CN114609210A - Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof - Google Patents

Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof Download PDF

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CN114609210A
CN114609210A CN202210235225.XA CN202210235225A CN114609210A CN 114609210 A CN114609210 A CN 114609210A CN 202210235225 A CN202210235225 A CN 202210235225A CN 114609210 A CN114609210 A CN 114609210A
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ppi
ppase
phe
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cps
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胡宇芳
秦玲霞
任信信
胡凯悦
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Shenzhen Wanzhida Technology Co ltd
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Ningbo University
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Abstract

The invention discloses a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof, which comprises the following steps: firstly, L-Phe is taken as a biological ligand, Cu (II) is taken as a metal node to obtain L-Phe-Cu (II) coordination polymers (L-Phe-Cu (II) CPs), and Cu (II) in-situ redox signals can be obtained through FSCV; secondly, when PPi exists, PPi has stronger complexation with Cu (II) to form PPi-Cu (II) complex, and L-Phe-Cu (II) CPs are prevented from forming; and the catalytic hydrolysis of PPi by PPase leads to the hydrolysis of PPi and the release of Cu (II), and L-Phe-Cu (II) CPs are better formed; meanwhile, when NaF is added, the ability to inhibit PPase activity eventually leads to the difficulty in forming L-Phe-Cu (II) CPs. Based on the method, the quantitative relation between the current response and the concentrations of Cu (II), PPi, PPase and NaF is established by the FSCV technology, and a simple, rapid, reliable and high-sensitivity electrochemical analysis method for Cu (II), PPi and PPase is constructed. Compared with the traditional method, the method has the advantages of simple and convenient operation, low cost, good specificity, high sensitivity and accurate and reliable result.

Description

Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof
Technical Field
The invention relates to a special electrochemical rapid scanning method and application thereof, in particular to a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and application thereof in copper ion, pyrophosphate and pyrophosphatase analysis sensing.
Background
Copper is a necessary trace element in human body, and accounts for less than 0.01% of the total weight of human body, and has a very small content in human body, but has strong biological effects, such as: participate in the metabolic processes of enzymes, hormones, vitamins and nucleic acids, and the physiological functions of the medicine are mainly expressed in assisting in conveying elements such as carbon, hydrogen, oxygen, nitrogen, calcium, magnesium, sodium and the like; as a component or activator of enzymes such as ceruloplasmin, cytochrome oxidase, dopamine beta-hydroxylase, tyrosinase, etc.; influence on nucleic acid metabolism, etc. In addition, it is indicated that copper deficiency may cause many diseases, such as congenital liver copper metabolism disorder, Wilson syndrome, knee valgus, atherosclerosis, increase of incidence rate of coronary heart disease, etc., whereas excessive copper has certain toxicity, which may cause hepatitis, gastroenteritis, pneumonia, etc., so monitoring of copper ion content is very important for human health. In addition, human body contains Pyrophosphate (PPi) which has excellent binding effect with copper ion, PPi in human body is widely existed inside and outside cells, intracellular PPi is mainly from mitochondria, extracellular PPi is mainly generated by adenosine triphosphate hydrolysis, PPi abnormality is related to diseases mainly based on vascular calcium, such as cartilage calcification disease, idiopathic infantile artery calcification disease, premature senility syndrome, etc. From this point of view, PPi analytical sensing, which is closely related to copper ions, is of great significance. On the other hand, Pyrophosphatase (PPase) is a hydrolase that specifically catalyzes the hydrolysis of PPi, and it acts on an acid anhydride in a double phosphate bond to catalyze the hydrolysis of one PPi molecule into two orthophosphate (Pi), which is a key factor in controlling the intracellular PPi concentration. Copper ions, PPi and PPase are closely related, so that the development of a sensitive and rapid detection method for the activity of copper ions (Cu (II)), PPi and PPase is of great significance for researching relevant biological processes and clinically developing diseases.
In modern analysis technologies, there are spectra, mass spectra, chromatograms, fluorescence methods, colorimetry, electrochemical methods, enzymatic methods and the like to detect activities of cu (ii), PPi and PPase, wherein the electrochemical methods are widely concerned due to their unique advantages, such as cheap instruments and cost, simple operation, short detection time and the like, while the traditional electrochemical methods have good sensitivity but limited stability and accuracy, in order to overcome the two defects, the subject group carries out a series of researches, and designs circuit elements by modifying and designing the electrochemical detection technology and the information engineering technology to construct a set of novel rapid scanning device, wherein the most classical method is rapid scanning cyclic voltammetry (FSCV), which is a variant of Cyclic Voltammetry (CV), is an electrochemical method presented at a high scanning speed for researching neurotransmitters and trace detection at specific positions, not only can the detection sensitivity be improved, but also the reliability of the analysis result can be increased. FSCV is a powerful electrochemical analysis technique, the most common method being the combination of electrodes with specially designed potentiostats, which allow the applied potential to be scanned at high scan speeds, yielding sub-second time resolution and sub-micron spatial resolution. The continuous development of FSCV solves the analysis challenge caused by biological needs, and can realize large-electrode application analysis, multi-station measurement and the like by combining with other technologies in the future.
The invention designs a new biosensing method based on a rapid scanning cyclic voltammetry technology and is applied to the activity analysis of Cu (II), PPi and PPase. The synthesized material is a new coordination polymer (L-Phe-Cu (II) CPs) formed by connecting inorganic metal ions Cu (II) and an organic ligand L-phenylalanine through coordination bonds, and the coordination polymer can be well combined with cucurbituril [7] (CB [7], a supermolecule compound with a hydrophobic cavity and a polar carbonyl port with certain sizes) through hydrogen bonds, coordination and ion-dipole effects so as to be attached to the surface of an electrode, and an electrochemical rapid scanning signal is generated through in-situ redox of copper. Based on the above, the patent constructs a novel biosensing platform aiming at three targets of Cu (II), PPi and PPase activity: (1) due to the strong binding capacity of PPi and Cu (II), after PPi and Cu (II) form a complex, the formation of L-Phe-Cu (II) CPs can be blocked, and the FSCV response of Cu (II) can be inhibited; (2) the introduction of PPase makes PPi hydrolyzed to Pi, PPi and Cu (II) complex unable to form, thereby facilitating the formation of L-Phe-Cu (II) CPs and leading to better FSCV response of Cu (II); (3) when the inhibitor NaF is added, PPase activity is inhibited, PPi cannot be catalyzed and hydrolyzed, L-Phe-Cu (II) CPs are finally prevented from being synthesized, and the screening of the PPase inhibitor is realized. At present, the reports of the electrochemical rapid scanning technology are rare, particularly, a high-sensitivity and high-selectivity analysis sensing method for constructing Cu (II), PPi and PPase activities by utilizing the electrochemical rapid scanning technology does not exist, the method has better innovation, and a new thought and a new method are provided for detecting metal ions, biological micromolecules and macromolecules in the cross fields of chemistry, medicine and biology.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of an electrochemical biosensor for detecting the activities of Cu (II), PPi and PPase based on a rapid scanning cyclic voltammetry technology and a coordination polymer, which has the advantages of convenient operation, high sensitivity, high specificity and low cost.
The technical scheme adopted by the invention for solving the technical problems is as follows: a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof comprise the following specific steps:
(1) preparation of L-Phe-Cu (II) CPs
And mixing 0.1-1 mL of L-phenylalanine solution with the concentration of 1-5 mu M and 0.1-1 mL of copper sulfate pentahydrate solution with the concentration of 10-50 mu M to obtain 0.1-2 mL of solution, and oscillating the solution vigorously at room temperature for 5-15 min to obtain the L-Phe-Cu (II) CPs.
(2) Preparation of electrochemical biosensor
a, Au: polishing and grinding a gold electrode (Au, the diameter of which is 2mm) on chamois leather for 1-5 min by using 1, 0.3 and 0.05 mu m of alumina powder in sequence, then ultrasonically washing the chamois leather for 1-5 min by using water and ethanol in sequence, and then drying the chamois leather by using nitrogen for later use, wherein the electrode is marked as Au.
CB [7 ]/Au: au is immersed in 0.1-0.2 mL cucurbituril [7] water solution (CB [7]) with concentration of 0.1-2 mM for 10-15 h, and then the electrode is rinsed slowly with ultrapure water to remove unbound CB [7], the electrode is labeled as CB [7 ]/Au.
CPs/CB [7 ]/Au: then, 5-10 μ L of L-Phe-Cu (II) CPs synthesized in step (1) is dropped on the surface of CB < 7 >/Au, and is kept stand for 0.1-2 h at room temperature, and the electrode is rinsed with ultrapure water to remove unbound CPs, and the electrode is marked as CPs/CB < 7 >/Au. And then scanning by using an electrochemical rapid scanning cyclic voltammetry (FSCV), setting the initial potential to be-1.5-0V, the termination potential to be 0.2-0.6V, and the scanning speed to be 10-400V/s, and using PBS (0.2M, pH 7.4) as an electrolyte solution.
Cu (ii) assay detection: in the step (1), the concentration (final concentration range: 0-1000 nM) of Cu (II) is changed, coordination polymerization is carried out on the Cu (II) and L-Phe according to different proportions to form a plurality of L-Phe-Cu (II) CPs, and the step (2) is kept unchanged, so that a series of Cu (II) sensors can be obtained and applied to Cu (II) sensing analysis.
And (3) analyzing and detecting PPi:
updating the step (1) as follows: sequentially adding 50-100 mu L of PPi solution with the final concentration of 0-50 nM and 50-100 mu L of Cu (II) solution with the final concentration of 0-100 nM, reacting for 0.1-2 h at room temperature, adding 50-100 mu L of L-Phe solution with the final concentration of 0-11.1 nM, and adding distilled water to prepare 100-200 mu L of solution. And (3) keeping the step (2) unchanged, and obtaining a series of PPi sensors based on the step (2) and applying the series of PPi sensors to PPi sensing analysis.
And (3) PPase analysis and detection:
updating the step (1) as follows: sequentially adding 5-10 mU L of PPase with the final concentration of 0-300 mU/mL, 5-10 mU L of PPi with the final concentration of 0-50 nM, 5-10 mU L of Cu (II) solution with the final concentration of 0-100 nM, 5-10 mU L of Mg (II) solution with the final concentration of 0-50 nM and 5-10 mU L of LTris-HCl buffer solution (1mM, pH 7.4), and vigorously shaking for 1-5 min to uniformly mix the solutions. Incubating the solution at 25-50 ℃ for 1-5 h, placing the solution in a water bath at 80-100 ℃ to inactivate the enzyme, and cooling the solution to room temperature. And then adding 5-10 mu L of L-Phe solution with the final concentration of 0-11.1 nM into the mixed solution, adding distilled water to prepare 50-100 mu L of solution, uniformly mixing, incubating at 25-50 ℃ for 5-15 min, and refrigerating and storing in a refrigerator at 4 ℃. And (3) keeping the step (2) unchanged, obtaining a series of PPase sensors based on the step, and applying the series of PPase sensors to PPase sensing analysis.
By utilizing the rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof, the rapid scanning cyclic voltammetry is adopted, the initial potential is set to be-1.5-0V, the final potential is set to be 0.2-0.6V, the scanning speed is 10-400V/s, various prepared modified electrodes are used as working electrodes, platinum electrodes are used as auxiliary electrodes, Ag/AgCl is used as a reference electrode, PBS (0.2M, pH 7.4) is used as an electrolyte solution to measure FSCV electrochemical signals corresponding to the activities of Cu (II), PPi and PPase with different concentrations, and the quantitative relation between the concentration and the electrochemical response is established.
The invention principle is as follows: the invention relates to a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof. Fixing a coordination polymer on the surface of an electrode through excellent combination of L-Phe and CB [7], obtaining an electrochemical signal of Cu (II) through an FSCV technology, changing the concentration of Cu (II), and changing the electrochemical signal, thereby obtaining a series of FSCV signal sizes corresponding to Cu (II) with different concentrations and realizing Cu (II) electrochemical detection; secondly, the complexation of Cu (II) and PPi and the catalytic hydrolysis of PPase to PPi are utilized to change the concentration of PPi or PPase, and the FSCV signal is also changed, so that a series of FSCV signal sizes corresponding to PPi (PPase) with different concentrations can be obtained; finally, when the inhibitor NaF is added, the PPase activity is well inhibited, PPi cannot be hydrolyzed into Pi, and L-Phe-Cu (II) CPs cannot be formed, so that the screening of the PPase inhibitor is realized. And establishing a quantitative relation between the current response and the concentrations of Cu (II), PPi, PPase and NaF by using an FSCV technology, and determining the contents of Cu (II), PPi, PPase and NaF in the sample to be detected according to the relation between the current response and the concentrations of Cu (II), PPi, PPase and NaF. Based on the aspects, a simple, rapid, reliable and high-sensitivity electrochemical analysis method for Cu (II), PPi and PPase is constructed.
Compared with the traditional low-scanning-rate electrochemical technology, the invention has the advantages that: the invention constructs a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof. First, a series of sensors were prepared by synthesizing an L-Phe-Cu (II) coordination polymer through coordination of L-Phe with Cu (II). Clearly, within a certain range, the greater the cu (ii) concentration, the more pronounced the FSCV response; the greater the PPi concentration, the more cu (ii) bound, the less cu (ii) free, the less coordination polymer formed, and the smaller the current response; after the PPase is introduced, the higher the concentration of the PPase, the more PPi capable of catalyzing hydrolysis, the more free Cu (II), and a large amount of L-Phe-Cu (II) coordination polymer can be generated with L-Phe, and obvious FSCV response is generated. In addition, in a fixed potential window, the number of electrons obtained and lost when all Cu (II) undergoes redox is a fixed value, namely the electric quantity Q, when the sweep rate is increased, the time t required by the test is reduced, and the conclusion that the corresponding current response is larger can be obtained according to the formula Q ═ it, so that the detection sensitivity is greatly improved. Experimental results show that the FSCV response magnitude and logarithm values of Cu (II), PPi and PPase concentrations are in a linear relation in a certain range, and the analysis and detection of Cu (II), PPi and PPase are well realized. The advantages are that:
(1) the sensor mechanism is novel: synthesizing coordination polymers (L-Phe-Cu (II) CPs) by adopting L-Phe as a biological ligand and Cu (II) as a metal node based on coordination between the L-Phe and the Cu (II); fixing coordination polymer on the surface of an electrode through excellent combination of L-Phe and CB [7], obtaining an electrochemical signal of Cu (II) through an FSCV technology, and constructing a series of sensors by utilizing the relation among Cu (II), PPi, PPase and NaF.
(2) The preparation is simple, the cost is low, the L-Phe and Cu (II) are common biological micromolecules and metal ions, and the synthesis steps of the coordination polymer are simple. The method has the advantages of low reagent consumption and convenient operation in the preparation and detection processes of the sensor, and can realize high-sensitivity detection on Cu (II), PPi and PPase by consuming a small amount of materials and reagents.
(3) The result has high reliability, the FSCV technology with high scanning speed can promote the in-situ oxidation reduction of Cu (II), the FSCV signal output is very stable, the repeatability is excellent, and the accuracy is excellent.
(4) The sensitivity is high, the invention utilizes the rapid scanning cyclic voltammetry technology to obtain the detection limit of Cu (II) of 1.3 multiplied by 10-11The detection limit of M, PPi is 3.3 multiplied by 10-12The detection limit of M and PPase is 0.033 mU/mL.
(5) The universality is good, and for Cu (II) detection: other reference substances Ag (I), Cd (II), Hg (II), Pb (II), Zn (II) and Mn (II), because other metal ions can also be coordinated and combined with phenylalanine, when other metal ions replace Cu (II) to carry out in-situ electrochemical signal output, FSCV signals with different sizes can be generated, and the universality of the system is shown.
(6) Good specificity, and detection of PPi: other control substances Na2CO3、NaHCO3、Na3PO4、Na2HPO4And NaH2PO4No interference is caused to the system; and (3) detecting PPase: other control substances such as Bovine Serum Albumin (BSA), glucose oxidase (GOx), Streptavidin (SA), Lysozyme (Lysozyme) and Trypsin (Trypsin) were not interfering with the system.
In conclusion, the invention constructs a rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof, has the advantages of simple operation, rapid analysis, high sensitivity, good selectivity, strong specificity, low cost and the like, can realize the detection of Cu (II), PPi and PPase with lower concentration, and has good application prospect.
Drawings
FIG. 1 is a diagram of a feasibility analysis of a sensor according to the present invention;
FIG. 2 is a graph of the linear dependence of the sensor response to Cu (II) current versus log concentration according to the present invention;
FIG. 3 is a graph of the linearity of the current response of a sensor of the present invention to PPi versus the log of concentration;
FIG. 4 is a graph of the current response of a sensor of the present invention to PPase and its inhibitor NaF versus the log concentration;
FIG. 5 is an experimental diagram of the sensor's universality to Cu (II);
FIG. 6 is a graph showing the selectivity of the sensor of the present invention to PPi and PPase;
FIG. 7 is a diagram of the anti-interference experiment of the sensor of the present invention on PPi and PPase.
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
EXAMPLE 1 preparation of L-Phe-Cu (II) CPs
Coordination Polymers (CPs) were prepared by sequentially taking an L-phenylalanine solution (1. mu.M, 0.1mL) and a copper sulfate pentahydrate solution (10. mu.M, 0.1 mL). Oscillating it vigorously at room temperature for 15min to obtain L-Phe-Cu (II) CPs.
EXAMPLE 2 preparation of electrochemical biosensor
a, Au: polishing and grinding a gold electrode (Au, the diameter of which is 2mm) on chamois leather for 2min by using 1, 0.3 and 0.05 mu m of alumina powder in sequence, then ultrasonically washing for 5min by using water and ethanol in sequence, and then blow-drying by using nitrogen for later use, wherein the electrode is marked as Au.
CB [7 ]/Au: au was immersed in 0.2mL of 1mM cucurbituril [7] in water (CB [7]) for 12h, and then the electrode, labeled CB [7]/Au, was slowly rinsed with ultrapure water to remove unbound CB [7 ].
CPs/CB [7 ]/Au: then, 5. mu.L of L-Phe-Cu (II) CPs synthesized in step (1) was dropped on the surface of CB [7]/Au, allowed to stand at room temperature for 1 hour, and the electrode, labeled as CPs/CB [7]/Au, was rinsed with ultrapure water to remove unbound CPs. And then scanning by using electrochemical Fast Scanning Cyclic Voltammetry (FSCV), setting a potential window to be-1.2-0.2V, and a scanning speed to be 200V/s, and using PBS (0.2M, pH 7.4) as an electrolyte solution.
Feasibility experiments:
d. as in example 1 above, 100. mu.L of PPi solutions (final concentration: 0 to 50nM) of different final concentrations and 50. mu.L of Cu (II) solution of 20nM final concentration were added in sequence, and reacted at room temperature for 1 hour, and 50. mu.L of L-Phe solution of 2.2nM final concentration and distilled water were added to prepare 200. mu.L of solution. Then 5 μ L of the above mixture was dropped on the electrode surface modified with CB [7], and the mixture was allowed to stand at room temperature for 1 hour, and the electrode surface was slowly rinsed with ultrapure water to remove unbound molecules. Other experimental steps were maintained, based on which a series of PPi sensors could be derived and applied to PPi sensing analysis.
e. mu.L of PPase solutions with different final concentrations (final concentration: 0-300 mU/mL), 10. mu.L of PPi with 10nM final concentration, 10. mu.L of Cu (II) solution with 20nM final concentration, 10. mu.L of Mg (II) solution with 5nM final concentration, 10. mu.L of Tris-HCl buffer (1mM, pH 7.4) and vigorously shaking for 5min are added in sequence to mix the solutions uniformly. The solution was incubated at 37 ℃ for 3h, then placed in a water bath at 85 ℃ to inactivate the enzyme, and cooled to room temperature. Then, 10. mu.L of L-Phe solution with a final concentration of 2.2nM was added to the above mixed solution, and distilled water was added to prepare 100. mu.L of solution, which was mixed well, incubated at 37 ℃ for 15min, and stored in a refrigerator at 4 ℃. Varying the PPase concentration enables a range of ALP sensors to be derived based thereon and applied to ALP sensing analysis.
As shown in FIG. 1, the experimental phenomenon shows that CPs modified c-electrode and PPase modified e-electrode have good FSCV electrochemical response, while bare gold a-electrode, CB [7] modified b-electrode and PPi modified d-electrode have almost no response. This demonstrates that the presence of copper ions in CPs is critical for the generation of electrochemical signals; on the other hand, the strong complexing ability of PPi with Cu (II) and the catalytic hydrolysis ability of PPase on PPi were also demonstrated, thus demonstrating that the experiment is theoretically and technically feasible.
Cu (ii) assay detection:
according to the preparation steps of the sensors in the steps of the example 1 and the example 2 c, the concentration of Cu (II) (final concentration: 0-1000 nM) is changed, and coordination polymerization is carried out on the concentration and the L-Phe according to different proportions to form a plurality of L-Phe-Cu (II) CPs, and the experimental step of the example 2 c is kept unchanged, so that a series of Cu (II) sensors can be obtained and applied to Cu (II) sensing analysis. The relation between the logarithm of Cu (II) concentration and the current is shown in figure 2, in a certain concentration range, the higher the Cu (II) concentration is, the more obvious the electrochemical signal is, the current response of the sensor to Cu (II) has a good linear relation with the logarithm of the concentration, and the linear range is 4.0 multiplied by 10-11～2.5×10-7M, limit of detection 1.3X 10-11M。
And (3) analyzing and detecting PPi:
a series of PPi sensors based on the above-described sensor preparation procedure of example 1 and d of example 2 were obtained by varying the PPi concentration (final concentration: 0 to 50nM) and applied to PPi sensing analysis. FSCV results are shown in FIG. 3, where the greater the PPi concentration, the more subtle the electrochemical signalWeak, the current response of the sensor to PPi is in good linear relation with the logarithm of the concentration, and the linear range is 1.0 multiplied by 10-11～1.5×10-8M, limit of detection is 3.3 × 10-12M。
PPase analysis detection and inhibitor screening:
for PPase: a series of PPase sensors based on the above steps of example 1 and the sensor preparation step of example 2 e were obtained by varying the concentration of PPase (final concentration: 0-300 mU/mL) and applied to PPase sensor analysis. As shown in FIG. 4A, in a certain concentration range, the larger the PPase concentration is, the stronger the electrochemical signal is, the good linear relationship between the current response of the sensor to the PPase and the logarithm of the concentration is, the linear range is 0.1-75 mU/mL, and the detection limit is 0.033 mU/mL.
For the inhibitor NaF: according to the preparation steps of the sensors of the above examples 1 and 2, 10. mu.L of NaF with a final concentration of 0 to 2000. mu.M and 10. mu.L of PPase with a final concentration of 150mU/mL are shaken at 37 ℃ for 5min to be uniformly mixed. Then, 10. mu.L of PPi with a final concentration of 10nM, 10. mu.L of Cu (II) solution with a final concentration of 20nM, 10. mu.L of Mg (II) solution with a final concentration of 5nM, and 10. mu.L of Tris-HCl buffer (1mM, pH 7.4) were added in this order, and the solution was mixed well by vigorous shaking for 5 min. The solution was incubated at 37 ℃ for 3h, then placed in a water bath at 85 ℃ to inactivate the enzyme, and cooled to room temperature. Then, 10. mu.L of L-Phe solution with a final concentration of 2.2nM was added to the above mixed solution, and distilled water was added to prepare 100. mu.L of solution, which was mixed well and incubated at 37 ℃ for 15 min. 5 μ L of the solution prepared above was dropped on the electrode surface modified with CB < 7 >, and the electrode surface was left to stand at room temperature for 1 hour, and then slowly rinsed with ultrapure water to remove unbound molecules. The results are shown in FIG. 4B, and it can be seen that the current response is reduced with the increase of the NaF concentration of the inhibitor, which indicates that NaF has good inhibitory effect on PPase activity, the half inhibitory concentration is 11.8 μ M/12.5 μ M, and PPase inhibitor screening is realized.
Example 3 Universal detection
To verify the universality of the sensor, the control substances of Cu (II) used in the system universality experiments are Ag (I), Cd (II), Hg (II), Pb (II), Zn (II) and Mn (II) with the same concentrations according to the preparation steps of the sensor in the above example 1 and example 2, the FSCV electrochemical response of the detection electrode is detected, and as a result, as shown in FIG. 5, other metal ions can be found to be coordinated and combined with phenylalanine and generate FSCV signals with different sizes.
Example 4 Selective detection
To verify the specificity of the sensor, the reagents used in the PPi selectivity experiments were the same concentration of Na according to the sensor preparation procedure of example 1 and example 2 above2CO3、NaHCO3、Na3PO4、Na2HPO4And NaH2PO4The FSCV electrochemical response of the electrode is detected. As a result, as shown in fig. 6A, it can be found that only PPi can bind cu (ii) to make the electrochemical signal very weak, while other salts have no effect on the system; the reagents used in the PPase selectivity experiment are Bovine Serum Albumin (BSA), glucose oxidase (GOx), Streptavidin (SA), Lysozyme (Lysozyme) and Trypsin (Trypsin) with the same concentration, and the specificity of the PPase of the sensor is detected by adopting FSCV. The results are shown in fig. 6B, and it can be seen that the electrochemical signals of the sensor are weak for other enzymes compared with PPase, indicating that the sensor has good specificity for PPase.
Example 5 tamper resistance detection
Following the sensor preparation procedure of examples 1 and 2 above, different salts were used in combination with PPi in PPi anti-interference experiments (Blank, PPi + Na, respectively)2CO3、PPi+NaHCO3、PPi+Na3PO4、PPi+Na2HPO4PPi+NaH2PO4) A series of electrochemical sensors were prepared, the results of the FSCV test are shown in FIG. 7A, and the sensors had substantially no difference in the FSCV electrochemical signals of the 5 interfering systems and the PPi-only system, except for the blank control, indicating that no interference was observed in the detection of PPi by other salts. In PPase anti-interference experiments, different biomolecules and PPase combinations (including Blank, PPase + BSA, PPase + GOx, PPase + SA, PPase + Lysozyme and PPase + Trypsin) are used to prepare a series of electrochemical sensors, the FSCV test result is shown in figure 7B, and the sensors are used for detecting 5 interference systems and electricity only with the PPase system except for Blank controlThere was essentially no difference in chemical signal, indicating that other biomolecules did not interfere with the detection of PPase.
It should be noted that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art will appreciate that various modifications, adaptations, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (3)

1. A rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof are disclosed, the mechanism is as follows: firstly, L-Phe is taken as a biological ligand, Cu (II) is taken as a metal node, and coordination polymers (L-Phe-Cu (II) CPs) are synthesized based on coordination between the L-Phe and the Cu (II). Fixing the coordination polymer on the surface of an electrode through the excellent combination effect of L-Phe and CB [7], obtaining an electrochemical signal of Cu (II) through an FSCV technology, changing the concentration of Cu (II), and changing the electrochemical signal, so as to obtain a series of FSCV signal sizes corresponding to Cu (II) with different concentrations, and realize Cu (II) electrochemical detection; secondly, the complexation of Cu (II) and PPi and the catalytic hydrolysis of PPase to PPi are utilized to change the concentration of PPi or PPase, and the FSCV signal is also changed, so that a series of FSCV signal sizes corresponding to PPi (PPase) with different concentrations can be obtained; finally, when the inhibitor NaF is added, the PPase activity is well inhibited, PPi cannot be hydrolyzed into Pi, and L-Phe-Cu (II) CPs cannot be formed, so that the screening of the PPase inhibitor is realized. And establishing a quantitative relation between the current response and the concentrations of Cu (II), PPi, PPase and NaF by using an FSCV technology, and determining the contents of Cu (II), PPi, PPase and NaF in the sample to be detected according to the relation between the current response and the concentrations of Cu (II), PPi, PPase and NaF. Based on the aspects, a simple, rapid, reliable and high-sensitivity electrochemical analysis method for Cu (II), PPi and PPase is constructed.
2. The rapid scanning sensing method and the biological application thereof based on the copper ion in-situ electrochemical signal output according to claim 1, characterized in that: the rapid scanning cyclic voltammetry technology, the coordination effect of L-Phe and Cu (II), the excellent combination effect of L-Phe and CB [7], the complex reaction of Cu (II) and PPi, the enzymatic reaction of PPi/PPase and the like are combined for the first time, and the sensitive and efficient analysis and detection of Cu (II), PPi and PPase are realized through the in-situ electrochemical signal output of copper ions.
3. The rapid scanning sensing method based on copper ion in-situ electrochemical signal output and the biological application thereof according to claims 1-2, characterized in that: setting a potential window of-1.2-0.2V and a sweep rate of 200V/s, analyzing and detecting Cu (II), PPi and PPase with different concentrations by using a rapid scanning cyclic voltammetry technology, and establishing a quantitative relation between the concentration of a target object and an FSCV signal-11The detection limit of M and PPi is 3.3 multiplied by 10-12M, ALP detection limit was 0.033 mU/mL.
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