a good linear relationship;

[0032] (3) An unknown concentration of ALP sample is dropped on the surface of the BiOBr / CuMOF / FTO sensor, incubated at 37℃ for 60-120min, placed in the above-mentioned three-electrode system, and the photoelectric current value is detected, and the linear curve of step (2) is substituted; the ALP concentration of the unknown sample is obtained.

[0033] In step (1), the concentration of AAP in the electrolyte solution is 50mM.

[0034] In step (2), the drop coating amount of the ALP solution is 20-30μL, and the concentration is 0.005U·L –1 -500U·L –1 .

[0035] The electrochemical experiment is implemented on a workstation of model CHI660e, and the light source is a xenon lamp parallel light source system instrument. The modified BiOBr / CuMOF / FTO electrode is used as a working electrode to build a three-electrode system, and is immersed in a PBS solution containing 50mM AAP. The light source is turned on, and the electrochemical workstation is connected. The I-t curve is used for real-time monitoring. When the baseline of the I-t curve is stable, the electrode is irradiated, and the change of the photocurrent response is observed. The bias voltage applied is 0V.

[0036] The beneficial effects of the present application are:

[0037] The BiOBr / CuMOF nanocomposite prepared in the present application is used as a photoelectric active material to successfully establish a photoelectrochemical sensing platform, and a signal inversion type photoelectrochemical detection method for alkaline phosphatase in human serum is established. The characteristics and advantages are as follows:

[0038] (1) The BiOBr / CuMOF nanocomposite prepared in the present application is used as a photoelectric active material to construct a photoelectrochemical sensor. The photocurrent signal of the nanocomposite is greatly enhanced relative to the monomer. This may be because the introduction of CuMOF can effectively broaden the light response range of BiOBr, promote charge separation, prolong the lifetime of photo-generated carriers, and provide abundant surface active sites, which helps to improve the photoactivity of the semiconductor material and improve the performance of the PEC sensor.

[0039] (2) The target and light cooperate to introduce oxygen vacancies into the BiOBr / CuMOF nanocomposite in the present application, which changes the electron transfer route and can realize the signal inversion of the photocurrent. The PEC sensor constructed based on the signal inversion can break through the limitation of the background signal, widen the detection width, eliminate the false negative or false positive signals caused by interfering substances, and effectively improve the accuracy of the PEC sensor.

[0040] (3) The present application constructs a PEC sensor for sensitive detection of ALP based on the excellent photoelectric performance of BiOBr / CuMOF nanocomposite and the signal inversion strategy. The sensor has a wider linear range (0.005U·L –1 ~ 500U·L –1 ), and a lower detection limit of 0.0017U·L –1 .

[0041] (4) Compared with the traditional PEC sensor, the biggest feature of the signal flip type PEC detection method of the ALP proposed in the application is that the switching characteristics of the photocurrent signal from the cathode to the anode are used to effectively eliminate the influence of the background signal and the interfering substances, and the sensitivity of the sensor can be greatly improved. The sensor also has the characteristics of more simple and flexible operation, simpler instrument equipment, lower detection cost, higher sensitivity and the like. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Figure 1 is a scanning electron microscope image of a BiOBr / CuMOF nanocomposite;

[0043] Figure 2 Figure 2 is an X-ray diffraction spectrum of a BiOBr / CuMOF nanocomposite;

[0044] Figure 3 Figure 3 is a signal diagram of an electrode modified with different materials;

[0045] Figure 4 Figure 4 is a photocurrent response diagram of a PEC sensor constructed by the application for detecting different concentrations of ALP (A); and a corresponding ALP detection linear curve (B).

[0046] Figure 5 Figure 5 is a photocurrent response performance diagram of a BiOBr / CuMOF nanocomposite with different proportions of CuMOF and BiOBr. DETAILED DESCRIPTION

[0047] The application will be described in detail below in conjunction with examples and the accompanying drawings, but the application is not limited to these examples.

[0048] Example 1:

[0049] (1) Preparation steps of BiOBr nanosheets:

[0050] 0.13g of bismuth nitrate pentahydrate was weighed and dissolved in 50mL of 2wt% HNO3, then the bismuth nitrate solution was added dropwise to 50mL of 8×10 -3 M CTAB solution, stirred and heated in a water bath at 80℃ for 3h, after the reaction was completed, washed with ethanol and water several times, then transferred to an oven and dried at 60℃ for 24h to obtain BiOBr nanosheets.

[0051] (2) Preparation steps of CuMOF nanosheets:

[0052] First, 3.6 mg of copper nitrate trihydrate, 10 mg of PVP were dissolved in 12 mL of a mixture of DMF and ethanol (volume ratio of 3:1), and then 10 μL of TFA (1.0 M) was added. Then, 4 mL of a mixture of DMF and ethanol (volume ratio of 3:1) containing 4 mg of TCPP was added to the above solution under stirring. Then the solution was mixed and heated to 80°C for 24 h; after cooling, it was washed several times with ethanol, centrifuged at 8000 rpm for 10 min, and freeze-dried overnight to obtain CuMOF nanosheets.

[0053] (3) Preparation of BiOBr / CuMOF nanocomposites

[0054] 0.1 g of BiOBr nanosheet powder was weighed into 60 mL of ethanol solvent and ultrasonically treated for 30 min to form a uniform dispersion. Then 3 mg of CuMOF was added to the above solvent and ultrasonically treated again for 15 min to form a uniformly dispersed suspension. Subsequently, the suspension was poured into a 100 mL round-bottom flask, the temperature of the heating oil bath was raised to 60°C, and the round-bottom flask was placed in the oil bath and continuously stirred for 12 h. Finally, the flask was removed from the oil bath and naturally cooled at room temperature, and the resulting solution was centrifuged at 13000 rad / s for 10 min, washed with ethanol and water three times, and dried at 60°C for 12 h.

[0055] Figure 1 FIG. 1 is a scanning electron microscope image and a transmission electron microscope image of BiOBr / CuMOF obtained in Example 1, and it can be seen that CuMOF nanosheets are attached to the surface of BiOBr, and the overall morphology retains the layered structure of BiOBr.

[0056] Figure 2 FIG. 2 is an X-ray diffraction spectrum of BiOBr / CuMOF obtained in Example 1, and the peaks of the two monomers appear in the XRD spectrum, and no other impurity peaks appear, which indicates that the composite material is indeed composed of CuMOF and BiOBr, and has high purity and contains few impurities.

[0057] And as shown in FIG. 3, the composite material has an improved photoelectric signal than CuMOF alone, and after the addition of the target, the photocurrent signal is effectively reversed. Figure 3

[0058] (4) Pretreatment of FTO electrode

[0059] ​Select 1*2 cm FTO as the base of the working electrode. FTO needs to be ultrasonically washed with toluene, acetone, ethanol and ultrapure water in turn for 30 min after cutting, the purpose is to remove the organic contaminants and some adhering impurities on the surface of FTO. Then, the dried FTO electrode is immersed in 1M ethanol / sodium hydroxide solution for 15 min, the purpose is to activate the surface of FTO. Finally, the FTO electrode is rinsed with ultrapure water again, and dried under nitrogen flow and then put into a dry dish for standby.

[0060] (5) Construction of photoelectrochemical (PEC) sensor for detecting ALP

[0061] Weigh 2mg BiOBr / CuMOF and disperse it in 1mL ethanol to obtain BiOBr / CuMOF dispersion, remove 20μL BiOBr / CuMOF dispersion and drop coat it on the nitrogen-dried FTO electrode with fixed area, place it under infrared lamp for drying, finally obtain BiOBr / CuMOF / FTO electrode. Further, add 20μL ALP solution with different concentrations to the surface of BiOBr / CuMOF / FTO electrode, prepare three electrodes for each concentration and mark them for testing. Incubate the BiOBr / CuMOF / FTO electrode with added ALP solution at 37℃ for 90min.

[0062] (6) PEC signal detection

[0063] The electrochemical experiment is carried out on a workstation with model CHI660e, and the light source is a xenon lamp parallel light source system instrument. The modified BiOBr / CuMOF / FTO electrode is used as the working electrode, the saturated calomel electrode is used as the reference electrode, and the platinum wire is used as the counter electrode, which is immersed in the PBS solution containing 50mM AAP, the light source is turned on, and the electrochemical workstation is connected, and the I-t curve is used for real-time monitoring. When the baseline of I-t curve is stable, irradiate the electrode with light, and observe the change of photocurrent response. The applied bias voltage is 0V.

[0064] As shown in Figure 4 , with the increase of ALP concentration, the photocurrent of the signal-inverted PEC sensor gradually increases, and in the concentration range of 0.005U·L –1 ~500U·L –1 , the photocurrent and the log value of ALP concentration show a good linear relationship, and the detection limit can reach 0.0017U·L –1 .

[0065] Example 2:

[0066] (1) Preparation of BiOBr / CuMOF nanocomposite

[0067] 0.1 g of BiOBr nanosheet powder was weighed and added to 60 mL of ethanol solvent. The mixture was sonicated for 30 min to form a uniform dispersion. Then, 9 mg of CuMOF was added to the solvent, and the mixture was sonicated again for 15 min to form a uniformly dispersed suspension. The suspension was then poured into a 100 mL round-bottom flask, and an oil bath was heated to 60 °C. The flask was placed in the oil bath and heated with continuous stirring for 12 h. Finally, the flask was removed from the oil bath and allowed to cool naturally at room temperature. The resulting solution was centrifuged at 13000 rad / s for 10 min, washed three times with ethanol and water, and dried at 60 °C for 12 h.

[0068] The other steps are the same as those in Example 1.

[0069] Example 3:

[0070] (1) Preparation of BiOBr / CuMOF nanocomposite

[0071] 0.1 g of BiOBr nanosheet powder was weighed and added to 60 mL of ethanol solvent. The mixture was sonicated for 30 min to form a uniform dispersion. Then, 1 mg of CuMOF was added to the solvent, and the mixture was sonicated again for 15 min to form a uniformly dispersed suspension. The suspension was then poured into a 100 mL round-bottom flask, and an oil bath was heated to 60 °C. The flask was placed in the oil bath and heated with continuous stirring for 12 h. Finally, the flask was removed from the oil bath and allowed to cool naturally at room temperature. The resulting solution was centrifuged at 13000 rad / s for 10 min, washed three times with ethanol and water, and dried at 60 °C for 12 h.

[0072] The other steps are the same as those in Example 1.

[0073] like Figure 5 As shown, when the mass ratio of CuMOF to BiOBr is 3wt%, the photocurrent response performance of the composite material exhibits the maximum value, which can be used to construct photoelectrochemical enzyme sensors with signal polarity reversal.

Claims

1. A method for constructing a photoelectrochemical enzyme sensor based on signal polarity inversion, characterized in that, The steps are as follows: Step 1, preparation of BiOBr nanosheets: 0.13 g of bismuth nitrate pentahydrate was dissolved in 50 mL of 2 wt% dilute nitric acid, and then the solution was added to 50 mL of 8 x 10 -3 M hexadecyl trimethyl ammonium bromide CTAB solution, stirred uniformly, and then transferred to a 80 °C water bath, and heated for 3 h under constant pressure and closed conditions. After the reaction was completed, it was washed with ethanol and water, and dried at 60 °C for 24 h to obtain BiOBr nanosheets; Step 2, preparation of CuMOF nanosheets: Dissolve copper nitrate trihydrate and polyvinylpyrrolidone PVP in a mixed solvent of N,N-dimethylformamide DMF and ethanol, then add a certain amount of trifluoroacetic acid TFA to the above solution; obtain solution A; Add ligand meso-tetra (4-carboxyphenyl) porphyrin TCPP to a mixed solvent of DMF and ethanol, ultrasonic dispersion to obtain solution B; Then add solution B to solution A, ultrasonic mixing to form a clear and transparent solution C; transfer solution C to a sealed glass bottle for solvothermal reaction, after the reaction is completed, wash, centrifuge, freeze-drying the obtained solid product to obtain CuMOF nanosheets; In step 2, In solution A, the amount ratio of copper nitrate trihydrate, PVP, mixed solvent of DMF and ethanol, and trifluoroacetic acid TFA is 3.6 mg:10 mg:12 mL:10 μL; wherein the concentration of TFA is 1.0M; In solution B, the amount ratio of TCPP, mixed solvent of DMF and ethanol is 4 mg:4 mL; The volume ratio of solution A and solution B is 3:1; In the mixed solvent of DMF and ethanol, the volume ratio of DMF and ethanol is 3:1; Step 3, preparation of BiOBr / CuMOF nanocomposite: Disperse the BiOBr nanosheets obtained in step 1 in ethanol, stir uniformly, then add the CuMOF nanosheets in step 2, ultrasonic uniformly to obtain a mixed solution D, transfer the mixed solution D to a three-necked flask for heating and reflux reaction, after the reaction is completed, wash, vacuum dry, the obtained solid product is BiOBr / CuMOF nanocomposite; Step 4, construction of signal flip detection ALP photoelectrochemical sensor: Disperse the BiOBr / CuMOF nanocomposite in anhydrous ethanol to obtain a BiOBr / CuMOF dispersion, take an appropriate amount of BiOBr / CuMOF dispersion and drop coat on the FTO electrode, place it under an infrared lamp for drying to obtain a BiOBr / CuMOF / FTO electrode, which constitutes a BiOBr / CuMOF / FTO photoelectrochemical sensor for detecting ALP.

2. The construction method of claim 1, wherein, In step 2, the temperature of the solvothermal reaction is 80℃, and the time is 24 h.

3. The construction method of claim 1, wherein, In step 3, the amount ratio of BiOBr nanosheets and CuMOF nanosheets is 100 mg:1~9 mg.

4. The construction method of claim 1, wherein, In step 3, the temperature of the reflux reaction is 60℃, and the time is 12~24 h.

5. The construction method of claim 1 wherein, In step 4, the concentration of the BiOBr / CuMOF dispersion is 2 mg / mL, and the drop coating amount is 20~30 μL.

6. Use of the photoelectrochemical enzyme sensor based on signal polarity flip obtained by the construction method of any one of claims 1~5 for detecting alkaline phosphatase ALP.

7. Use according to claim 6, characterized in that, The detection steps are as follows: (1) The signal inversion type photoelectrochemical sensor for detecting alkaline phosphatase is constructed by taking BiOBr / CuMOF / FTO electrode as a working electrode, saturated calomel electrode as a reference electrode, platinum wire as a counter electrode, and PBS solution containing AAP as an electrolyte solution. The photoelectric current value is used to reflect the change of ALP concentration by detecting the photoelectric current value under the irradiation of xenon lamp and in the nitrogen atmosphere through the three-electrode system of electrochemical workstation; (2) A series of ALP solutions with different concentrations are dropped on the surface of BiOBr / CuMOF / FTO sensor, and incubated at 37℃ for 60-120 min. The photoelectric current value is detected by placing the sensor in the above three-electrode system. The results show that the ALP concentration and the photoelectric current value have a good linear relationship; (3) The ALP sample with unknown concentration is dropped on the surface of BiOBr / CuMOF / FTO sensor, and incubated at 37℃ for 60-120 min. The photoelectric current value is detected by placing the sensor in the above three-electrode system. The ALP concentration of the unknown sample is obtained by substituting the linear curve of step (2).

8. The use according to claim 7, characterized in that, in step (1), the concentration of AAP in the electrolyte solution is 50 mM; In step (2), the ALP solution is dropped at an amount of 20-30 μL and a concentration of 0.005 U·L  –1 ~500  U·L –1 .

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