CN114518397B - Construction method and application of electrochemical detection device for trace elements in milk powder - Google Patents
Construction method and application of electrochemical detection device for trace elements in milk powder Download PDFInfo
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Abstract
The invention belongs to the technical field of food quality safety detection, and particularly relates to a construction method and application of an electrochemical detection device for trace elements in milk powder; the method mainly comprises the steps of preparing a rod-shaped MOF probe and constructing an integrated electrochemical detection device; based on the preparation of the rod-shaped MOF probe, the application of the MOF biomimetic enzyme with high adsorption capacity and multiple surface active sites in electrochemical detection is realized, and the MOF biomimetic enzyme has the advantages of stronger current signal and more accurate measurement result; the construction of the integrated electrochemical detection device realizes micro-nano of the electrochemical DNA sensor and the integration of the sensor and the detection circuit, and has the advantages of simple and convenient operation, portability and capability of carrying out real-time micro-area quantitative analysis on trace elements.
Description
Technical Field
The invention belongs to the technical field of food quality safety detection, and particularly relates to a construction method and application of an electrochemical detection device for trace elements in milk powder.
Background
The milk powder can become a kind of high-nutrition and multi-effect food, on one hand, because the milk powder contains rich protein, carbohydrate, vitamin and other essential nutrient components, and on the other hand, the milk powder contains copper, zinc and other trace elements essential to life activities. However, the trace element level in liquid milk materials such as cow's milk or sheep's milk is difficult to meet the needs of special people, so that a certain amount of trace elements are usually added in the milk powder processing process to improve the nutritional value of the milk powder. Wherein, the trace elements added in the feeding link are uniformly distributed in the raw materials, which directly affects the quality of the milk powder; meanwhile, when trace elements are excessively added, normal metabolism of consumers can be disturbed and even various diseases can be caused. The national standard infant formula for food safety issued by the Ministry of health of China (GB 10765-2021) clearly specifies the content standard of different microelements in infant formula milk powder: the content of zinc in each 100mL of infant formula milk powder is 0.3-1.05 mg, and the content of copper is 36-84 mug. Therefore, in the processing process of the liquid milk raw material, the uniform and quantitative feeding of the microelements in each micro-area is strictly controlled, and the method has remarkable economic and social values for guaranteeing the product quality and further promoting the sustainable development of the dairy industry in China. Therefore, the development of a sensitive and rapid analysis method for realizing in-situ micro-area monitoring of the feeding level of microelements in the milk powder production process is particularly important.
The electrochemical sensor combines the electrochemical high-sensitivity characteristic with the specificity of sensing analysis, and provides a very potential method for detecting microelements in a milk powder complex matrix. In the construction process of an electrochemical sensing system, the signal probe plays an important role in realizing the specific identification and in-situ catalysis of the object to be detected. However, the biological enzyme for preparing the signal probe is easy to inactivate and has poor stability, and the requirements of trace element real-time monitoring in the links of homogenizing, pasteurizing, spray drying and the like in the milk powder processing process are difficult to be met. In recent years, metal-organic framework Materials (MOFs) have the advantages of strong stability and simple preparation, and are widely applied to sensor construction. However, the existing spherical MOF bionic enzyme has a limited number of surface active sites, so that the general popularization of the spherical MOF bionic enzyme in electrochemical sensing sensitive analysis is affected; meanwhile, the pretreatment process of the working electrode for electrochemical determination is complex and a special signal output device is needed, so that the application of the technology in real-time and in-situ detection of a multi-component sample is limited to a great extent.
Disclosure of Invention
Aiming at the problems, the rod-shaped MOF bionic enzyme with high adsorption capacity and multiple surface active sites is prepared, and the electrochemical micro-nano sensor assisted by the smart phone is constructed for real-time sensitive detection of micro-regions of multi-component microelements in milk powder.
In order to achieve the technical purpose, the invention comprises the following specific steps:
step one: the preparation of the rod-shaped MOF-based probe comprises the following steps:
process one: cerium nitrate and 1,3,5 trimesic acid (H) are weighed 3 BTC) is dispersed in a mixed solution of ethanol and water to obtain a mixed solution A;
and a second process: transferring the mixed solution A obtained in the first process into a reaction kettle with polytetrafluoroethylene as a lining, then placing the mixed solution A into an oil bath kettle for heating reaction, centrifuging to obtain white precipitate after the heating reaction is finished, respectively cleaning the white precipitate with deionized water and ethanol, then performing vacuum drying to obtain a rod-shaped MOF material, and finally dispersing the rod-shaped MOF material into a Tris-HCl buffer solution to obtain a rod-shaped MOF solution;
and a third process: adding a certain amount of nano gold (AuNPs) solution into the rod-shaped MOF solution prepared in the second process, stirring for a period of time, and then washing a precipitate obtained by centrifugation with ethanol to obtain an MOF composite material with the AuNPs, wherein the MOF composite material is denoted as Au@MOF material; dispersing the obtained Au@MOF material into a Tris-HCl buffer solution to obtain an Au@MOF solution;
and a process IV: determining the types of trace elements according to the requirements to select DNA2 containing corresponding specific recognition sites; diluting the DNA2 stock solution with sterilized water to obtain DNA2 diluent, and adding the DNA2 diluent into tris- (2-formylethyl) phosphonium hydrochloride (TCEP) solution for activating reaction for a period of time to obtain activated DNA2; then mixing the activated DNA2 with a certain amount of Au@MOF solution, and incubating at room temperature to obtain DNA2@Au@MOF mixed solution;
And a fifth process: adding bovine serum albumin into the DNA2@Au@MOF mixed solution obtained in the fourth step for reacting for a period of time, and dispersing a product obtained by centrifugation into a Tris-HCl buffer solution to obtain a rod-shaped MOF-based probe solution;
further, in the first step, the dosage ratio of cerium nitrate, 1,3,5 trimesic acid, ethanol and water is 0.01-0.05 g: 0.005-0.05 g: 10-30 mL: 10-30 mL.
Further, in the second step, the temperature of the heating reaction is 60-120 ℃ and the reaction time is 1-3 h; the temperature of the vacuum drying is 60-70 ℃; the Tris-HCl buffer solution had a concentration of 20mM and a pH of 7.4.
Further, in the third step, the volume ratio of the nano gold solution to the rod-shaped MOF solution is 2-8: 0.5 to 5; the concentration of the nano gold solution is 0.05-0.1 mg/mL; the concentration of the rod-shaped MOF solution is 50-70 mg/mL; the stirring time is 3-5 h; the centrifugation conditions are as follows: 5000-15000 rpm, 5-20 min; the amount of the Tris-HCl buffer solution is 400-600 mu L, the concentration is 20mM, and the pH is 7.4.
Further, in the fourth step, the DNA2 stock solution is an HPLC purified oligonucleotide purchased from nanjing gold stree bioengineering limited (nanjing); the concentration of the DNA2 diluent is 5-10 mu M; the concentration of TCEP is 5mM; the dosage relation of the DNA2 diluent and the TCEP is 50-400 mu L: 10-80 mu L; the activation reaction is carried out for 2-3 hours; the dosage relation of the activated DNA2 and the Au@MOF solution is 50-400 mu L, 100-800 mu L, and the concentration of the Au@MOF solution is 60-80 mg/mL; the incubation time at room temperature was 14h.
Further, the sequence of the DNA2 is determined by the kind of the detected trace element; for example, when detecting copper, a sequence of G containing a specific recognition site of copper ions is adopted; when detecting zinc, a sequence of rA containing a specific recognition site of zinc ion is used.
Further, in the fifth step, the dosage relationship of the dna2@au@mof mixed solution and the bovine serum albumin is 100 to 200 μl: 80-100 mu L of bovine serum albumin with the mass fraction of 1%; the reaction time is 0.5-1.5 h; the centrifugation conditions are as follows: 5000-15000 rpm, 5-20 min; the concentration of the Tris-HCl buffer solution is 20mM, and the pH is 7.4; the concentration of the rod-shaped MOF probe solution is 50-100 mg/mL.
Step two: the construction of the integrated electrochemical detection device comprises the following steps:
process one: preparation of screen printing electrode: overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode layer by layer on the surface of a PET substrate by utilizing a screen printing technology to obtain a screen printing electrode of a three-electrode system;
and a second process: according to the species of trace elements to be determined, selecting DNA1 containing corresponding specific recognition sites, and performing base pairing with DNA 2; after DNA1 is selected, diluting the DNA1 stock solution with sterilized water to obtain DNA1 diluent, and adding the DNA1 diluent into TCEP solution for activation reaction to obtain activated DNA1;
And a third process: immersing the screen-printed electrode in sulfuric acid (H) 2 SO 4 ) In the solution, an electrochemical workstation adopts cyclic voltammetry scanning to complete electrode activation, and deionized water is used for ultrasonic cleaning and natural airing is carried out at room temperature; then, a certain amount of AuNPs solution is dripped on the surface of the working electrode of the activated screen printing electrode, and the mixture is stood for a period of time, so that a layer of uniform gold nano film is formed on the surface of the working electrode; finally, 1 drop of activated DNA in the second process is taken to react on the surface of the AuNPs modified working electrode for a period of time, and a treated working electrode is obtained;
and a process IV: dripping the rod-shaped MOF-based probe solution in the first step on the surface of the working electrode after the treatment in the third step, incubating for a period of time at room temperature, and then cleaning with Tris-HCl buffer solution to remove unbound probes, so as to obtain an electrochemical micro-nano sensor;
and a fifth process: and (3) constructing an integrated electrochemical detection device:
the integrated electrochemical detection device consists of an electrochemical micro-nano sensor, a power supply module, an impedance converter module, a microcontroller module and a Bluetooth module; the electrochemical micro-nano sensor is electrically connected with the power supply module, the power supply module is electrically connected with the impedance converter module, the impedance converter module is electrically connected with the microcontroller module, the microcontroller module is electrically connected with the Bluetooth module, the Bluetooth module is connected with the intelligent terminal, and the control is realized through signal input and output among the Bluetooth module and the intelligent terminal, so that the integrated electrochemical detection device is jointly formed.
Preferably, in the first step of the second step, the reference electrode of the screen printing electrode is a 1/6 ring, the auxiliary electrode is a 2/3 ring, the openings are opposite, the middle area is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not contacted with each other; the conductive silver rail is three parallel conductive strips, wherein one end of each of the three conductive strips is respectively connected with the working electrode, the auxiliary electrode and the reference electrode; and (3) each layer of printing is dried in a drying oven at 70 ℃, the next layer of printing is dried and cooled, and finally, the rectangular insulating ink pattern is obtained after the printing insulating ink is dried, and the rectangular insulating ink covers the printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of the conductive silver rail.
Further, in the second step, the DNA1 is an HPLC purified oligonucleotide purchased from Nanjing Jinsri bioengineering Co., ltd. (Nanj); the concentration of the DNA1 diluent is 0.5-5 mu M; the concentration of TCEP is 5mM; the time of the activation reaction is 2-3 hours; the dosage relation of the DNA1 diluent and the TCEP is 50-400 mu L: 10-80 mu L;
further, the sequence of the DNA1 is determined by the detected trace element species and the DNA2 together; for example, when detecting copper, sequences containing G for the specific recognition site of copper ion are used, and selected sequences can be linked to base pairing in DNA 2; when detecting zinc, a sequence of rA containing a specific recognition site for zinc ions is used, and the selected sequence can be linked to base pairing in DNA 2;
Further, in the third step of the second step, H is 2 SO 4 The concentration of the solution is 1mol/L; the dosage of the AuNPs solution is 1-10 mu L, and the concentration is 0.05mg/mL; the dosage of the DNA1 solution is 1-10 mu L; standing for 0.5-5 h; the reaction time is 0.5-3 h.
Further, in the fourth step, the amount of the rod-shaped MOF probe solution is 1 to 10 mu L; the incubation time is 0.5-5 h.
The invention also provides an application of the electrochemical detection device for detecting trace elements in milk powder, which specifically comprises the following steps:
process one: firstly, preparing a series of microelement standard solutions with the concentration of Q respectively 1 、Q 2 、Q 3 、……、Q n-1 、Q n N concentration gradients in total, n being a positive integer; then respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the step twoIn the standard solution, one device corresponds to one concentration of solution;
and a second process: the intelligent terminal is connected with the microcontroller module through Bluetooth, electrochemical detection software installed on the intelligent terminal is opened, start-stop voltage for controlling the electrochemical micro-nano sensor is set on the software, a corresponding command is generated by the microcontroller module, and the command is transmitted to the impedance converter module and further signals are sent to the electrochemical micro-nano sensor; the electrochemical micro-nano sensor obtains preset voltage, a generated current response value is measured, the current response value is collected by the impedance converter module and is transmitted back to the intelligent terminal through the microcontroller module and Bluetooth, then a standard curve is established according to the correlation between the concentration of trace elements and the response current, the equation of the standard curve is y=ax+b, wherein x represents the logarithm of the concentration of the trace elements, y represents the response current value, and a and b are constants;
And a third process: firstly preparing sample liquid, immersing an electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the sample liquid, substituting a response current value of the trace element in the sample liquid measured on electrochemical detection software into the mathematical model to calculate the concentration of the trace element according to the mathematical model established in the second step, and thus realizing in-situ quantitative detection of the trace element in the sample liquid.
Further, the concentration of the trace elements in the first process is in the range of 0 to 3 multiplied by 10 -7 M; the trace elements include copper or zinc.
Further, in the second process, the electrochemical detection software includes PSTrace, PSTouch, CHI or GPES; the intelligent terminal comprises a mobile phone, a computer or a tablet personal computer.
After the result is measured, the judgment can be carried out according to the national standard, and the trace elements of copper and zinc are taken as examples: if the measured copper content m Cu Below 5.6X10) -6 M, zinc content M Zn Below 4.6X10) -5 M, the content of trace elements in the milk powder is lower than the national standard, and corresponding trace element salt solution is required to be continuously added in the processing process; if the measured copper content m Cu Higher than 1.3X10 -5 M, zinc content M Zn Higher than 1.6X10 -4 M indicates that the trace elements in the milk powder containThe amount is higher than the national standard, and the corresponding raw milk should be added in the processing process.
Compared with the prior art, the invention has the beneficial effects that:
(1) The rod-shaped MOF material provided by the invention has the characteristics of high adsorption capacity, high stability, multiple surface active sites and the like.
(2) The sensor combines MOF in-situ catalysis and DNA specific recognition, and can effectively overcome the interference of other components in the complex milk powder matrix.
(3) The electrochemical micro-nano electrode is combined with the intelligent terminal equipment, so that portability and practicability of electrochemical measurement are improved.
(4) The electrochemical detection equipment provided by the invention can realize real-time detection of micro-areas of multi-component microelements and simultaneous determination of different areas.
(5) According to the method, by measuring the dynamic change of the trace elements in the processing process of the liquid milk raw material, the feeding and distribution conditions of the trace elements are evaluated in situ rapidly, so that a manufacturer is facilitated to adjust the processing procedure of the milk powder in time, and meanwhile, the real-time rapid analysis method is improved for quality supervision and supervision of the milk powder quality.
Drawings
FIG. 1 is a scanning electron microscope image of a rod-shaped MOF.
FIG. 2 is a transmission electron micrograph of a rod MOF.
Fig. 3 is a schematic diagram of a screen printing electrode modification process, wherein 1 is a working electrode, 2 is a reference electrode, 3 is an auxiliary electrode, and 4 is a conductive silver rail.
FIG. 4 is a schematic diagram of a smart phone assisted integrated electrochemical detection device; wherein 9 is an electrochemical micro-nano sensor, 5 is a power module, 6 is an impedance converter module, 7 is a microcontroller module, and 8 is a Bluetooth module.
Detailed Description
The invention is further described below with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto; the milk powder used in this example was purchased from a flying crane official flagship, and specifically was a flying crane star sail infant formula milk powder.
Example 1:
step one: the preparation of the rod-shaped MOF-based probe comprises the following steps:
process one: weigh 0.01g cerium nitrate and 0.005. 0.005g H 3 BTC is dispersed in a mixed solution of 10mL of ethanol and 10mL of water to obtain a mixed solution A;
and a second process: transferring the mixed solution A into a reaction kettle with polytetrafluoroethylene as a lining, putting the reaction kettle into an oil bath kettle, heating the reaction kettle at 60 ℃ for 3 hours, centrifuging to obtain white precipitate after heating, respectively centrifuging and cleaning the white precipitate by deionized water and ethanol, vacuum drying the white precipitate at 60 ℃ to obtain a rod-shaped MOF material, and finally dispersing the rod-shaped MOF material into a 400 mu L, pH 7.4.4 Tris-HCl (20 mM) buffer solution to obtain a rod-shaped MOF solution;
and a third process: adding 2mL of nano gold (AuNPs) solution with the concentration of 0.05mg/mL into 0.5mL of rod-shaped MOF solution, stirring for 3h, and then washing a precipitate obtained after centrifugation (5000 rpm,20 min) with ethanol to obtain an MOF composite material with the AuNPs, which is marked as Au@MOF material; dispersing the obtained Au@MOF material into 400 mu L of Tris-HCl (20 mM) buffer solution with pH of 7.4 to obtain Au@MOF solution, and storing at 4 ℃ for later use;
And a process IV: determining the types of trace elements according to the requirements to select DNA2 containing corresponding specific recognition sites; after DNA2 was selected, the DNA2 stock solution was diluted with sterilized water to give a DNA2 dilution with a concentration of 5. Mu.M, and the 50. Mu.L of the DNA2 dilution was added to 10. Mu.L of a 5mM TCEP solution and activated for 2 hours to give activated DNA2. Mixing 50 mu L of activated DNA2 with 100 mu L of Au@MOF solution, and incubating for 14 hours at room temperature to obtain DNA2@Au@MOF mixed solution; wherein the DNA2 sequence for detecting copper is 3' -HS- (CH) 2 ) 6 GATTATGCCTGGCGCTTCTCGTTTTTTACGAGAAG-5'; the DNA2 sequence for detecting zinc is 5' -AATGCTTCCGTCCACCrAGGAAGCCACCACGATT- (CH) 2 ) 6 -SH-3’；
And a fifth process: to 100. Mu.L of DNA2@Au@MOF mixture was added 80. Mu.L of 1% by mass bovine serum albumin to react for 0.5h, and the resultant was centrifuged (5000 rpm,20 min) to obtain a product which was dispersed in 500. Mu.L of Tris-HCl (20 mM) buffer solution to obtain a rod-like MOF-based probe solution, and stored at 4℃for use.
FIG. 1 is a scanning electron microscope image of a rod-shaped MOF material, from which it can be seen that the rod-shaped MOF material has a smooth surface and exhibits a more regular rod-shaped structure.
FIG. 2 is a transmission electron micrograph of a rod-shaped MOF material with an average diameter of about 100nm.
Step two: the construction of the integrated electrochemical detection device comprises the following steps:
Process one: preparation of screen printing electrode: overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode layer by layer on the surface of a PET substrate by utilizing a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is a 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings are opposite, the middle area is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not contacted with each other; the conductive silver rail is three parallel conductive strips, wherein one end of each of the three conductive strips is respectively connected with the working electrode, the auxiliary electrode and the reference electrode; each layer is printed, the printed layers are dried in a drying oven at 70 ℃, the next layer is printed after drying and cooling, and finally, rectangular insulating ink patterns are obtained after printing insulating ink and drying, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of conductive silver tracks;
and a second process: according to the species of trace elements to be determined, selecting DNA1 containing corresponding specific recognition sites, and performing base pairing with DNA 2; diluting the DNA1 stock solution with sterilized water to obtain 0.5. Mu.M DNA1 dilution, adding 50. Mu.L of the DNA1 dilution to 10. Mu.L of 5mM TCEP solution, and activating for 2 hours to obtain activated DNA1; wherein the DNA1 sequence for detecting copper is 5' -HS- (CH) 2 ) 6 AGCAAGCTAATACGGCTTATTTCTCGTTCG-3'; the DNA1 sequence for detecting zinc is 5' -CCACCACCCTCTCTTAATGCTTCCGACCrAGGAAGAGATGGTGGTGG- (CH) 2 ) 6 -SH-3’；
And a third process: the screen-printed electrode was immersed in 5mL of 1mol/L H 2 SO 4 In the solution, an electrochemical workstation adopts cyclic voltammetry scanning to complete electrode activation, and deionized water is used for ultrasonic cleaning and natural airing at room temperature; screen printing after activationDropwise adding 1 mu L of AuNPs solution on the surface of a working electrode of the electrode, and standing for 0.5h, so that a layer of uniform gold nano film is formed on the surface of the working electrode; finally, taking 1 mu L of activated DNA1 solution drops in the second process to react on the surface of the AuNPs modified working electrode for 0.5h to obtain a treated working electrode;
and a process IV: dripping 1 mu L of the rod-shaped MOF probe solution prepared in the step one on the surface of the working electrode after the treatment of the step three, incubating for 0.5h at room temperature, and then washing with Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in figure 3);
and a fifth process: and (3) constructing an integrated electrochemical detection device: the detection device consists of an electrochemical micro-nano sensor 9, a power module 5, an impedance converter module 6, a microcontroller module 7 and a Bluetooth module 8; the electrochemical micro-nano sensor 9 is electrically connected with the power module 5, the power module 5 is electrically connected with the impedance converter module 6, the impedance converter module 6 is electrically connected with the microcontroller module 7, and the microcontroller module 7 is electrically connected with the Bluetooth module 8; the whole detection device is connected with the smart phone through the Bluetooth module 8, and is controlled through signal input and signal output, so that the detection device is jointly constructed into an integrated electrochemical detection device (shown in fig. 4).
Step three: the establishment and application of the microelement mathematical model comprise the following processes:
process one: preparing a series of copper ion standard solutions with the concentration of 0 and 1 multiplied by 10 respectively -13 M、1×10 -12 M、1×10 -11 M、1×10 -10 M、1×10 -9 M、1×10 -8 M、1×10 -7 M; immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the standard solution respectively, wherein one device corresponds to one concentration solution;
and a second process: firstly, PSTouch software at the smart phone end is opened, the PSTouch software is connected with a micro-controller module 7 through Bluetooth, start-stop voltage for controlling an electrochemical micro-nano sensor 9 is set on the software, a corresponding command is generated by the micro-controller module 7, and the command is transmitted to an impedance converter module 6 and further sensed to the electrochemical micro-nano sensorThe device 9 sends out a signal; the electrochemical micro-nano sensor 9 then obtains preset voltage to obtain a response current value of copper ions, the response current value is collected by the impedance converter module 6 and is transmitted back to the smart phone through the microcontroller module 7 and the Bluetooth module 8, and a mathematical model y=1.5x+22.73 (R 2 ＝0.9986)；
According to the same operation of the first and second processes in the third step, the difference is that the trace element zinc is measured; finally, a mathematical model y=10.531x+141.34 (R) is constructed based on the trace element zinc ion concentration and current value 2 ＝0.9923)；
The mathematical model of other microelements can also be constructed according to the operations of the first and second processes in the third step;
and a third process: firstly, accurately brewing purchased milk powder according to a specification, immersing an electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step, and taking the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the response current values according to the mathematical model established in the second step; the results show that m Cu 9.2X10 -6 M，m Zn 8.1X10 times -5 M, namely the content of copper and zinc is within a specified range.
Example 2:
step one: the preparation of the rod-shaped MOF-based probe comprises the following steps:
process one: weigh 0.03g cerium nitrate and 0.025g H 3 The BTC is dispersed in a mixed solution of 20mL of ethanol and 20mL of water to obtain a mixed solution A;
and a second process: transferring the mixed solution A into a reaction kettle with polytetrafluoroethylene as a lining, putting the reaction kettle into an oil bath kettle, heating the reaction kettle at 90 ℃ for 2 hours, respectively cleaning white precipitate obtained by centrifugation after heating by deionized water and ethanol, vacuum-drying the obtained rod-shaped MOF material at 65 ℃, and dispersing the rod-shaped MOF material into 500 mu L of Tris-HCl (20 mM) buffer solution with pH of 7.4 to obtain rod-shaped MOF solution;
And a third process: adding 5mL of nano gold (AuNPs) solution with the concentration of 0.08mg/mL into 0.3mL of rod-shaped MOF solution, stirring for 4h, and then washing a precipitate obtained after centrifugation (10000 rpm,15 min) with ethanol to obtain an MOF composite material with the AuNPs, which is marked as Au@MOF material; dispersing the obtained Au@MOF material into 500 mu L of Tris-HCl (20 mM) buffer solution with pH of 7.4 to obtain Au@MOF solution, and storing at 4 ℃ for later use;
and a process IV: determining the types of trace elements according to the requirements to select DNA2 containing corresponding specific recognition sites; after DNA2 was selected, the DNA2 stock solution was diluted with sterilized water to give a DNA2 dilution with a concentration of 8. Mu.M, and 200. Mu.L of the DNA2 dilution was added to 50. Mu.L of a 5mM TCEP solution and activated for 2.5 hours to give activated DNA2. 200 mu L of activated DNA2 is mixed with 500 mu L of Au@MOF solution, and incubated for 14 hours at room temperature to obtain DNA2@Au@MOF mixed solution; wherein the DNA2 sequence for detecting copper is 3' -HS- (CH) 2 ) 6 The DNA2 sequence of-GTAATTGCCTGGCGCTTCTCGTTTTTTACG AGAAG-5 'zinc detection is 5' -AATCGTTCCGGAGGrACCAAGCCACCACGAT- (CH) 2 ) 6 -SH-3’；
And a fifth process: to 150. Mu.L of DNA2@Au@MOF mixture was added 90. Mu.L of 1% bovine serum albumin to react for 1h, and the resultant was centrifuged (10000 rpm,15 min) to obtain a product which was dispersed in 500. Mu.L of Tris-HCl (20 mM) buffer solution to obtain a rod-like MOF-based probe solution, and stored at 4℃for use.
Step two: the construction of the integrated electrochemical detection device comprises the following steps:
process one: preparation of screen printing electrode: overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode layer by layer on the surface of a PET substrate by utilizing a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is a 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings are opposite, the middle area is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not contacted with each other; the conductive silver rail is three parallel conductive strips, wherein one end of each of the three conductive strips is respectively connected with the working electrode, the auxiliary electrode and the reference electrode; each layer is printed, the printed layers are dried in a drying oven at 70 ℃, the next layer is printed after drying and cooling, and finally, rectangular insulating ink patterns are obtained after printing insulating ink and drying, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of conductive silver tracks;
and a second process: according to the species of trace elements to be determined, selecting DNA1 containing corresponding specific recognition sites, and performing base pairing with DNA 2; diluting the DNA1 stock solution with sterilized water to obtain 3. Mu.M DNA1 dilution, adding 200. Mu.L of the DNA1 dilution to 50. Mu.L of 5mM TCEP solution, and activating for 2.5 hours to obtain activated DNA1; wherein the DNA1 sequence for detecting copper is 5' -HS- (CH) 2 ) 6 The DNA1 sequence of-AGCAAGCATTAACGGCTTATTTCTCGTTCG-3 'zinc detection is 5' -CCACCAGCCTCTCTTAATGCTTCCGAGGrACCAAGAGATGGTGGTGG- (CH) 2 ) 6 -SH-3’；
And a third process: the screen-printed electrode was immersed in 5mL of 1mol/L H 2 SO 4 In the solution, an electrochemical workstation adopts cyclic voltammetry scanning to complete electrode activation, and deionized water is used for ultrasonic cleaning and natural airing at room temperature; dripping 5 mu L of AuNPs solution on the surface of the working electrode of the activated screen printing electrode, and standing for 3 hours, so that a layer of uniform gold nano film is formed on the surface of the working electrode; finally, taking 5 mu L of activated DNA1 solution drop to react on the surface of the AuNPs modified working electrode for 1.5 hours to obtain a treated working electrode;
and a process IV: dropwise adding 5 mu L of the rod-shaped MOF-based probe solution prepared in the step one on the surface of a working electrode after treatment, incubating for 3 hours at room temperature, and then washing with Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in figure 3);
and a fifth process: and (3) constructing an integrated electrochemical detection device: the detection device consists of an electrochemical micro-nano sensor 9, a power module 5, an impedance converter module 6, a microcontroller module 7 and a Bluetooth module 8; the electrochemical micro-nano sensor 9 is electrically connected with the power module 5, the power module 5 is electrically connected with the impedance converter module 6, the impedance converter module 6 is electrically connected with the microcontroller module 7, and the microcontroller module 7 is electrically connected with the Bluetooth module 8; the whole detection device is connected with the smart phone through the Bluetooth module 8, and is controlled through signal input and output between the detection device and the smart phone, so that the detection device is formed into an integrated electrochemical detection device (shown in fig. 4);
Step three: the establishment and application of the microelement mathematical model comprise the following processes:
process one: preparing a series of copper ion standard solutions with the concentration of 0 and 2 multiplied by 10 respectively -13 M、2×10 -12 M、2×10 -11 M、2×10 -10 M、2×10 -9 M、2×10 -8 M、2×10 -7 M; immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the standard solution respectively, wherein one device corresponds to one concentration solution;
and a second process: firstly, PSTouch software of a smart phone end is opened, the PSTouch software is connected with a micro-controller module 7 through Bluetooth, start-stop voltage for controlling an electrochemical micro-nano sensor 9 is set on the software, a corresponding command is generated by the micro-controller module 7, and the command is transmitted to an impedance converter module 6 and further signals are sent to the electrochemical micro-nano sensor 9; the electrochemical micro-nano sensor 9 then obtains preset voltage to obtain a response current value of copper ions, the response current value is collected by the impedance converter module 6 and is transmitted back to the smart phone through the microcontroller module 7 and the Bluetooth module 8, and a mathematical model y=1.478x+20.15 (R 2 ＝0.9985)；
According to the same operation as the first and second processes in the third step, the trace element zinc is measured, and finally, a mathematical model y=10.531x+141.34 (R 2 ＝0.9923)；
And a third process: firstly, accurately brewing purchased milk powder according to a specification, then immersing an electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the milk powder, and taking the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the response current values according to the mathematical model established in the second step. The results show that m Cu 9.8X10 -6 M，m Zn Is 1.1X10 -4 M, namely the content of copper and zinc is within a specified range.
Example 3:
step one: the preparation of the rod-shaped MOF-based probe comprises the following steps:
Procedureand (3) a step of: weigh 0.05g cerium nitrate and 0.05g H 3 The BTC is dispersed in a mixed solution of 30mL of ethanol and 30mL of water to obtain a mixed solution A;
and a second process: transferring the mixed solution A into a reaction kettle with polytetrafluoroethylene as a lining, putting the reaction kettle into an oil bath kettle, heating the reaction kettle at 120 ℃ for 3 hours, respectively cleaning white precipitate obtained by centrifugation after heating by deionized water and ethanol, vacuum-drying the obtained rod-shaped MOF material at 70 ℃, and dispersing the rod-shaped MOF material into 600 mu L of Tris-HCl (20 mM) buffer solution with pH of 7.4 to obtain rod-shaped MOF solution;
and a third process: adding 8mL of nano gold (AuNPs) solution with the concentration of 0.1mg/mL into 5mL of rod-shaped MOF solution, stirring for 5h, and then washing a precipitate obtained after centrifugation (15000 rpm,5 min) with ethanol to obtain an MOF composite material with the AuNPs, which is marked as Au@MOF material; dispersing the obtained Au@MOF material into 600 mu L of Tris-HCl (20 mM) buffer solution with pH of 7.4 to obtain Au@MOF solution, and storing at 4 ℃ for later use;
And a process IV: determining the types of trace elements according to the requirements to select DNA2 containing corresponding specific recognition sites; after DNA2 was selected, the DNA2 stock solution was diluted with sterilized water to give a DNA2 dilution with a concentration of 10. Mu.M, and 400. Mu.L of the DNA2 dilution was added to 80. Mu.L of a 5mM TCEP solution and activated for 3 hours to give activated DNA2. Mixing 400 mu L of activated DNA2 with 800 mu L of Au@MOF solution, and incubating for 14h at room temperature to obtain DNA2@Au@MOF mixed solution; wherein the DNA2 sequence for detecting copper is 3' -HS- (CH) 2 ) 6 The DNA2 sequence of-GTAATTGCGTGGCGCTTCTCGAATTTTACG AGAAG-5 'zinc detection is 5' -AATCCTCTCCGGRAGGRACCACACCCACGCGAT- (CH) 2 ) 6 -SH-3’；
And a fifth process: to 200. Mu.L of DNA2@Au@MOF mixture was added 100. Mu.L of 1% bovine serum albumin to react for 1.5 hours, and the resultant product was dispersed in 500. Mu.L of Tris-HCl (20 mM) buffer solution by centrifugation (15000 rpm,5 min) to give a rod-like MOF-based probe solution, and stored at 4℃for use.
Step two: the construction of the integrated electrochemical detection device comprises the following steps:
process one: preparation of screen printing electrode: overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode layer by layer on the surface of a PET substrate by utilizing a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is a 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings are opposite, the middle area is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not contacted with each other; the conductive silver rail is three parallel conductive strips, wherein one end of each of the three conductive strips is respectively connected with the working electrode, the auxiliary electrode and the reference electrode; each layer is printed, the printed layers are dried in a drying oven at 70 ℃, the next layer is printed after drying and cooling, and finally, rectangular insulating ink patterns are obtained after printing insulating ink and drying, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of conductive silver tracks;
And a second process: according to the species of trace elements to be determined, selecting DNA1 containing corresponding specific recognition sites, and performing base pairing with DNA 2; diluting the DNA1 stock solution with sterilized water to obtain 5. Mu.M DNA1 dilution, adding 400. Mu.L of the DNA1 dilution to 80. Mu.L of 5mM TCEP solution, and activating for 3 hours to obtain activated DNA1; wherein the DNA1 sequence for detecting copper is 5' -HS- (CH) 2 ) 6 The DNA1 sequence of-AGCAAGCATTAACGGCTTATTGCTCGTTCG-3 'zinc detection is 5' -CCACCragCCTCCTCTAATGCTTCCGAGGrACCAAGAGATGGTGGTGG- (CH) 2 ) 6 -SH-3’；
And a third process: the screen-printed electrode was immersed in 5mL of 1mol/L H 2 SO 4 In the solution, an electrochemical workstation adopts cyclic voltammetry scanning to complete electrode activation, and deionized water is used for ultrasonic cleaning and natural airing at room temperature; dripping 10 mu L of AuNPs solution on the surface of the working electrode of the activated screen printing electrode, and standing for 5 hours, so that a layer of uniform gold nano film is formed on the surface of the working electrode; finally, taking 10 mu L of activated DNA1 solution drop to react on the surface of the AuNPs modified working electrode for 3 hours to obtain a treated working electrode;
and a process IV: dripping 10 mu L of the rod-shaped MOF-based probe solution prepared in the step one on the surface of a working electrode after treatment, incubating for 5 hours at room temperature, and then washing with Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in figure 3);
And a fifth process: and (3) constructing an integrated electrochemical detection device: the detection device consists of an electrochemical micro-nano sensor 9, a power module 5, an impedance converter module 6, a microcontroller module 7 and a Bluetooth module 8; the electrochemical micro-nano sensor 9 is electrically connected with the power module 5, the power module 5 is electrically connected with the impedance converter module 6, the impedance converter module 6 is electrically connected with the microcontroller module 7, and the microcontroller module 7 is electrically connected with the Bluetooth module 8; the whole detection device is connected with the smart phone through the Bluetooth module 8, and is controlled through signal input and output between the detection device and the smart phone, so that the detection device is formed into an integrated electrochemical detection device (shown in fig. 4);
step three: the establishment and application of the microelement mathematical model comprise the following processes:
process one: preparing a series of copper ion standard solutions with the concentration of 0 and 3 multiplied by 10 respectively -13 M、3×10 -12 M、3×10 -11 M、3×10 -10 M、3×10 -9 M、3×10 -8 M、3×10 -7 M; immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the standard solution respectively, wherein one device corresponds to one concentration solution;
and a second process: firstly, PSTouch software of a smart phone end is opened, the PSTouch software is connected with a micro-controller module 7 through Bluetooth, start-stop voltage for controlling an electrochemical micro-nano sensor 9 is set on the software, a corresponding command is generated by the micro-controller module 7, and the command is transmitted to an impedance converter module 6 and further signals are sent to the electrochemical micro-nano sensor 9; the electrochemical micro-nano sensor 9 then obtains a preset voltage to obtain a response current value of copper ions, the response current value is collected by the impedance converter module 6 and is transmitted back to the smart phone through the microcontroller module 7 and the Bluetooth module 8, and a mathematical model y=1.512x+19.84 (R 2 ＝0.9983)；
The same operation as in the first and second processes in the third step is performed except that the trace element zinc is measured, and finally, a mathematical model y=10.4815x+140 is constructed based on the concentration of the trace element zinc ion and the current value.87(R 2 ＝0.9921)；
And a third process: firstly, accurately brewing purchased milk powder according to a specification, then immersing an electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the milk powder, and taking the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the response current values according to the mathematical model established in the second step. The results show that m Cu Is 1.1X10 -5 M，m Zn 9.8X10 -5 M, namely the content of copper and zinc is within a specified range.
In summary, the invention is a high-sensitivity and high-specificity portable electrochemical sensor constructed based on a rod-shaped MOF material as a bionic catalyst, and is combined with a smart phone to be used for real-time in-situ micro-area detection of trace elements in milk powder. The detection technology is simple and convenient to operate, the microelements in the micro-area can be quantitatively analyzed, and meanwhile, the established multichannel micro-nano sensing system can realize the detection of various microelements and the simultaneous multi-area measurement of samples. The sensing system can monitor the trace element condition in the milk powder processing process in real time, thereby providing basis for the milk powder production enterprises to adjust the processing technology in time; meanwhile, a real-time rapid analysis method is provided for milk powder quality supervision and supervision departments, and the method has important economic value and social significance.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (1)
1. The application of the electrochemical detection device for detecting trace elements in milk powder is characterized by comprising the following steps:
step one: preparing a rod-shaped MOF probe;
process one: cerium nitrate and 1,3,5 trimesic acid are weighed and dispersed in a mixed solution of ethanol and water to obtain a mixed solution A;
and a second process: transferring the mixed solution A obtained in the first process into a reaction kettle with polytetrafluoroethylene as a lining, then placing the mixed solution A into an oil bath kettle for heating reaction, centrifuging to obtain white precipitate after the heating reaction is finished, respectively cleaning the white precipitate with deionized water and ethanol, then performing vacuum drying to obtain a rod-shaped MOF material, and finally dispersing the rod-shaped MOF material into a Tris-HCl buffer solution to obtain a rod-shaped MOF solution;
and a third process: adding a certain amount of nano gold solution into the rod-shaped MOF solution prepared in the second process, stirring for a period of time, and then washing a precipitate obtained by centrifugation with ethanol to obtain an MOF composite material with AuNPs, wherein the MOF composite material is denoted as Au@MOF material; dispersing the obtained Au@MOF material into a Tris-HCl buffer solution to obtain an Au@MOF solution;
And a process IV: determining the types of trace elements according to the requirements to select DNA2 containing corresponding specific recognition sites; diluting the DNA2 stock solution with sterilized water to obtain DNA2 diluent, and adding the DNA2 diluent into tris- (2-formylethyl) phosphorus hydrochloride solution for activating reaction for a period of time to obtain activated DNA2; then mixing the activated DNA2 with a certain amount of Au@MOF solution, and incubating at room temperature to obtain DNA2@Au@MOF mixed solution;
and a fifth process: adding bovine serum albumin into the DNA2@Au@MOF mixed solution obtained in the fourth step for reacting for a period of time, and dispersing a product obtained by centrifugation into a Tris-HCl buffer solution to obtain a rod-shaped MOF-based probe solution;
step two: constructing an integrated electrochemical detection device;
process one: preparation of screen printing electrode: overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode layer by layer on the surface of a PET substrate by utilizing a screen printing technology to obtain a screen printing electrode of a three-electrode system;
and a second process: according to the species of trace elements to be determined, selecting DNA1 containing corresponding specific recognition sites, and performing base pairing with DNA2; after DNA1 is selected, diluting the DNA1 stock solution with sterilized water to obtain DNA1 diluted solution, wherein the concentration is 0.5-5 mu M; adding the DNA1 diluent into TCEP solution for activation reaction for 2-3 h to obtain activated DNA1; in the second step, the DNA1 is an oligonucleotide purified by HPLC; the concentration of TCEP is 5mM; the dosage relation of the DNA1 diluent and the TCEP is 50-400 mu L: 10-80 mu L;
The sequence of the DNA1 is determined by the detected trace element species and the DNA2 together; for example, when detecting copper, sequences containing G for the specific recognition site of copper ion are used, and selected sequences can be linked to base pairing in DNA 2; when detecting zinc, a sequence of rA containing a specific recognition site for zinc ions is used, and the selected sequence can be linked to base pairing in DNA 2;
and a third process: immersing the screen-printed electrode in sulfuric acid (H) 2 SO 4 ) In the solution, an electrochemical workstation adopts cyclic voltammetry scanning to complete electrode activation, and deionized water is used for ultrasonic cleaning and natural airing is carried out at room temperature; then, a certain amount of AuNPs solution is dripped on the surface of the working electrode of the activated screen printing electrode, and the mixture is stood for a period of time, so that a layer of uniform gold nano film is formed on the surface of the working electrode; finally, 1 drop of activated DNA in the second process is taken to react on the surface of the AuNPs modified working electrode for a period of time, and a treated working electrode is obtained;
and a process IV: dripping the rod-shaped MOF-based probe solution prepared in the step one on the surface of the working electrode after the treatment of the step three, incubating for a period of time at room temperature, and then cleaning with Tris-HCl buffer solution to remove unbound probes, so as to obtain an electrochemical micro-nano sensor;
And a fifth process: and (3) constructing an integrated electrochemical detection device:
the integrated electrochemical detection device consists of an electrochemical micro-nano sensor, a power supply module, an impedance converter module, a microcontroller module and a Bluetooth module; the electrochemical micro-nano sensor is electrically connected with the power supply module, the power supply module is electrically connected with the impedance converter module, the impedance converter module is electrically connected with the microcontroller module, the microcontroller module is electrically connected with the Bluetooth module, the Bluetooth module is connected with the intelligent terminal, and the control is realized through the mutual signal input and output, so that the integrated electrochemical detection device is jointly formed;
step three: detecting;
process one: first, a first is preparedSeries of microelement standard solutions with the concentration of Q respectively 1 、Q 2 、Q 3 、……、Q n-1 、Q n N concentration gradients in total, n being a positive integer; then, respectively immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the standard solution, wherein one device corresponds to one concentration solution;
and a second process: the intelligent terminal is connected with the microcontroller module through Bluetooth, electrochemical detection software installed on the intelligent terminal is opened, start-stop voltage for controlling the electrochemical micro-nano sensor is set on the software, a corresponding command is generated by the microcontroller module, and the command is transmitted to the impedance converter module and further signals are sent to the electrochemical micro-nano sensor; the electrochemical micro-nano sensor obtains preset voltage, a generated current response value is measured, the current response value is collected by the impedance converter module and is transmitted back to the intelligent terminal through the microcontroller module and Bluetooth, then a standard curve is established according to the correlation between the concentration of trace elements and the response current, the equation of the standard curve is y=ax+b, wherein x represents the logarithm of the concentration of the trace elements, y represents the response current value, and a and b are constants;
And a third process: firstly preparing a sample liquid, immersing an electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the sample liquid, substituting a response current value of the trace element in the sample liquid measured on electrochemical detection software into the mathematical model to calculate the concentration of the trace element according to the mathematical model established in the second step, and thus realizing in-situ quantitative detection of the trace element in the sample liquid;
in the first step, the dosage ratio of cerium nitrate, 1,3,5 trimesic acid, ethanol and water is 0.01-0.05 g: 0.005-0.05 g: 10-30 mL: 10-30 mL;
in the second process, the temperature of the heating reaction is 60-120 ℃ and the reaction time is 1-3 h; the temperature of the vacuum drying is 60-70 ℃; the concentration of the Tris-HCl buffer solution is 20mM, and the pH is 7.4;
in the third process, the volume ratio of the nano gold solution to the rod-shaped MOF solution is 2-8: 0.5 to 5; the concentration of the nano gold solution is 0.05-0.1 mg/mL; the concentration of the rod-shaped MOF solution is 50-70 mg/mL; the stirring time is 3-5 h; the centrifugation conditions are as follows: 5000-15000 rpm, 5-20 min; the dosage of the Tris-HCl buffer solution is 400-600 mu L, the concentration is 20mM, and the pH is 7.4;
In the fourth step, the DNA2 stock solution is the oligonucleotide purified by HPLC; the concentration of the DNA2 diluent is 5-10 mu M; the concentration of TCEP is 5mM; the dosage relation of the DNA2 diluent and the TCEP is 50-400 mu L: 10-80 mu L; the activation reaction is carried out for 2-3 hours; the dosage relation of the activated DNA2 and the Au@MOF solution is 50-400 mu L, 100-800 mu L, and the concentration of the Au@MOF solution is 60-80 mg/mL; the incubation time at room temperature is 14h;
the sequence of the DNA2 is determined by the detected trace element; for example, when detecting copper, a sequence of G containing a specific recognition site of copper ions is adopted; when detecting zinc, adopting a sequence rA containing a zinc ion specific recognition site;
in the fifth process, the dosage relationship of the DNA2@Au@MOF mixed solution and the bovine serum albumin is 100-200 mu L: 80-100 mu L of bovine serum albumin with the mass fraction of 1%; the reaction time is 0.5-1.5 h; the centrifugation conditions are as follows: 5000-15000 rpm, 5-20 min; the concentration of the Tris-HCl buffer solution is 20mM, and the pH is 7.4; the concentration of the rod-shaped MOF probe solution is 50-100 mg/mL;
in the first step, the reference electrode of the screen printing electrode is a 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings are opposite, the middle area is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not contacted with each other; the conductive silver rail is three parallel conductive strips, wherein one end of each of the three conductive strips is respectively connected with the working electrode, the auxiliary electrode and the reference electrode; each layer is printed, the printed layers are dried in a drying oven at 70 ℃, the next layer is printed after drying and cooling, and finally, rectangular insulating ink patterns are obtained after printing insulating ink and drying, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of conductive silver tracks; procedure three, H 2 SO 4 The concentration of the solution is 1mol/L; the dosage of the AuNPs solution is 1-10 mu L, and the concentration is 0.05mg/mL; the saidThe dosage of the DNA1 solution is 1-10 mu L; standing for 0.5-5 h; the reaction time is 0.5-3 h; in the fourth process, the dosage of the rod-shaped MOF probe solution is 1-10 mu L; the incubation time is 0.5-5 h;
the concentration range of the trace elements in the first step of the third step is 0-3 multiplied by 10 -7 M; the microelements comprise copper or zinc; in the second process, the electrochemical detection software comprises PSTrace, PSTouch, CHI or GPES; the intelligent terminal comprises a mobile phone, a computer or a tablet personal computer.
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