CN113000079A - Electrochemical micro-fluidic sensing chip for heavy metal ion detection and preparation method thereof - Google Patents
Electrochemical micro-fluidic sensing chip for heavy metal ion detection and preparation method thereof Download PDFInfo
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- CN113000079A CN113000079A CN202010489991.XA CN202010489991A CN113000079A CN 113000079 A CN113000079 A CN 113000079A CN 202010489991 A CN202010489991 A CN 202010489991A CN 113000079 A CN113000079 A CN 113000079A
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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Abstract
The invention discloses an electrochemical microfluidic sensing chip for heavy metal ion detection, which is characterized by comprising a substrate layer and a PDMS layer, wherein the PDMS layer is fixed above the substrate layer through physical adsorption and is provided with a plurality of detection units which are symmetrically distributed; each detection unit comprises a heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material marked heavy metal ion probe chain, a working electrode with a conducting layer, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove; the counter electrode pool and the reference electrode pool are respectively arranged at two sides of the working electrode pool and are communicated through the micro-flow channel groove, and the counter electrode and the reference electrode are respectively inserted into the counter electrode pool and the reference electrode pool. The microfluidic electrochemical sensing chip has the advantages of high detection speed, high precision, good stability, small sampling amount and convenience in carrying.
Description
Technical Field
The technology belongs to the field of genetic engineering, and particularly relates to an electrochemical microfluidic sensing chip, a preparation method thereof and a heavy metal ion detection method.
Background
With the rapid development of the industry, the pollution problem caused by the emission of heavy metal ions in large quantities becomes more serious. Heavy metal ions are highly likely to bind to proteins in the body and undergo irreversible changes, thereby affecting the function of tissue cells. For example, in clinical practice, excessive blood lead concentration may impair the neurological development of children, cause hypertension and anemia in adults, and even cause death. The currently common heavy metal ion detection methods mainly comprise: atomic absorption spectrometry, inductively coupled plasma mass spectrometry, titration, and gravimetric methods. Although the detection methods are developed mature and accurate in result, the equipment is expensive and not easy to carry, the detection process is long in time consumption, the technical requirements on operators are high, and the detection requirements in vast remote rural areas and under emergency conditions cannot be met. In order to realize timely control of heavy metal ion pollution, it is necessary to design a quick and portable sensing device with low cost, high sensitivity and high accuracy.
The micro-fluidic chip is used as a novel detection platform, and the characteristics of miniaturization, integration and portability of the micro-fluidic chip enable the micro-fluidic chip to be widely applied to the fields of disease diagnosis, environmental detection and the like. The classification of the material according to the base material mainly comprises: silicon chip, paper-based chip, glass chip. Among them, the silicon material is incompatible with the conventional detection technology due to its semiconductor characteristics such as poor high voltage resistance, opacity and brittleness. The paper-based chip which is cheap in raw materials and simple to process and manufacture has the advantages of being foldable and designable, and indeed meets some practical application requirements. However, the fiber structure inside the paper chip cannot be completely copied, and the paper fiber is easy to damage and absorb moisture, so that the accuracy of the detection result is easily affected. However, the glass material has many advantages such as good electroosmosis property and optical property, and the surface and the inside can be modified by physical and chemical means to meet the application requirements. Therefore, the carrier can be used as a carrier of the microfluidic chip, and the controllability, the reproducibility, the sensitivity and the accuracy of a detection result can be effectively improved.
The analysis method established on the microfluidic chip analysis device at present mainly comprises the following steps: colorimetric method, optical detection method, electrochemical detection method, etc. Among them, the electrochemical sensor constructed by the three-electrode system is favored by researchers because of its high sensitivity, wide detection range, simple operation and rapid detection. However, the common electrochemical detection method mostly relies on biological enzyme as a load label for catalytic reaction to realize signal conversion. The biological enzyme has the characteristics of difficult extraction, difficult storage, easy inactivation and the like, so that the preparation and storage conditions of the sensor are improved, and the inaccurate detection result is caused. With the development and development of nano materials, researchers find that a plurality of nano materials have the characteristics of large specific surface area, good biocompatibility, enzyme-like catalysis and the like. Proper nano materials without biological activity are selected to be introduced into the electrochemical construction, so that the accuracy of the detection result of the sensor is hopefully improved, and the cost consumption caused by storage is reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation process of an electrochemical micro-fluidic sensing chip and a heavy metal ion detection method.
The electrochemical micro-fluidic sensing chip for detecting the heavy metal ions comprises a substrate layer and a PDMS layer, wherein the PDMS layer is fixed above the substrate layer through physical adsorption, and a plurality of detection units which are symmetrically distributed are planned; each detection unit comprises a heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material marked heavy metal ion probe chain, a working electrode with a conducting layer, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove; the counter electrode pool and the reference electrode pool are respectively arranged at two sides of the working electrode pool and are communicated through the micro-flow channel groove, and the counter electrode and the reference electrode are respectively inserted into the counter electrode pool and the reference electrode pool.
Further, the conducting layer is a gold nano conducting layer, the preparation method is any one of an evaporation method, a sputtering method, electroplating, electrochemical deposition and a chemical growth method, the length of the gold nano conducting layer is 6-16mm, and the width of the gold nano conducting layer is 2-6 mm.
Further, the heavy metal ion probe chain marked by the heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material is characterized in that the heavy metal ion specific reaction enzyme and mercaptohexanol are respectively fixed on the gold nano conductive layer through gold-sulfur bonds; the cerium dioxide-gold nano composite material is fixed on the surface of the gold nano conductive layer in a complementary pairing mode of a heavy metal ion probe chain and a heavy metal ion specific reaction enzyme.
Furthermore, during detection, a proper amount of buffer solution of o-phenylenediamine and hydrogen peroxide is added into the working electrode pool, the counter electrode pool and the reference electrode pool are communicated, the chip is connected into an electrochemical workstation to detect current signals, wherein the cerium dioxide-gold nano composite material catalyzes the reaction of the hydrogen peroxide and the o-phenylenediamine to obtain the strongest current signals, and when the heavy metal ion specific reaction enzyme is activated by the heavy metal ions to catalyze the corresponding heavy metal ion probe chains to break, the cerium dioxide-gold nano composite material falls off from the surface of the working electrode to reduce the current signals, so that the detection of the heavy metal ions is realized.
A preparation method of an electrochemical micro-fluidic sensing chip for heavy metal ion detection comprises the following specific preparation steps:
s1, selecting a proper insulating material as a substrate layer, immersing the substrate layer in a Micro90 solution, boiling and washing, ultrasonically cleaning by using deionized water, and finally drying by using nitrogen for later use;
s2, designing and manufacturing a mask, and covering one side of the basal layer with the manufactured mask;
s3, preparing a gold nano conductive layer;
s4, mixing PDMS and a curing agent in a ratio of 10: 1, continuously vacuumizing for 1-2h, taking out, pouring into a mold, heating for 1-3h at 60-80 ℃, cooling, taking out PDMS from the mold to obtain 76 x 18 x 3mm cuboid PDMS, punching the PDMS by using a puncher, dividing a plurality of groups of working electrode pools, counter electrode pools and reference electrode pools, and cutting grooves on the non-bonding surface of the PDMS to serve as micro-flow channel grooves;
s5, bonding the bonding surface of the PDMS micro-runner-free groove with the substrate bonding surface of the gold nano conductive layer;
s6, dropwise adding 15-25 mu L of heavy metal ion specific reaction enzyme solution to a working electrode pool, incubating at room temperature for 10-15h, washing the working electrode pool with a buffer solution, and drying; dropwise adding mercaptohexanol of 15-25 mu L into a working electrode pool, incubating at room temperature for 1.5-2.5h, washing the working electrode pool with a buffer solution, and drying; and (3) dropwise adding 15-25 mu L of cerium dioxide-gold nano composite material modified probe chain solution onto the surface of the working electrode, incubating at 30-40 ℃ for 1.5-2.5h, cleaning the working electrode pool with buffer solution, drying, and completing the preparation of the chip.
Further, the synthesis steps of the cerium dioxide-gold nano composite material are as follows:
s61.80-120mL of 1M chloroauric acid is boiled under stirring, 8-10mL of 38.8mM sodium citrate solution is added, the color of the mixed solution is changed from light yellow to mauve, heating and stirring are continued for 8-15min, then heating is stopped, reaction is stopped after stirring for 3-5min, centrifugation is carried out for 10min at the rotation speed of 8000rpm, and the obtained solid is washed by deionized water, so that the gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone into a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180 mu L of ammonia water, continuously stirring for 15min to form a white colloid, adding 100 mu L of 30% hydrogen peroxide, continuing stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating in a 120-170 ℃ oven for 4-12h, after the reaction is finished, removing the reaction kettle out of the oven, naturally cooling to room temperature, washing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium dioxide nano material;
s63, redispersing the gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium dioxide dispersion into 0.5-1.5mL of 1% bovine serum albumin, stirring for 3-5h at 0-8 ℃, redispersing in 2-5mL of gold nanoparticles, incubating for 8-14h, centrifuging at 8000-16000rpm, and washing with deionized water to obtain the cerium dioxide-gold nanoparticle composite material.
Further, the specific synthesis steps of the cerium dioxide-gold nanocomposite labeled heavy metal ion probe chain are as follows: 4-8 mu L of 5 mu M probe chain and 100-200 mu L of cerium dioxide-gold nano material are added into a buffer solution to be uniformly dispersed, the mixture is incubated for 10-18h under magnetic stirring, the obtained compound is centrifuged and redispersed to remove free probe chain, and then the mixture is stored at 4 ℃ for further use.
Further, the liquid range of the conduction of the working electrode cell, the counter electrode cell and the reference electrode cell in the detection unit is 36-70 muL.
Further, the heavy metal ion specific reaction enzyme is any one of a lead ion polymerase chain, a zinc ion polymerase chain, a copper ion polymerase chain, a cadmium ion polymerase chain or a magnesium ion polymerase chain, and the corresponding probe chain is one of a lead ion probe chain, a zinc ion probe chain, a copper ion probe chain, a cadmium ion probe chain or a magnesium ion probe chain.
Advantageous effects
1. The invention introduces the micro-fluidic chip into the electrochemical sensor, so that the electrochemical sensor has the advantages of miniaturization, integration and convenient carrying, and meets the application requirements on pollution prevention and control and disease diagnosis in vast remote rural areas;
2. the glass is used as a substrate material, and compared with a silicon material and a paper-based material, the glass has the advantages of difficulty in damage, accurate detection and good stability;
3. the groove design of the PDMS layer effectively avoids complicated chip processing technologies such as photoetching, reduces the manufacturing cost of the chip, simplifies the manufacturing technology and shortens the preparation time;
4. according to the invention, the PDMS layer is used for dividing a plurality of groups of detection units, so that the detection of a plurality of groups of samples can be realized, the utilization efficiency of the chip is improved, the use cost is greatly reduced, and the popularization and the application of the glass-based microfluidic chip are facilitated;
5. according to the invention, the cerium dioxide-gold nano composite material is used as a load label, the cerium dioxide is a mesoporous material, the specific surface area is larger, the catalytic performance is good, and the catalytic efficiency of the material is further improved by the load of the gold particles, so that the stability, the sensitivity and the accuracy of a detection result of the microfluidic electrochemical sensing chip are improved.
Drawings
FIG. 1 is a schematic view of a mask according to the present invention;
FIG. 2 is a schematic view of a gold nano-conductive layer structure according to the present invention;
FIG. 3 is a scanning electron microscope image of a gold nanoconducting layer according to the present invention;
FIG. 4 is a top view of a PDMS structure according to the present invention;
FIG. 5 is a top view of the electrochemical micro-fluidic sensing chip structure according to the present invention;
FIG. 6 is a front view of the electrochemical micro-fluidic sensing chip structure according to the present invention;
FIG. 7 is a scanning electron microscope image of a cerium oxide-gold nanocomposite material according to the present invention;
FIG. 8 is a graph of current intensity versus lead ion concentration according to the present invention;
FIG. 9 is a plot of current intensity versus log lead ion concentration according to the present invention;
FIG. 10 is an interference contrast bar graph according to the present invention;
FIG. 11 is a schematic diagram of the overall structure of an electrochemical microfluidic sensor chip according to the present invention;
FIG. 12 is a schematic diagram of lead ion detection of the electrochemical microfluidic sensor chip according to the present invention;
description of the reference numerals
1-mask, 2-gold nano conductive layer, 3-micro flow channel groove, 4-counter electrode pool, 5-working electrode pool, 6-reference electrode pool, 7-basal layer, 8-electrochemical workstation, 9-PDMS layer, 10-working electrode, 11-reference electrode and 12-counter electrode.
Detailed description of the invention
The following further description of the technology, in conjunction with the accompanying figures 1-12 and the specific embodiments, is provided to assist in understanding the present invention.
An electrochemical micro-fluidic sensing chip for detecting heavy metal ions comprises a substrate layer 7 and a PDMS layer 9; the PDMS layer 9 is fixed above the substrate layer 7 through physical adsorption, and a plurality of detection units which are symmetrically distributed are planned after the PDMS layer and the substrate layer are combined; each detection unit comprises a heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material marked heavy metal ion probe chain, a working electrode 10 with a conducting layer, a reference electrode 11, a counter electrode 12, a working electrode pool 5, a counter electrode pool 4, a reference electrode pool 6 and a micro-channel groove 3; wherein the counter electrode pool 4 and the reference electrode pool 6 are respectively arranged at two sides of the working electrode pool 5 and are communicated through the micro-flow channel groove 3, and the counter electrode 12 and the reference electrode 11 are respectively inserted into the counter electrode pool 4 and the reference electrode pool 6.
The conducting layer is a gold nano conducting layer 2, the preparation method is any one of an evaporation method, a sputtering method, electroplating, electrochemical deposition and a chemical growth method, and the length of the gold nano conducting layer 2 is 11mm, and the width of the gold nano conducting layer is 4 mm.
The heavy metal ion probe chain marked by the heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material is characterized in that the heavy metal ion specific reaction enzyme and mercaptohexanol are respectively fixed on the gold nano conductive layer 2 through gold-sulfur bonds; the cerium dioxide-gold nano composite material is fixed on the surface of the gold nano conducting layer 2 in a complementary pairing mode of a heavy metal ion probe chain and a heavy metal ion specific reaction enzyme.
During detection, a proper amount of buffer solution of o-phenylenediamine and hydrogen peroxide is added into a working electrode pool 5, the working electrode pool 5, a counter electrode pool 4 and a reference electrode pool 6 are communicated, a chip is connected into an electrochemical workstation 8 to detect current signals, wherein the cerium dioxide-gold nano composite material catalyzes the reaction of the hydrogen peroxide and the o-phenylenediamine to obtain the strongest current signals, when the heavy metal ion specific reaction enzyme is activated by heavy metal ions to catalyze the corresponding heavy metal ion probe chains to break, the cerium dioxide-gold nano composite material falls off from the surface of the working electrode to reduce the current signals, and the detection of the heavy metal ions is realized.
The heavy metal ion specific reaction enzyme is any one of lead ion reaction enzyme or zinc ion reaction enzyme or copper ion reaction enzyme or cadmium ion enzyme or magnesium ion enzyme, and the corresponding probe chain is one of a lead ion probe chain or a zinc ion probe chain or a copper ion probe chain or a cadmium ion probe chain or a magnesium ion probe chain. Wherein,
lead ion reactive enzyme: 5' -SH- (T)7CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG AGT-3’
Lead ion probe chain: 5' -SH-ACT CAC TAT rA GGA AGA GAT G-3
Zinc ion reactive enzyme: 5' -CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG AGT (A)9-(CH2)6-SH-3′
Zinc ion probe chain: 5 '-ACT CAC TAT rA GGA AGA GAT G-SH-3'
Copper ion-reactive enzyme: 5' -GGT AAG CCT GGG CCT CTT TCT TTT TAA GAA AGA AC (A)9-(CH2)6-SH-3′
Copper ion probe chain: 5 '-AGC TTC TTT CTA ATA CGG CTT ACC-SH-3'
Cadmium ion reactive enzyme: 5 '-TTT CGC CAT CTT CCT TCG ATA GTT AAA ATA GTG ACT CGT GAC-SH-3'
Cadmium ion probe chain: 5 '-GTC ACG AGT CAC TAT rA GGA AGA TGG CGA AA-SH-3'
Magnesium ion-reactive enzyme: 5 '-SH-TTT GAG GAT CAA GCG ATC TGG AAC AGC ACC CAT GTC CTT GGG GGC C-3'
Magnesium ion probe chain: 5 '-SH-GGA CGU GGA CGU AGA CGU GGA CGU G-3'
The chip is provided with three to ten groups of independent detection units, and can detect ions of different heavy metals each time.
A preparation method of an electrochemical micro-fluidic sensing chip for heavy metal ion detection comprises the following specific preparation steps:
s1, selecting a glass slide with the size of 76 x 26mm as a substrate layer 7, immersing the substrate layer 7 in a Micro90 solution, boiling and washing, ultrasonically cleaning with deionized water, and finally drying with nitrogen for later use;
s2, designing a mask pattern on a computer by utilizing Ledit software, manufacturing a mask 1, wherein the mask 1 pattern is shown in figure 1, immersing the mask in 1-3% of Micro90 solution, boiling and washing for 2-3h, ultrasonically cleaning for 5-10min by using deionized water, and finally drying by blowing nitrogen, wherein the manufactured mask 1 covers one side of a substrate layer 7;
s3, sputtering 2-5nm of titanium and then 40-60nm of gold to one side covered with the mask 1 by using an electron beam evaporation coating machine to obtain a gold nano conductive layer 2, wherein the shape of the gold nano conductive layer is shown in figure 2, and the scanning electron microscope image of the gold nano conductive layer is shown in figure 3; the gold nano conductive layer 2 of each detection unit has a length of 11mm and a width of 4 mm.
S4, mixing PDMS and a curing agent in a ratio of 10: 1, continuously vacuumizing for 1-2h, taking out and pouring into a mould, heating for 1-3h at 60-80 ℃, cooling and taking out PDMS from the mould to obtain a 76X 18X 3mm rectangular PDMS layer 9, punching the PDMS layer by using a 4X 4mm square puncher with a diameter of 1mm, dividing a plurality of groups of working electrode pools, counter electrode pools and reference electrode pools, cutting grooves on the non-bonding surfaces of the PDMS to serve as micro-flow channel grooves 3, wherein the conducting liquid range of the working electrode pools, the counter electrode pools and the reference electrode pools is 36-70 muL.
S5, bonding the bonding surface of the PDMS layer 9 without the micro-runner groove with the substrate bonding surface of the gold nano conductive layer 2;
s6, dropwise adding 20 mu L of heavy metal ion specific reaction enzyme solution to the working electrode pool 5, incubating at room temperature for 12h, washing the working electrode pool 5 with buffer solution, and drying; dropwise adding 20 mu L of mercaptohexanol into the working electrode pool 5, incubating at room temperature for 2h, washing the working electrode pool 5 with a buffer solution, and drying; and (3) dropwise adding 20 mu L of cerium dioxide-gold nano composite material modified probe chain solution onto the surface of the working electrode 5, incubating for 2h at 37 ℃, cleaning the working electrode pool 5 by using buffer solution, drying and finishing the preparation of the chip.
S61.80-120mL of 1M chloroauric acid is boiled under stirring, 8-10mL of 38.8mM sodium citrate solution is added, the color of the mixed solution is changed from light yellow to mauve, heating and stirring are continued for 8-15min, then heating is stopped, reaction is stopped after stirring for 3-5min, centrifugation is carried out for 10min at the rotation speed of 8000rpm, and the obtained solid is washed by deionized water, so that the gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone into a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180 mu L of ammonia water, continuously stirring for 15min to form a white colloid, adding 100 mu L of 30% hydrogen peroxide, continuing stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating in a 120-170 ℃ oven for 4-12h, after the reaction is finished, removing the reaction kettle out of the oven, naturally cooling to room temperature, washing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium dioxide nano material;
s63, redispersing the gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium dioxide dispersion into 0.5-1.5mL of 1% bovine serum albumin, stirring for 4h at 4 ℃, redispersing in 2mL of gold nanoparticles, incubating for 8-14h, centrifuging at 8000-16000rpm, and washing with deionized water to obtain the cerium dioxide-gold nanoparticle composite material.
The specific synthesis steps of the cerium dioxide-gold nano composite material marked heavy metal ion probe chain are as follows: 4-8 mu L of 5 mu M probe chain and 100-200 mu L of cerium dioxide-gold nano material are added into a buffer solution to be uniformly dispersed, the mixture is incubated for 10-18h under magnetic stirring, the obtained compound is centrifuged and redispersed to remove free probe chain, and then the mixture is stored at 4 ℃ for further use. The buffer solution comprises PBS buffer solution, acetic acid buffer solution and Tris-HCl buffer solution.
A heavy metal ion detection method of an electrochemical micro-fluidic sensing chip takes lead ion detection as an example and comprises the following steps,
p1. adding 10-30 μ L Tris-HCl solution containing lead ions at certain concentration to the surface of gold electrode, incubating at 37 deg.C, washing with 10mM Tris-HCl solution with pH of 7.4, and drying;
p2. weighing 0.011g o-phenylenediamine, dissolving in 10mL 10mM pH 7.4Tris-HCl, adding 8 μ L30% hydrogen peroxide, mixing, adding 40-60 μ L above mixture dropwise onto the surface of gold electrode, and flowing into reference electrode pool and counter electrode pool via micro flow channel;
p3. inserting counter electrode, silver/silver chloride reference electrode and working electrode of electrochemical workstation 8 into counter electrode cell 4, reference electrode cell 6 and working electrode cell 5 of the chip respectively, and recording current intensity I, wherein the overall structure diagram is shown in FIG. 11;
p4., drawing a relation curve of current intensity and different lead ion concentrations, as shown in figure 7, wherein the current intensity value is in direct proportion to the logarithm of the concentration, as shown in figure 8, the regression equation is I17.8975 lgc-89.0982, and the detection result is as low as 3.1pM, which indicates that the chip has good sensitivity;
p5. adding 20 μ L of Tris-HCl solution of heavy metal ion sample to be detected onto gold electrode surface, incubating at 37 deg.C, washing with 10mM Tris-HCl with pH of 7.4, and drying;
p6. repeating the above steps p2 and p3 in turn, and recording the current intensity value I of the sampleSample (A)Substituting into the regression equation established in p4 to calculate the lead ion concentration cSample (A)Completing the sample detection;
the prepared chip is used for detecting a plurality of potential interfering ion solutions with the concentration of 10 mu M, and comprises the following components: zn2+、Cr2+、Cu2 +、Mn2+、Co2+、Fe3+、K+、Mg2+The amount of change in the measured current intensity was plotted as an interference contrast bar chart with 1. mu.M of lead ions and the above-mentioned mixed solution. See figure 10, wherein the current intensity variation of the interfering ions is lower, and has a larger difference compared with the lead ions and the mixed solution, and the current intensity values of the lead ions and the mixed solution are similar, which shows that the chip has better specificity, is less influenced by interference, and has a wider practical application prospect.
As shown in fig. 12, when the solution to be detected contains lead ions, the reaction enzyme on the surface of the working electrode is activated by the lead ions, the catalytic probe chain is broken, so that the ceria/gold nanocomposite falls off from the surface of the electrode, the signal is reduced, and the detection is realized;
when the liquid to be detected has no lead ions, the surface of the working electrode has no change, and the electrochemical workstation detects a stronger current signal due to the good catalytic action of the cerium dioxide/gold nano composite material.
The detection method can also be used for detecting any one of lead ions, zinc ions, cadmium ions, copper ions and magnesium ions.
When other heavy metal ions need to be measured, specific reaction enzyme corresponding to the heavy metal ions is incubated in an electrode pool, a cerium dioxide-gold nano composite material modified and matched probe chain is complementarily paired with a base of the probe chain, and the probe reaction is carried out for detection.
Of course, the above description is not intended to limit the present technology, and the present technology is not limited to the above examples, and those skilled in the art may make variations, modifications, additions or substitutions within the spirit and scope of the present invention.
Claims (9)
1. The electrochemical microfluidic sensing chip for detecting the heavy metal ions is characterized by comprising a substrate layer and a PDMS layer, wherein the PDMS layer is fixed above the substrate layer through physical adsorption, and a plurality of detection units which are symmetrically distributed are planned; each detection unit comprises a heavy metal ion specific reaction enzyme/mercaptohexanol/cerium dioxide-gold nano composite material marked heavy metal ion probe chain, a working electrode with a conducting layer, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove; the counter electrode pool and the reference electrode pool are respectively arranged at two sides of the working electrode pool and are communicated through the micro-flow channel groove, and the counter electrode and the reference electrode are respectively inserted into the counter electrode pool and the reference electrode pool.
2. The electrochemical micro-fluidic sensing chip for detecting heavy metal ions according to claim 1, wherein the conductive layer is a gold nano conductive layer, the length of the gold nano conductive layer is 6-16mm, and the width of the gold nano conductive layer is 2-6 mm.
3. The electrochemical micro-fluidic sensor chip for detecting heavy metal ions according to claim 2, wherein the heavy metal ion probe chain marked by the heavy metal ion specific reaction enzyme/mercaptohexanol/ceria-gold nanocomposite is formed by fixing the heavy metal ion specific reaction enzyme and mercaptohexanol on a gold nano conductive layer through gold-sulfur bonds respectively; the cerium dioxide-gold nano composite material is fixed on the surface of the gold nano conductive layer in a complementary pairing mode of a heavy metal ion probe chain and a heavy metal ion specific reaction enzyme.
4. The electrochemical micro-fluidic sensing chip for detecting heavy metal ions according to claim 3, wherein during detection, a proper amount of buffer solution of o-phenylenediamine and hydrogen peroxide is added into the working electrode pool, so that the working electrode pool, the counter electrode pool and the reference electrode pool are communicated, the chip is connected into an electrochemical workstation to detect a current signal, wherein the cerium dioxide-gold nano composite material catalyzes the reaction of the hydrogen peroxide and the o-phenylenediamine to obtain a strongest current signal, when the heavy metal ion specific reaction enzyme is activated by the heavy metal ions to catalyze the corresponding heavy metal ion probe chains to break, the cerium dioxide-gold nano composite material falls off from the surface of the working electrode to reduce the current signal, and the detection of the heavy metal ions is realized.
5. A preparation method of an electrochemical micro-fluidic sensing chip for heavy metal ion detection is characterized by comprising the following specific preparation steps:
s1, selecting a proper insulating material as a substrate layer, immersing the substrate layer in a Micro90 solution, boiling and washing, ultrasonically cleaning by using deionized water, and finally drying by using nitrogen for later use;
s2, designing and manufacturing a mask, and covering one side of the basal layer with the manufactured mask;
s3, preparing a gold nano conductive layer;
s4, mixing PDMS and a curing agent in a ratio of 10: 1, continuously vacuumizing for 1-2h, taking out, pouring into a mold, heating for 1-3h at 60-80 ℃, cooling, taking out PDMS from the mold to obtain 76 x 18 x 3mm cuboid PDMS, punching the PDMS by using a puncher, dividing a plurality of groups of working electrode pools, counter electrode pools and reference electrode pools, and cutting grooves on the non-bonding surface of the PDMS to serve as micro-flow channel grooves;
s5, bonding the bonding surface of the PDMS micro-runner-free groove with the substrate bonding surface of the gold nano conductive layer;
s6, dropwise adding 15-25 mu L of heavy metal ion specific reaction enzyme solution to a working electrode pool, incubating at room temperature for 10-15h, washing the working electrode pool with a buffer solution, and drying; dropwise adding mercaptohexanol of 15-25 mu L into a working electrode pool, incubating at room temperature for 1.5-2.5h, washing the working electrode pool with a buffer solution, and drying; and (3) dropwise adding 15-25 mu L of cerium dioxide-gold nano composite material modified probe chain solution onto the surface of the working electrode, incubating at 30-40 ℃ for 1.5-2.5h, cleaning the working electrode pool with buffer solution, drying, and completing the preparation of the chip.
6. The preparation method of the electrochemical micro-fluidic sensing chip for detecting heavy metal ions according to claim 5, wherein the synthesis step of the cerium dioxide-gold nano composite material is as follows:
s61.80-120mL of 1M chloroauric acid is boiled under stirring, 8-10mL of 38.8mM sodium citrate solution is added, the color of the mixed solution is changed from light yellow to mauve, heating and stirring are continued for 8-15min, then heating is stopped, reaction is stopped after stirring for 3-5min, centrifugation is carried out for 10min at the rotation speed of 8000rpm, and the obtained solid is washed by deionized water, so that the gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone into a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180 mu L of ammonia water, continuously stirring for 15min to form a white colloid, adding 100 mu L of 30% hydrogen peroxide, continuing stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating in a 120-170 ℃ oven for 4-12h, after the reaction is finished, removing the reaction kettle out of the oven, naturally cooling to room temperature, washing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium dioxide nano material;
s63, redispersing the gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium dioxide dispersion into 0.5-1.5mL of 1% bovine serum albumin, stirring for 3-5h at 0-8 ℃, redispersing in 2-5mL of gold nanoparticles, incubating for 8-14h, centrifuging at 8000-16000rpm, and washing with deionized water to obtain the cerium dioxide-gold nanoparticle composite material.
7. The preparation method of the electrochemical micro-fluidic sensor chip for heavy metal ion detection according to claim 5, wherein the specific synthesis steps of the ceria-gold nanocomposite-labeled heavy metal ion probe chain are as follows: 4-8 mu L of 5 mu M probe chain and 100-200 mu L of cerium dioxide-gold nano material are added into a buffer solution to be uniformly dispersed, the mixture is incubated for 10-18h under magnetic stirring, the obtained compound is centrifuged and redispersed to remove free probe chain, and then the mixture is stored at 4 ℃ for further use.
8. The method for preparing the electrochemical micro-fluidic sensor chip for detecting the heavy metal ions according to claim 5, wherein the conducting liquid range of the working electrode cell, the counter electrode cell and the reference electrode cell in the detection unit is 36-70 μ L.
9. The method according to claim 5, wherein the heavy metal ion specific reaction enzyme is any one of a lead ion polymerase chain, a zinc ion polymerase chain, a copper ion polymerase chain, a cadmium ion polymerase chain, or a magnesium ion polymerase chain, and the corresponding probe chain is one of a lead ion probe chain, a zinc ion probe chain, a copper ion probe chain, a cadmium ion probe chain, or a magnesium ion probe chain.
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