CN114518397A - 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 PDF

Info

Publication number
CN114518397A
CN114518397A CN202210040769.0A CN202210040769A CN114518397A CN 114518397 A CN114518397 A CN 114518397A CN 202210040769 A CN202210040769 A CN 202210040769A CN 114518397 A CN114518397 A CN 114518397A
Authority
CN
China
Prior art keywords
solution
mof
concentration
trace elements
electrode
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210040769.0A
Other languages
Chinese (zh)
Other versions
CN114518397B (en
Inventor
张新爱
周悦
石吉勇
邹小波
黄晓玮
胡雪桃
李志华
翟晓东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu University
Original Assignee
Jiangsu University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu University filed Critical Jiangsu University
Priority to CN202210040769.0A priority Critical patent/CN114518397B/en
Publication of CN114518397A publication Critical patent/CN114518397A/en
Application granted granted Critical
Publication of CN114518397B publication Critical patent/CN114518397B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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 MOF biomimetic enzyme with high adsorption capacity and multiple surface active sites in electrochemical detection is realized, and the method has the advantages of stronger current signals and more accurate determination results; the construction of the integrated electrochemical detection device realizes the micro-nano of the electrochemical DNA sensor and the integration of the sensor and a 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

Construction method and application of electrochemical detection device for trace elements in milk powder
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 be a high-nutrition multifunctional food, on one hand, the milk powder contains rich nutritional ingredients such as protein, carbohydrate, vitamins and the like which are necessary for human bodies, and on the other hand, the milk powder contains trace elements such as copper, zinc and the like which are indispensable to life activities. However, the trace element level in liquid milk raw materials such as cow milk or goat milk is difficult to meet the needs of special people, so a certain amount of trace elements are usually added in the milk powder processing process to improve the nutritional value. Wherein, the uniform distribution degree of the added trace elements in the raw materials in the feeding link can directly influence the quality of the milk powder; meanwhile, when the trace elements are added in excess, the normal metabolism of consumers can be disturbed and even various diseases can be caused. The infant formula food of national food safety standard issued by the ministry of health of China (GB 10765-2021) definitely stipulates the content standards of different trace elements in the infant formula milk powder: in the ready-to-eat state of the infant formula milk powder, the content of zinc in each 100mL is 0.3-1.05 mg, and the content of copper is 36-84 mu g. Therefore, in the processing process of the liquid milk raw material, the uniform and quantitative feeding of the trace elements in each micro-area is strictly controlled, so that the method has obvious economic and social values for ensuring the product quality and further promoting the sustainable development of the dairy industry in China. Therefore, it is particularly important to develop a sensitive and rapid analysis method to realize in-situ micro-area monitoring of the feeding level of trace elements in the milk powder production process.
The electrochemical sensor combines the high sensitivity of electrochemistry with the specificity of sensing analysis, and provides a potential method for detecting trace elements in the complex matrix of the milk powder. In the construction process of an electrochemical sensing system, the signal probe plays an important role in realizing the specific recognition 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 requirement of monitoring trace elements in real time in links of homogenization, pasteurization, spray drying and the like in the milk powder processing process is difficult to meet. 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 number of active sites on the surface of the existing spherical MOF (metal organic framework) biomimetic enzyme is limited, so that the general popularization of the spherical MOF biomimetic enzyme in electrochemical sensing sensitive analysis is influenced; meanwhile, the pretreatment process of the working electrode for electrochemical determination is complex and needs a special signal output device, so that the application of the technology in real-time and in-situ detection of multi-component samples is limited to a great extent.
Disclosure of Invention
Aiming at the problems, the invention prepares the rod-shaped MOF biomimetic enzyme with high adsorption capacity and many surface active sites, and constructs the electrochemical micro-nano sensor assisted by the smart phone for real-time sensitive detection of micro-regions of multi-component trace elements in milk powder.
In order to achieve the technical purpose, the method comprises the following specific steps:
the method comprises the following steps: the preparation of the rod-shaped MOF probe comprises the following processes:
the first process is as follows: weighing cerium nitrate and 1,3, 5-benzenetricarboxylic acid (H)3BTC) 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 step into a reaction kettle with polytetrafluoroethylene as an inner lining, putting the reaction kettle into an oil bath pot for heating reaction, centrifuging after the heating reaction is finished to obtain a white precipitate, respectively washing with deionized water and ethanol, then carrying out 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;
the third process: adding a certain amount of nanogold (AuNPs) solution into the rod-shaped MOF solution prepared in the second process, stirring for a period of time, then washing a precipitate obtained by centrifugation with ethanol, and obtaining an MOF composite material attached with AuNPs after washing, wherein the MOF composite material is marked as an Au @ MOF material; dispersing the obtained Au @ MOF material into a Tris-HCl buffer solution to obtain an Au @ MOF solution;
the process four is as follows: selecting DNA2 containing corresponding specific recognition sites according to the type of the trace elements to be detected; selecting DNA2, diluting the DNA2 stock solution with sterilized water to obtain a DNA2 diluent, and then adding the DNA2 diluent into a tris- (2-formylethyl) phosphate (TCEP) solution for activation reaction for a period of time to obtain activated DNA 2; then mixing the activated DNA2 with a certain amount of Au @ MOF solution, and incubating at room temperature to obtain a DNA2@ Au @ MOF mixed solution;
and a fifth process: adding bovine serum albumin into the DNA2@ Au @ MOF mixed solution obtained in the fourth process for reaction for a period of time, and dispersing the product obtained by centrifugation into a Tris-HCl buffer solution to obtain a rod-shaped MOF probe-based solution;
further, in the first step, the dosage ratio of the cerium nitrate, the 1,3, 5-trimesic acid, the ethanol and the water is 0.01-0.05 g: 0.005-0.05 g: 10-30 mL: 10-30 mL.
Further, in the second process of the first step, the heating reaction temperature is 60-120 ℃, and the reaction time is 1-3 hours; the temperature of the vacuum drying is 60-70 ℃; the concentration of the Tris-HCl buffer solution was 20mM, pH 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 were: 5000-15000 rpm for 5-20 min; the dosage of the Tris-HCl buffer solution is 400-600 mu L, the concentration is 20mM, and the pH value is 7.4.
Further, in the fourth step, the stock solution of DNA2 is HPLC purified oligonucleotide purchased from Nanjing Kinsley bioengineering, Inc. (Nanjing); the concentration of the DNA2 diluent is 5-10 mu M; the concentration of TCEP is 5 mM; the dosage relationship between the DNA2 diluent and TCEP is 50-400 mu L: 10-80 μ L; the activation reaction lasts for 2-3 h; the dosage relation of the activated DNA2 and the Au @ MOF solution is 50-400 muL and 100-800 muL, and the concentration of the Au @ MOF solution is 60-80 mg/mL; the incubation time at room temperature was 14 h.
Further, the sequence of the DNA2 is determined by the types of the trace elements detected; when detecting copper, adopting a sequence of G containing a specific recognition site of copper ions; when detecting zinc, the sequence of rA containing zinc ion specific recognition site is adopted.
Further, in the fifth step, the dosage relation of the DNA2@ Au @ MOF mixed solution and the bovine serum albumin is 100-200 μ L: 80-100 mu L of bovine serum albumin, wherein the mass fraction of the bovine serum albumin is 1%; the reaction time is 0.5-1.5 h; the centrifugation conditions were: 5000-15000 rpm for 5-20 min; the concentration of the Tris-HCl buffer solution is 20mM, and the pH value is 7.4; the concentration of the rod-shaped MOF probe solution is 50-100 mg/mL.
Step two: the integrated electrochemical detection device is constructed by the following processes:
the first process comprises the following steps: preparing a screen printing electrode: the method comprises the steps of overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode on the surface of a PET substrate layer by using a screen printing technology to obtain a screen printing electrode of a three-electrode system;
and a second process: DNA1 containing a corresponding specific recognition site is selected according to the kind of the trace element to be determined, and it can base-pair with DNA 2; selecting DNA1, diluting the DNA1 stock solution with sterilized water to obtain a DNA1 diluent, and adding the DNA1 diluent into a TCEP solution for activation reaction to obtain activated DNA 1;
the third process: dipping the screen-printed electrode into sulfuric acid (H)2SO4) In the solution, an electrochemical workstation is used for scanning by adopting a cyclic voltammetry method to complete electrode activation, and then deionized water is used for ultrasonic cleaning and natural drying at room temperature; then, dropwise adding a certain amount of AuNPs solution on the surface of the working electrode of the activated screen printing electrode, and standing for a period of time, thereby forming a layer of uniform gold nano-film on the surface of the working electrode; finally, dripping the activated DNA1 obtained in the second step on the surface of the AuNPs modified working electrode to react for a period of time to obtain the finished working electrode;
the process four is as follows: dropwise adding the rod-shaped MOF probe solution obtained in the first step to the surface of a working electrode treated in the third step, incubating for a period of time at room temperature, and then washing with a Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor;
and a fifth process: the integrated electrochemical detection device is constructed as follows:
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 a power module, the power module is electrically connected with an impedance converter module, the impedance converter module is electrically connected with a microcontroller module, the microcontroller module is electrically connected with a Bluetooth module, the Bluetooth module is connected with an intelligent terminal, control is realized through signal input and output between the Bluetooth module and the intelligent terminal, and the electrochemical micro-nano sensor and the power module are jointly built into an integrated electrochemical detection device.
Preferably, in the first step, the reference electrode of the screen-printed electrode is an 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings of the reference electrode and the auxiliary electrode are opposite, the middle area of the reference electrode is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not in contact with each other; the conductive silver track is three parallel conductive strips, wherein one ends of the three conductive strips are respectively connected with the working electrode, the auxiliary electrode and the reference electrode; and (3) drying one layer of the printed layer in a 70 ℃ drying oven, drying and cooling the printed layer, printing the next layer of the printed layer, and finally drying the printed insulating ink to obtain a rectangular insulating ink pattern, wherein 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 track.
Further, in the second step, the DNA1 is HPLC purified oligonucleotide and purchased from Nanjing Kinsley bioengineering, Inc. (Nanjing); the concentration of the DNA1 diluent is 0.5-5 mu M; the concentration of TCEP is 5 mM; the activation reaction time is 2-3 h; the dosage relationship between the DNA1 diluent and TCEP is 50-400 mu L: 10-80 μ L;
further, the sequence of the DNA1 is determined by the detected trace element types and the DNA 2; when copper is detected, for example, a sequence of G containing a specific recognition site for copper ions is used, and the selected sequence can be ligated to base pairing in DNA 2; when detecting zinc, a sequence of rA containing a zinc ion specific recognition site is used, and the selected sequence can be connected with base pairing in DNA 2;
further, the process of step two is the process of step three, the process of H2SO4The concentration of the solution is 1 mol/L; the above-mentionedThe dosage of the AuNPs solution is 1-10 mu L, and the concentration is 0.05 mg/mL; the using amount of the DNA1 solution is 1-10 mu L; standing for 0.5-5 h; the reaction time is 0.5-3 h.
Further, the dosage of the rod-shaped MOF probe solution in the fourth step is 1-10 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 processes:
the first process is as follows: firstly, preparing a series of trace element standard solutions with the concentrations of Q1、Q2、Q3、……、Qn-1、QnN concentration gradients, n being a positive integer; secondly, respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the second step into the standard solutions, wherein one device corresponds to a solution with one concentration;
and a second process: the intelligent terminal is connected with the microcontroller module through Bluetooth, electrochemical detection software installed on the intelligent terminal is started, starting and stopping voltage for controlling the electrochemical micro-nano sensor is set on the software, a corresponding command is generated by the microcontroller module, transmitted to the impedance converter module and further sent to the electrochemical micro-nano sensor; then, the electrochemical micro-nano sensor obtains a 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 the Bluetooth, then a standard curve is established according to the correlation between the concentration of the 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 value of the response current, and a and b are constants;
the third process: firstly preparing a sample solution, then immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step in the sample solution, and substituting the response current value of the trace elements in the sample solution measured on the electrochemical detection software into the mathematical model to calculate the concentration of the trace elements according to the mathematical model established in the second step, thereby realizing the in-situ quantitative detection of the trace elements in the sample solution.
Further, the method can be used for preparing a novel liquid crystal displayIn the first step, the concentration of the trace elements is in the range of 0 to 3X 10-7M; the trace elements include copper or zinc.
Further, 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 computer.
The determination can be carried out according to the national standard after the result is measured, and the trace elements of copper and zinc are taken as examples: if the measured copper content mCuLess than 5.6X 10-6M, zinc content MZnLess than 4.6X 10-5M, indicating that the content of the trace elements in the milk powder is lower than the national standard, and continuously adding corresponding trace element salt solution in the processing process; if the measured copper content mCuHigher than 1.3X 10-5M, zinc content MZnHigher than 1.6X 10-4And M indicates that the content of the trace elements in the milk powder is higher than the national standard, and corresponding raw milk is 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, many 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 matrix of the milk powder.
(3) The electrochemical micro-nano electrode is combined with the intelligent terminal equipment, so that the portability and the practicability of the electrochemical measurement are improved.
(4) The electrochemical detection equipment can realize the real-time detection of micro-areas of multi-component trace elements and the simultaneous determination of different areas.
(5) The method rapidly evaluates the feeding and distribution conditions of the trace elements in situ by measuring the dynamic change of the trace elements in the processing process of the liquid milk raw material, is beneficial to manufacturers to adjust the processing procedures of the milk powder in time, and simultaneously improves the real-time rapid analysis method for quality supervision and supervision of the milk powder quality.
Drawings
FIG. 1 is a scanning electron micrograph of a rod-shaped MOF.
FIG. 2 is a transmission electron micrograph of a rod-shaped 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 structure of a smart phone assisted integrated electrochemical detection device; the electrochemical micro-nano sensor is 9, the power module is 5, the impedance converter module is 6, the microcontroller module is 7, and the Bluetooth module is 8.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific embodiments, but the scope of the invention is not limited thereto; the milk powder used in this example was purchased from the official flagship store of the flying crane, specifically, the infant formula milk powder of flying crane star and flying sail.
Example 1:
the method comprises the following steps: the preparation of the rod-shaped MOF probe comprises the following processes:
the first process is as follows: 0.01g of cerium nitrate and 0.005g H g of cerium nitrate were weighed3BTC 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 an inner lining, putting the reaction kettle into an oil bath pan, heating for 3 hours at 60 ℃, after heating, respectively centrifugally cleaning white precipitates obtained by centrifugation with deionized water and ethanol, then carrying out vacuum drying at 60 ℃ to obtain a rod-shaped MOF material, and finally dispersing the rod-shaped MOF material into a Tris-HCl (20mM) buffer solution with the concentration of 400 mu L, pH 7.4.4 to obtain a rod-shaped MOF solution;
the third process: adding 2mL of nanogold (AuNPs) solution with the concentration of 0.05mg/mL into 0.5mL of rod-shaped MOF solution, stirring for 3h, then washing a precipitate product obtained after centrifugation (5000rpm for 20min) with ethanol to obtain an MOF composite material attached with AuNPs, and marking as an Au @ MOF material; dispersing the obtained Au @ MOF material into 400 mu L of Tris-HCl (20mM) buffer solution with the pH value of 7.4 to obtain Au @ MOF solution, and storing the Au @ MOF solution at 4 ℃ for later use;
the process four is as follows: selecting the specific recognition site according to the type of trace element to be detectedSpotted DNA 2; after selecting DNA2, the original DNA2 solution was diluted with sterilized water to obtain a 5. mu.M diluted DNA2 solution, and 50. mu.L of the diluted DNA2 solution was added to 10. mu.L of a 5mM TCEP solution to activate the solution for 2 hours, thereby obtaining activated DNA 2. Then mixing 50 mu L of the activated DNA2 with 100 mu L of Au @ MOF solution, and incubating for 14h at room temperature to obtain a DNA2@ Au @ MOF mixed solution; wherein the sequence of DNA2 for detecting copper is 3' -HS- (CH)2)6-GATTATGCCTGGCGCTTCTCGTTTTTTACGAGAAG-5'; the DNA2 sequence for detecting zinc is 5' -AATGCTTCCGACCrAGGAAGCCACGCGCGAG- (CH)2)6-SH-3’；
And a fifth process: mu.L of the mixture of DNA2@ Au @ MOF was reacted with 80. mu.L of bovine serum albumin at a mass concentration of 1% for 0.5h, and the product obtained by centrifugation (5000rpm, 20min) was dispersed in 500. mu.L of Tris-HCl (20mM) buffer to obtain a rod-like MOF probe-based solution, which was stored at 4 ℃ until use.
FIG. 1 is a scanning electron micrograph of a rod-shaped MOF material, from which it can be seen that the surface of the rod-shaped MOF material is smooth and exhibits a more regular rod-shaped structure.
FIG. 2 is a transmission electron micrograph of a rod-shaped MOF material having an average diameter of about 100 nm.
Step two: the integrated electrochemical detection device is constructed by the following processes:
the first process comprises the following steps: preparing a screen printing electrode: the method comprises the steps of overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode on the surface of a PET substrate layer by using a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is an 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings of the reference electrode and the auxiliary electrode are opposite, the middle area of the reference electrode is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not in contact with each other; the conductive silver track is three parallel conductive strips, wherein one ends of the three conductive strips are respectively connected with the working electrode, the auxiliary electrode and the reference electrode; drying one layer of the printed layer in a 70 ℃ drying oven, drying and cooling the printed layer, printing the next layer of the printed layer, and finally drying the printed insulating ink to obtain a rectangular insulating ink pattern, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of the conductive silver tracks;
and a second process: DNA1 containing a corresponding specific recognition site is selected according to the kind of the trace element to be determined, and it can base-pair with DNA 2; diluting the stock solution of DNA1 with sterilized water to obtain 0.5 μ M diluted solution of DNA1, adding 50 μ L diluted solution of DNA1 into 10 μ L of 5mM TCEP solution, and activating for 2h to obtain activated DNA 1; wherein the sequence of DNA1 for detecting copper is 5' -HS- (CH)2)6-AGCAAGCTAATACGGCTTATTTCTCGTTCG-3'; the DNA1 sequence for detecting zinc is 5' -CCACCACGCTCTCCTTAATGCTTCCGAGCCAGGAAGAGAGAGATGTGGG- (CH)2)6-SH-3’；
The third process: a screen-printed electrode was immersed in 5mL of 1mol/L H2SO4In the solution, an electrochemical workstation is used for scanning by adopting a cyclic voltammetry method to complete electrode activation, and then deionized water is used for ultrasonic cleaning and natural drying at room temperature; dripping 1 mu L of AuNPs solution on the surface of a working electrode of the activated screen printing electrode, and standing for 0.5h, thereby forming a layer of uniform gold nano-film on the surface of the working electrode; finally, dripping 1 mu L of the activated DNA1 solution in the second process on the surface of the AuNPs modified working electrode for reaction for 0.5h to obtain the finished working electrode;
the process four is as follows: dripping 1 μ L of the rod-shaped MOF probe solution prepared in the first step on the surface of the working electrode treated in the third step, incubating for 0.5h at room temperature, and then washing with a Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in FIG. 3);
and a fifth process: the integrated electrochemical detection device is constructed as follows: 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 realizes control through mutual signal input and output, and the whole detection device is jointly constructed into an integrated electrochemical detection device (as shown in figure 4).
Step three: the establishment and application of the microelement mathematical model comprise the following processes:
the first process comprises the following steps: preparing a series of copper ion standard solutions with the concentrations of 0 and 1 multiplied by 10 respectively-13M、1×10-12M、1×10-11M、1×10-10M、1×10-9M、1×10-8M、1×10-7M; respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the second step into the standard solutions, wherein one device corresponds to a solution with one concentration;
and a second process: firstly, opening PSTouch software at a smart phone end, connecting the PSTouch software with a microcontroller module 7 through Bluetooth, setting starting and stopping voltage for controlling an electrochemical micro-nano sensor 9 on the software, generating a corresponding command by using the microcontroller module 7, transmitting the corresponding command to an impedance converter module 6 and further sending a signal to the electrochemical micro-nano sensor 9; then the electrochemical micro-nano sensor 9 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 is established based on the concentration and the current value of the trace element copper ions to be 1.415x +22.73(R is the concentration of the trace element copper ions and the current value2＝0.9986)；
According to the same operation of the first process and the second process in the third step, the difference is that the trace element zinc is measured; finally, a mathematical model y of 10.531x +141.34 (R) is constructed based on the concentration and the current value of the trace element zinc ions2＝0.9923)；
Similarly, mathematical models of other trace elements can be constructed according to the operations of the first process and the second process in the third step;
the third process: accurately brewing the purchased milk powder according to the specification, immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step in the milk powder, and substituting the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the copper and zinc according to the mathematical model established in the second step; the results show that mCuIs 9.2X 10-6M，mZnIs 8.1 × 10-5The contents of M, i.e., copper and zinc are within the specified ranges.
Example 2:
the method comprises the following steps: the preparation of the rod-shaped MOF probe comprises the following processes:
the first process is as follows: 0.03g of cerium nitrate and 0.025g H g of cerium nitrate were weighed3BTC is dispersed in a mixed solution of 20mL ethanol and 20mL 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 for 2 hours at 90 ℃, respectively cleaning white precipitates obtained by centrifugation after heating with deionized water and ethanol, carrying out vacuum drying at 65 ℃ to obtain a rod-shaped MOF material, and dispersing into 500 mu L of Tris-HCl (20mM) buffer solution with the pH value of 7.4 to obtain a rod-shaped MOF solution;
the third process: adding 5mL of nanogold (AuNPs) solution with the concentration of 0.08mg/mL into 0.3mL of rod-shaped MOF solution, stirring for 4h, then washing a precipitate obtained after centrifugation (10000rpm, 15min) with ethanol to obtain an MOF composite material attached with AuNPs, and marking as an Au @ MOF material; dispersing the obtained Au @ MOF material into 500 mu L of Tris-HCl (20mM) buffer solution with pH7.4 to obtain Au @ MOF solution, and storing the Au @ MOF solution at 4 ℃ for later use;
the process four is as follows: selecting DNA2 containing corresponding specific recognition sites according to the type of the trace elements to be detected; after selecting DNA2, the original DNA2 solution was diluted with sterilized water to obtain 8. mu.M diluted DNA2, and 200. mu.L of the diluted DNA2 was added to 50. mu.L of 5mM TCEP solution to activate for 2.5 hours to obtain activated DNA 2. Mixing 200 mu L of activated DNA2 with 500 mu L of Au @ MOF solution, and incubating for 14h at room temperature to obtain a DNA2@ Au @ MOF mixed solution; wherein the sequence of DNA2 for detecting copper is 3' -HS- (CH)2)6-GTAATTGCCTGGCGCTTCTCGTTTTTTACG AGAAG-5 'DNA 2 sequence for detecting zinc is 5' -AATCGTTCCGAGGrACCAAGCCACGCGAG- (CH)2)6-SH-3’；
And a fifth process: mu.L of 1% bovine serum albumin was added to 150. mu.L of the mixture of DNA2@ Au @ MOF for reaction for 1 hour, and the product obtained by centrifugation (10000rpm, 15min) was dispersed in 500. mu.L of Tris-HCl (20mM) buffer to obtain a rod-like MOF probe-based solution, which was stored at 4 ℃ until use.
Step two: the integrated electrochemical detection device is constructed by the following processes:
the first process is as follows: preparing a screen printing electrode: the method comprises the steps of overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode on the surface of a PET substrate layer by using a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is an 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings of the reference electrode and the auxiliary electrode are opposite, the middle area of the reference electrode is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not in contact with each other; the conductive silver track is three parallel conductive strips, wherein one ends of the three conductive strips are respectively connected with the working electrode, the auxiliary electrode and the reference electrode; drying one layer of the printed layer in a 70 ℃ drying oven, drying and cooling the printed layer, printing the next layer of the printed layer, and finally drying the printed insulating ink to obtain a rectangular insulating ink pattern, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of the conductive silver tracks;
and a second process: DNA1 containing a corresponding specific recognition site is selected according to the kind of the trace element to be determined, and it can base-pair with DNA 2; diluting the stock solution of DNA1 with sterilized water to obtain 3 μ M diluted solution of DNA1, adding 200 μ L diluted solution of DNA1 into 50 μ L of 5mM TCEP solution, and activating for 2.5h to obtain activated DNA 1; wherein the sequence of DNA1 for detecting copper is 5' -HS- (CH)2)6-AGCAAGCATTAACGGCTTATTTCTCGTTCG-3 'DNA 1 sequence for detecting zinc is 5' -CCACCAGCCTCTTAATTGCTTCCGAGGArACCAAGAGATGTGGTGGTGG- (CH)2)6-SH-3’；
The third process: the screen-printed electrode was immersed in 5mL of 1mol/L H2SO4In the solution, an electrochemical workstation is used for scanning by adopting a cyclic voltammetry method to complete electrode activation, and then deionized water is used for ultrasonic cleaning and natural drying at room temperature; dripping 5 mu L of AuNPs solution on the surface of a working electrode of the activated screen printing electrode, and standing for 3h, thereby forming a layer of uniform gold nano-film on the surface of the working electrode; finally, 5 mu L of activated DNA1 solution is dropped on the surface of the AuNPs modified working electrode to react for 1.5h, and the finished working electrode is obtained;
the fourth process is as follows: dripping 5 mu L of the rod-shaped MOF probe solution prepared in the first step on the surface of the processed working electrode, incubating for 3h at room temperature, and then washing with a Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in figure 3);
and a fifth process: the integrated electrochemical detection device is constructed as follows: 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 realizes control through mutual signal input and output, and the whole detection device is jointly constructed into an integrated electrochemical detection device (as shown in figure 4);
step three: the establishment and application of the microelement mathematical model comprise the following processes:
the first process is as follows: preparing a series of copper ion standard solutions with the concentrations of 0 and 2 multiplied by 10 respectively-13M、2×10-12M、2×10-11M、2×10-10M、2×10-9M、2×10-8M、2×10-7M; respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the second step into the standard solutions, wherein one device corresponds to a solution with one concentration;
and a second process: firstly, opening PSTouch software at a smart phone end, connecting the PSTouch software with a microcontroller module 7 through Bluetooth, setting starting and stopping voltage for controlling an electrochemical micro-nano sensor 9 on the software, generating a corresponding command by using the microcontroller module 7, transmitting the corresponding command to an impedance converter module 6 and further sending a signal to the electrochemical micro-nano sensor 9; then the electrochemical micro-nano sensor 9 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 is established based on the concentration and the current value of the trace element copper ions, wherein the mathematical model y is 1.478x +20.15(R is the concentration of the trace element copper ions and the current value2＝0.9985)；
The same operation is carried out according to the procedures one and two in the step threeThe difference is that trace element zinc is measured, and finally, a mathematical model y is constructed based on the concentration and the current value of the trace element zinc ion, wherein the mathematical model y is 10.531x +141.34(R2＝0.9923)；
The third process: firstly, accurately brewing the purchased milk powder according to the specification, then immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the milk powder, and substituting the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the copper and zinc according to the mathematical model established in the second step. The results show that mCuIs 9.8 multiplied by 10-6M，mZnIs 1.1X 10-4The contents of M, i.e., copper and zinc are within the specified ranges.
Example 3:
the method comprises the following steps: the preparation of the rod-shaped MOF probe comprises the following processes:
the first process is as follows: 0.05g of cerium nitrate and 0.05g H g of cerium nitrate were weighed3BTC 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 pan, heating for 3 hours at 120 ℃, respectively cleaning white precipitates obtained by centrifugation after heating with deionized water and ethanol, carrying out vacuum drying at 70 ℃ to obtain a rod-shaped MOF material, and dispersing into 600 mu L of Tris-HCl (20mM) buffer solution with the pH of 7.4 to obtain a rod-shaped MOF solution;
the third process: adding 8mL of nanogold (AuNPs) solution with the concentration of 0.1mg/mL into 5mL of rod-shaped MOF solution, stirring for 5h, then centrifuging (15000rpm, 5min), and cleaning a precipitate product with ethanol to obtain an MOF composite material attached with AuNPs, wherein the MOF composite material is marked as an Au @ MOF material; dispersing the obtained Au @ MOF material into 600 mu L of Tris-HCl (20mM) buffer solution with the pH value of 7.4 to obtain Au @ MOF solution, and storing the Au @ MOF solution at 4 ℃ for later use;
the process four is as follows: selecting DNA2 containing corresponding specific recognition sites according to the type of the trace elements to be detected; after selecting DNA2, the original DNA2 solution was diluted with sterilized water to obtain a 10. mu.M diluted DNA2 solution, and the diluted DNA2 solution was added to 80. mu.L of 5mM TCEP solution to activate the solution for 3 hours to obtain activated DNA 2. 400 mu.L of the active ingredientsMixing the well-dissolved DNA2 with 800 mu L of Au @ MOF solution, and incubating for 14h at room temperature to obtain a DNA2@ Au @ MOF mixed solution; wherein the sequence of the DNA2 for detecting copper is 3' -HS- (CH)2)6-GTAATTGCGTGGCGCTTCTCGAATTTTACG AGAAG-5 'DNA 2 sequence for detecting zinc is 5' -AATCCTTCCGAGGRACCAACCACCACGCGCG- (CH)2)6-SH-3’；
And a fifth process: mu.L of 1% bovine serum albumin was added to 200. mu.L of the mixture of DNA2@ Au @ MOF for 1.5h, and the product obtained by centrifugation (15000rpm, 5min) was dispersed in 500. mu.L of Tris-HCl (20mM) buffer to obtain a rod-like MOF probe-based solution, which was stored at 4 ℃ until use.
Step two: the integrated electrochemical detection device is constructed by the following processes:
the first process is as follows: preparing a screen printing electrode: the method comprises the steps of overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode on the surface of a PET substrate layer by using a screen printing technology to obtain a screen printing electrode of a three-electrode system; the reference electrode of the screen printing electrode is an 1/6 circular ring, the auxiliary electrode is a 2/3 circular ring, the openings of the reference electrode and the auxiliary electrode are opposite, the middle area of the reference electrode is a circular working electrode, and the working electrode, the auxiliary electrode and the reference electrode are not in contact with each other; the conductive silver track is three parallel conductive strips, wherein one ends of the three conductive strips are respectively connected with the working electrode, the auxiliary electrode and the reference electrode; drying one layer of the printed layer in a 70 ℃ drying oven, drying and cooling the printed layer, printing the next layer of the printed layer, and finally drying the printed insulating ink to obtain a rectangular insulating ink pattern, wherein the rectangular insulating ink covers printing areas of the working electrode, the reference electrode and the auxiliary electrode and part of the conductive silver tracks;
and a second process: DNA1 containing a corresponding specific recognition site is selected according to the kind of the trace element to be determined, and it can base-pair with DNA 2; diluting the stock solution of DNA1 with sterilized water to obtain 5 μ M diluted solution of DNA1, adding 400 μ L diluted solution of DNA1 into 80 μ L5 mM TCEP solution, and activating for 3h to obtain activated DNA 1; wherein the sequence of DNA1 for detecting copper is 5' -HS- (CH)2)6-AGCAAGCATTAACGGCTTATTGCTCGTTCG-3 'DNA 1 sequence for detecting zinc is 5' -CCACCRAGCCCTCTTAATTGCTTCCGAGGArACCAAGAGATGTGGTGG-(CH2)6-SH-3’；
The third process: the screen-printed electrode was immersed in 5mL of 1mol/L H2SO4In the solution, an electrochemical workstation is used for scanning by adopting a cyclic voltammetry method to complete electrode activation, and then deionized water is used for ultrasonic cleaning and natural drying at room temperature; dripping 10 mu L of AuNPs solution on the surface of a working electrode of the activated screen printing electrode, and standing for 5h, thereby forming a layer of uniform gold nano-film on the surface of the working electrode; finally, 10 mu L of activated DNA1 solution is dropped on the surface of the AuNPs modified working electrode to react for 3h, and the finished working electrode is obtained;
the process four is as follows: dripping 10 mu L of the rod-shaped MOF probe solution prepared in the first step on the surface of the processed working electrode, incubating for 5h at room temperature, and then washing with a Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor (shown in figure 3);
and a fifth process: the integrated electrochemical detection device is constructed as follows: 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 realizes control through mutual signal input and output, and the whole detection device is jointly constructed into an integrated electrochemical detection device (as shown in figure 4);
step three: the establishment and application of the microelement mathematical model comprise the following processes:
the first process is as follows: preparing a series of copper ion standard solutions with the concentrations of 0 and 3 multiplied by 10 respectively-13M、3×10-12M、3×10-11M、3×10-10M、3×10-9M、3×10-8M、3×10-7M; respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the second step into the standard solutions, wherein one device corresponds to a solution with one concentration;
and a second process: firstly, opening PSTouch software at a smart phone end, connecting the PSTouch software with a microcontroller module 7 through Bluetooth, setting starting and stopping voltage for controlling an electrochemical micro-nano sensor 9 on the software, generating a corresponding command by using the microcontroller module 7, transmitting the corresponding command to an impedance converter module 6 and further sending a signal to the electrochemical micro-nano sensor 9; then the electrochemical micro-nano sensor 9 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 is established based on the concentration and the current value of the trace element copper ions and is 1.512x +19.84(R is equal to 1.512x + 19.84)2＝0.9983)；
The same procedure as in the first and second steps except that the trace element zinc is measured, and finally, a mathematical model y of 10.485x +140.87 is constructed based on the concentration and current value of the trace element zinc ions (R is2＝0.9921)；
The third process: firstly, accurately brewing the purchased milk powder according to the specification, then immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step into the milk powder, and substituting the measured response current values of copper and zinc into the constructed mathematical model to calculate the concentration of the copper and zinc according to the mathematical model established in the second step. The results show that mCuIs 1.1X 10-5M，mZnIs 9.8 multiplied by 10-5The contents of M, i.e., copper and zinc are within the specified ranges.
In conclusion, the invention is based on that a rod-shaped MOF material is used as a bionic catalyst to construct a portable electrochemical sensor with high sensitivity and high specificity, which is combined with a smart phone and used for real-time in-situ micro-area detection of trace elements in milk powder. The detection technology is simple and convenient to operate, can carry out quantitative analysis on trace elements in micro-regions, and a multi-channel micro-nano sensing system established at the same time can realize detection of various trace elements and multi-region simultaneous determination of samples. The sensing system can monitor the trace element condition in the milk powder processing process in real time, so as to provide a basis for 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-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (10)

1. A construction method of an electrochemical detection device for trace elements in milk powder is characterized by comprising the following steps:
the method comprises the following steps: preparing a rod-shaped MOF probe;
the first process is as follows: weighing cerium nitrate and 1,3, 5-trimesic acid, and dispersing 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 step into a reaction kettle with polytetrafluoroethylene as an inner lining, putting the reaction kettle into an oil bath pot for heating reaction, centrifuging after the heating reaction is finished to obtain a white precipitate, respectively washing with deionized water and ethanol, then carrying out 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;
the 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, then washing a precipitate obtained by centrifugation with ethanol, and obtaining an MOF composite material attached with AuNPs after washing, wherein the MOF composite material is marked as an Au @ MOF material; dispersing the obtained Au @ MOF material into a Tris-HCl buffer solution to obtain an Au @ MOF solution;
the process four is as follows: selecting DNA2 containing corresponding specific recognition sites according to the type of the trace elements to be detected; selecting DNA2, diluting a DNA2 stock solution with sterilized water to obtain a DNA2 diluent, and then adding the DNA2 diluent into a tris- (2-formylethyl) phosphate solution for activation reaction for a period of time to obtain activated DNA 2; then mixing the activated DNA2 with a certain amount of Au @ MOF solution, and incubating at room temperature to obtain a DNA2@ Au @ MOF mixed solution;
and a fifth process: adding bovine serum albumin into the DNA2@ Au @ MOF mixed solution obtained in the fourth process for reaction for a period of time, and dispersing the product obtained by centrifugation into a Tris-HCl buffer solution to obtain a rod-shaped MOF probe-based solution;
step two: the method comprises the steps of (1) building an integrated electrochemical detection device;
the first process is as follows: preparing a screen printing electrode: the method comprises the steps of overprinting a conductive silver rail, a working electrode, an auxiliary electrode and an Ag/AgCl reference electrode on the surface of a PET substrate layer by using a screen printing technology to obtain a screen printing electrode of a three-electrode system;
and a second process: DNA1 containing a corresponding specific recognition site is selected according to the kind of the trace element to be determined, and it can base-pair with DNA 2; selecting DNA1, diluting a DNA1 stock solution with sterilized water to obtain a DNA1 diluent, adding the DNA1 diluent into a TCEP solution, and performing an activation reaction to obtain activated DNA 1;
the third process: dipping the screen-printed electrode into sulfuric acid (H)2SO4) In the solution, an electrochemical workstation is used for scanning by adopting a cyclic voltammetry method to complete electrode activation, and then deionized water is used for ultrasonic cleaning and natural drying at room temperature; then, dropwise adding a certain amount of AuNPs solution on the surface of the working electrode of the activated screen printing electrode, and standing for a period of time, thereby forming a layer of uniform gold nano-film on the surface of the working electrode; finally, dripping the activated DNA1 obtained in the second step on the surface of the working electrode modified by AuNPs for reaction for a period of time to obtain the processed working electrode;
the process four is as follows: dropwise adding the rod-shaped MOF probe solution prepared in the first step to the surface of the working electrode treated in the third step, incubating for a period of time at room temperature, and then washing with a Tris-HCl buffer solution to remove unbound probes to obtain an electrochemical micro-nano sensor;
and a fifth process: the integrated electrochemical detection device is constructed as follows:
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, control is realized through mutual signal input and output, and the electrochemical micro-nano sensor and the power supply module are jointly built into an integrated electrochemical detection device.
2. The construction method of the electrochemical detection device for trace elements in milk powder according to claim 1, wherein in the first step, the dosage ratio of the cerium nitrate, the 1,3, 5-trimesic acid, the ethanol and the water is 0.01-0.05 g: 0.005-0.05 g: 10-30 mL: 10-30 mL;
in the second process, the heating reaction temperature 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 value is 7.4.
3. The construction method of the electrochemical detection device for trace elements in milk powder according to claim 1, wherein in the third step, the volume ratio of the nanogold 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 were: 5000-15000 rpm for 5-20 min; the dosage of the Tris-HCl buffer solution is 400-600 mu L, the concentration is 20mM, and the pH value is 7.4.
4. The method for constructing the device for electrochemically detecting trace elements in milk powder according to claim 1, wherein in the fourth step, the DNA2 stock solution is an oligonucleotide purified by HPLC; the concentration of the DNA2 diluent is 5-10 mu M; the concentration of TCEP is 5 mM; the dosage relationship between the DNA2 diluent and TCEP is 50-400 mu L: 10-80 μ L; the activation reaction lasts for 2-3 h; the dosage relation of the activated DNA2 and the Au @ MOF solution is 50-400 muL and 100-800 muL, and the concentration of the Au @ MOF solution is 60-80 mg/mL; the incubation time at room temperature is 14 h;
the sequence of the DNA2 is determined by the type of trace element detected; when detecting copper, adopting a sequence of G containing a specific recognition site of copper ions; when detecting zinc, the sequence of rA containing zinc ion specific recognition site is adopted.
5. The construction method of the electrochemical detection device for trace elements in milk powder according to claim 1, wherein in the fifth step, the dosage relationship between the DNA2@ Au @ MOF mixed solution and the bovine serum albumin is 100-200 μ L: 80-100 mu L of bovine serum albumin, wherein the mass fraction of the bovine serum albumin is 1%; the reaction time is 0.5-1.5 h; the centrifugation conditions were: 5000-15000 rpm for 5-20 min; the concentration of the Tris-HCl buffer solution is 20mM, and the pH value is 7.4; the concentration of the rod-shaped MOF probe solution is 50-100 mg/mL.
6. The method for constructing a device for electrochemically detecting trace elements in milk powder according to claim 1, wherein in the first step, the reference electrode of the screen-printed electrode is 1/6 circular ring, the auxiliary electrode is 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 track is three parallel conductive strips, wherein one ends of the three conductive strips are respectively connected with the working electrode, the auxiliary electrode and the reference electrode; and (3) drying one layer of the printed layer in a 70 ℃ drying oven, drying and cooling the printed layer, printing the next layer of the printed layer, and finally drying the printed insulating ink to obtain a rectangular insulating ink pattern, wherein 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 track.
7. The method for constructing a device for electrochemically detecting trace elements in milk powder according to claim 1, wherein in the second step, the DNA1 is an oligonucleotide purified by HPLC; the concentration of the DNA1 diluent is 0.5-5 mu M; the concentration of TCEP is 5 mM; the activation reaction time is 2-3 h; the dosage relationship between the DNA1 diluent and TCEP is 50-400 mu L: 10-80 μ L;
the sequence of the DNA1 is determined by the detected trace element types and the DNA 2; when copper is detected, for example, a sequence of G containing a specific recognition site for copper ions is used, and the selected sequence can be ligated to base pairing in DNA 2; when detecting zinc, a sequence of rA containing a zinc ion specific recognition site is used, and the selected sequence can be ligated to base pairing in DNA 2.
8. The method for constructing an electrochemical detection device for trace elements in milk powder according to claim 1, wherein H is added in the third step2SO4The concentration of the solution is 1 mol/L; the dosage of the AuNPs solution is 1-10 mu L, and the concentration is 0.05 mg/mL; the using amount 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 process IV of the step II, the dosage of the rod-shaped MOF probe-based solution is 1-10 mu L; the incubation time is 0.5-5 h.
9. The use of the electrochemical detection device constructed according to the method of any one of claims 1 to 8 for detecting trace elements in milk powder is characterized by comprising the following steps:
the first process comprises the following steps: firstly, preparing a series of trace element standard solutions with the concentrations of Q1、Q2、Q3、……、Qn-1、QnN concentration gradients, n being a positive integer; secondly, respectively immersing the electrochemical micro-nano sensors in the integrated electrochemical detection device prepared in the second step into the standard solutions, wherein one device corresponds to a solution with one concentration;
and a second process: the intelligent terminal is connected with the microcontroller module through Bluetooth, electrochemical detection software installed on the intelligent terminal is started, starting and stopping voltage for controlling the electrochemical micro-nano sensor is set on the software, a corresponding command is generated by the microcontroller module, transmitted to the impedance converter module and further sent to the electrochemical micro-nano sensor; then, the electrochemical micro-nano sensor obtains a 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 the Bluetooth, then a standard curve is established according to the correlation between the concentration of the 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;
the third process: firstly preparing a sample solution, then immersing the electrochemical micro-nano sensor in the integrated electrochemical detection device prepared in the second step in the sample solution, and substituting the response current value of the trace elements in the sample solution measured on the electrochemical detection software into the mathematical model to calculate the concentration of the trace elements according to the mathematical model established in the second step, thereby realizing the in-situ quantitative detection of the trace elements in the sample solution.
10. The use according to claim 9, wherein the concentration of the trace elements in the first process is in the range of 0 to 3 x 10-7M; the trace elements include 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 computer.
CN202210040769.0A 2022-01-14 2022-01-14 Construction method and application of electrochemical detection device for trace elements in milk powder Active CN114518397B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210040769.0A CN114518397B (en) 2022-01-14 2022-01-14 Construction method and application of electrochemical detection device for trace elements in milk powder

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210040769.0A CN114518397B (en) 2022-01-14 2022-01-14 Construction method and application of electrochemical detection device for trace elements in milk powder

Publications (2)

Publication Number Publication Date
CN114518397A true CN114518397A (en) 2022-05-20
CN114518397B CN114518397B (en) 2023-09-26

Family

ID=81596363

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210040769.0A Active CN114518397B (en) 2022-01-14 2022-01-14 Construction method and application of electrochemical detection device for trace elements in milk powder

Country Status (1)

Country Link
CN (1) CN114518397B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101216454A (en) * 2008-01-16 2008-07-09 中国科学院长春应用化学研究所 Electrochemical analyzer with wireless transmission function
CN107525834A (en) * 2017-07-18 2017-12-29 济南大学 A kind of DNA aptamer sensors of Cu MOF marks are used for the method for detecting Acetamiprid
CN109655502A (en) * 2017-10-11 2019-04-19 中山大学 A kind of portable multi-channel electrochemical detection system
CN210990263U (en) * 2019-09-06 2020-07-14 吴迪 Intelligent wearable assembly of self-powered flexible electrode and intelligent wearable system
US20210247349A1 (en) * 2019-08-27 2021-08-12 Qingdao University METHOD FOR PREPARING RATIOMETRIC ELECTROCHEMICAL miR3123 APTASENSOR BASED ON METAL-ORGANIC FRAMEWORK COMPOSITE

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101216454A (en) * 2008-01-16 2008-07-09 中国科学院长春应用化学研究所 Electrochemical analyzer with wireless transmission function
CN107525834A (en) * 2017-07-18 2017-12-29 济南大学 A kind of DNA aptamer sensors of Cu MOF marks are used for the method for detecting Acetamiprid
CN109655502A (en) * 2017-10-11 2019-04-19 中山大学 A kind of portable multi-channel electrochemical detection system
US20210247349A1 (en) * 2019-08-27 2021-08-12 Qingdao University METHOD FOR PREPARING RATIOMETRIC ELECTROCHEMICAL miR3123 APTASENSOR BASED ON METAL-ORGANIC FRAMEWORK COMPOSITE
CN210990263U (en) * 2019-09-06 2020-07-14 吴迪 Intelligent wearable assembly of self-powered flexible electrode and intelligent wearable system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
蒋艳娟: "基于MOFs电化学DNA传感器的构建及其对乳制品中金属离子汞铜锌的检测研究" *

Also Published As

Publication number Publication date
CN114518397B (en) 2023-09-26

Similar Documents

Publication Publication Date Title
CN106841335B (en) A kind of preparation method of antibiotic self energizing aptamer sensor
CN107037102B (en) Nano composite material and preparation method and application thereof
CN107576707A (en) Analyte testing sensor and its system and the method for measuring at least one analyte
AU2006208484A1 (en) Method, system and device for obtaining electrochemical measurements
US20220308005A1 (en) Single-cell-based Electrochemical Sensor based on Functionalized Nano-probe and Application thereof
CN104502432A (en) Sensitive membrane, screen-printed electrode containing sensitive membrane and application thereof
Wang et al. Electrochemical immunosensor based on AuNPs/Zn/Ni-ZIF-8-800@ graphene for rapid detection of aflatoxin B1 in peanut oil
CN110632146A (en) Enzyme-free glucose electrochemical sensor and detection method thereof
CN108120761A (en) Electrochemica biological sensor based on the peptidomimetics with electro catalytic activity detects for acetylcholinesterase
Huang et al. A novel electrochemical immunosensor based on hydrogen evolution inhibition by enzymatic copper deposition on platinum nanoparticle-modified electrode
CN113484391B (en) Construction method of self-reference ratio electrochemical biosensor and application of self-reference ratio electrochemical biosensor in aflatoxin B1 detection
Jia et al. Battery-free and wireless tag for in situ sensing of urinary albumin/creatinine ratio (ACR) for the assessment of albuminuria
CN110487872A (en) A kind of electrochemica biological sensor and its method for sensing based on spatula
CN114518397A (en) Construction method and application of electrochemical detection device for trace elements in milk powder
CN113358726A (en) Electrode, test paper and preparation method thereof for detecting creatinine by electrochemical method
CN106442665B (en) A kind of preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue
CN115950938B (en) Manufacturing method of electrochemical biosensor and electrochemical detector
CN103926297A (en) Preparation method of glucose sensor based on composite material
CN104931565A (en) Preparation of screen-printed electrode aptamer sensor for detecting tetracycline residue
CN203923432U (en) Microelectrode array electroplanting device based on testing impedance
CN215493305U (en) Electrode, test paper and biosensor for detecting creatinine by electrochemical method
CN109470761B (en) Electrochemical DNA aptamer sensor for zearalenone ZEN detection and ZEN detection method
CN114813868A (en) Flexible sweat multi-marker electrochemical sensor based on metal organic framework derivative and application thereof
CN104730130B (en) Determine the preparation method of GPR70 acceptors and the electrochemica biological sensor of its aglucon interaction strength
JP2000131264A (en) Enzyme sensor

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant