CN113000079B - Electrochemical microfluidic sensing chip for heavy metal ion detection and preparation method thereof - Google Patents
Electrochemical microfluidic sensing chip for heavy metal ion detection and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical microfluidic sensing chip for detecting heavy metal ions, 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 a plurality of symmetrically distributed detection units are planned; each detection unit comprises a heavy metal ion probe chain marked by heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite, and a working electrode, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove which are provided with conducting layers; 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-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 portability.
Description
Technical Field
The technology belongs to the field of genetic engineering, and in particular relates to an electrochemical microfluidic sensing chip and a preparation method and a heavy metal ion detection method thereof.
Background
With the rapid development of industry, pollution problems caused by heavy metal ion emission are increasingly serious. Heavy metal ions are very easy to combine with proteins in organisms, and generate irreversible changes, thereby affecting the functions of tissue cells. For example, in clinical practice, lead concentration exceeding the standard may impair the neurological development of children, cause hypertension and anemia in adults, and even more cause death. The current common heavy metal ion detection method mainly comprises the following steps: atomic absorption spectrometry, inductively coupled plasma mass spectrometry, titration and gravimetric methods. Although the detection methods are developed and mature, the results are accurate, the detection methods are expensive, the detection methods are not easy to carry, the detection process is long in time consumption, and have high technical requirements on operators, so that the detection requirements in wide remote rural areas and emergency situations can not 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 precision.
The micro-fluidic chip is used as an emerging detection platform, and has the characteristics of miniaturization, integration and portability, so that the micro-fluidic chip is widely applied to the fields of disease diagnosis, environment detection and the like. The method is mainly characterized by classifying the materials according to the substrate materials: silicon chip, paper-based chip, glass chip. Among them, silicon materials are incompatible with conventional inspection techniques due to their semiconductor characteristics of intolerance to high pressure, opacity, and brittleness. The paper-based chip with cheap raw materials and simple processing and manufacturing has the advantages of being foldable and designable, and meets the requirements of some practical applications. However, the fiber structure inside the paper chip cannot be completely duplicated, and the paper fiber is easy to break and absorb moisture, so that the accuracy of the detection result is easy to influence. However, glass materials have many advantages such as good electroosmotic and optical properties, and can be modified both surface and interior by physical and chemical means to meet application needs. Therefore, the method can be used as a micro-fluidic chip carrier, and can effectively improve the controllability, reproducibility, sensitivity and accuracy of detection results.
The analysis method established on the microfluidic chip analysis device at present mainly comprises the following steps: colorimetric, optical, electrochemical, and the like. 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 is mostly dependent on biological enzyme as a load label for catalyzing reaction to realize signal conversion. The biological enzyme has the characteristics of difficult extraction, difficult storage, easy inactivation and the like, not only improves the preparation and storage conditions of the sensor, but also causes inaccurate detection results. 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 catalytic property and the like. Proper bioactive nano material is selected to be introduced into the electrochemical construction, so that the accuracy of the detection result of the sensor is expected to be improved, and the cost consumption caused by storage is expected to be reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation process of an electrochemical microfluidic sensing chip and a heavy metal ion detection method, and the microfluidic sensing chip has the advantages of portability, simple equipment, rapid operation, low cost, high sensitivity and the like.
The electrochemical microfluidic sensing chip for detecting 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 symmetrically distributed detection units are planned; each detection unit comprises a heavy metal ion probe chain marked by heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite, and a working electrode, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove which are provided with conducting layers; 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-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 conductive layer is a gold nano conductive layer, the preparation method is any one of an evaporation method, a sputtering method, electroplating, electrochemical deposition and chemical growth method, and the length of the gold nano conductive layer is 6-16mm, and the width of the gold nano conductive layer is 2-6mm.
Further, the heavy metal ion probe chain marked by the heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite is respectively fixed on the gold nano conductive layer through gold-sulfur bonds; the ceria-gold nanocomposite is fixed on the surface of the gold nano conductive layer in a way of complementary pairing of a heavy metal ion probe chain and heavy metal ion specific reaction enzyme.
Furthermore, during detection, a proper amount of buffer solution of o-phenylenediamine and hydrogen peroxide is added into a working electrode pool, so that the working electrode pool, a counter electrode pool and a reference electrode pool are communicated, a chip is connected into an electrochemical workstation to detect a current signal, wherein the ceria-gold nanocomposite catalyzes the hydrogen peroxide to react with the o-phenylenediamine to obtain the strongest current signal, when the heavy metal ion specific reaction enzyme is activated by heavy metal ions, the heavy metal ion specific reaction enzyme catalyzes the corresponding heavy metal ion probe chain to break, and the ceria-gold nanocomposite falls off from the surface of the working electrode to reduce the current signal, so that the detection of heavy metal ions is realized.
The preparation method of the electrochemical microfluidic 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 with deionized water, and finally drying with nitrogen for later use;
s2, designing and manufacturing a mask, and covering the manufactured mask on one side of the basal layer;
s3, preparing a gold nano conductive layer;
s4, mixing PDMS and a curing agent according to a proportion of 10:1, vacuumizing until no obvious bubbles are generated in the colloid, continuously vacuumizing for 1-2 hours, taking out, pouring into a mold, heating for 1-3 hours at 60-80 ℃, cooling, taking out PDMS from the mold to obtain cuboid PDMS with the thickness of 76 multiplied by 18 multiplied by 3mm, punching the cuboid 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-channel grooves;
s5, bonding the bonding surface of the PDMS non-micro flow channel groove with the bonding surface of the substrate of the gold nano conductive layer;
s6, taking 15-25 mu L of heavy metal ion specific reaction enzyme solution, dripping the solution into a working electrode pool, incubating the solution for 10-15 hours at room temperature, and then cleaning the working electrode pool by using a buffer solution and drying the working electrode pool; dripping 15-25 mu L of mercapto hexanol into a working electrode pool, incubating for 1.5-2.5h at room temperature, washing the working electrode pool with a buffer solution, and drying; and (3) dripping 15-25 mu L of the ceria-gold nanocomposite modified probe chain solution onto the surface of the working electrode, incubating for 1.5-2.5 hours at 30-40 ℃, cleaning the working electrode pool with a buffer solution, drying, and preparing the chip.
Further, the synthesis steps of the ceria-gold nanocomposite 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 purple red, heating is stopped after the mixed solution is continuously heated and stirred for 8-15min, the reaction is stopped after the mixed solution is stirred for 3-5min, the mixture is centrifuged for 10min at a rotating speed of 8000rpm, and the obtained solid is washed by deionized water, so that gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone in a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180-220 mu L of ammonia water, continuously stirring for 15min to form white colloid, adding 100 mu L of 30% hydrogen peroxide, continuously stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating for 4-12h in a 120-170 ℃ oven, removing the reaction kettle from the oven, naturally cooling to room temperature, flushing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium oxide nanomaterial;
s63, redispersing gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium oxide dispersion liquid into 0.5-1.5mL of 1% bovine serum albumin, stirring for 3-5h at the temperature of 0-8 ℃, redispersing the mixture into 2-5mL of gold nanoparticles, incubating for 8-14h, centrifuging at the speed of 8000-16000rpm, and washing with deionized water to obtain the cerium oxide-gold nanocomposite.
Further, the specific synthesis steps of the heavy metal ion probe chain marked by the ceria-gold nanocomposite are as follows: 4-8. Mu.L of 5. Mu.M probe chains and 100-200. Mu.L of cerium oxide-gold nanomaterial are added into a buffer solution to be uniformly dispersed, hatching reaction is carried out for 10-18 hours under magnetic stirring, and the obtained complex is centrifuged and redispersed to remove free probe chains, and then stored at 4 ℃ for further use.
Further, the liquid conducting range of the working electrode pool, the counter electrode pool and the reference electrode pool in the detection unit is 36-70 mu L.
Further, the heavy metal ion specific reaction enzyme is any one of a lead ion reaction enzyme chain, a zinc ion reaction enzyme chain, a copper ion reaction enzyme chain, a cadmium ion enzyme chain or a magnesium ion enzyme 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. According to the invention, the microfluidic chip is introduced into the electrochemical sensor, so that the electrochemical sensor has the advantages of miniaturization, integration and portability, and meets the application requirements of pollution prevention and control and disease diagnosis in vast remote rural areas;
2. compared with silicon materials and paper-based materials, the glass is used as a base material, and has the advantages of being not easy to damage, accurate in detection and good in stability;
3. the groove design of the PDMS layer effectively avoids complex chip processing technologies such as photoetching and the like, 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 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 application of the glass-based microfluidic chip are facilitated;
5. according to the invention, the ceria-gold nanocomposite is used as a load label, and the ceria is a mesoporous material, has a large specific surface area and good catalytic performance, and the load of gold particles enables the catalytic efficiency of the material to be further improved, 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 diagram of a mask structure according to the present invention;
FIG. 2 is a schematic diagram of the structure of the gold nano-conductive layer according to the present invention;
FIG. 3 is a scanning electron microscope image of a gold nano-conductive 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 an electrochemical microfluidic sensor chip according to the present invention;
FIG. 6 is a front view of the structure of an electrochemical microfluidic sensor chip according to the present invention;
FIG. 7 is a scanning electron microscope image of a ceria-gold nanocomposite according to the invention;
FIG. 8 is a graph showing current intensity versus lead ion concentration for various lead ions according to the present invention;
FIG. 9 is a plot of current intensity versus lead ion concentration plotted in accordance with the present invention;
FIG. 10 is a bar graph of interference contrast 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 an 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 preferred embodiments
The techniques are further described below in conjunction with figures 1-12 and the specific embodiments to aid in understanding the present invention.
An electrochemical microfluidic sensing chip for detecting heavy metal ions comprises a basal layer 7 and a PDMS layer 9; the PDMS layer 9 is fixed above the basal layer 7 through physical adsorption, and a plurality of symmetrically distributed detection units are planned after the PDMS layer and the basal layer are combined; each detection unit comprises a heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite labeled 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 cell 4 and the reference electrode cell 6 are respectively positioned at two sides of the working electrode cell 5 and are communicated through the micro-channel groove 3, and the counter electrode 12 and the reference electrode 11 are respectively inserted into the counter electrode cell 4 and the reference electrode cell 6.
The conductive layer is a gold nano conductive layer 2, the preparation method is any one of an evaporation method, a sputtering method, electroplating, electrochemical deposition and chemical growth method, and the length of the gold nano conductive layer 2 is 11mm and the width is 4mm.
The heavy metal ion probe chain marked by the heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite is respectively fixed on the gold nano conductive layer 2 through gold-sulfur bonds; the ceria-gold nanocomposite is fixed on the surface of the gold nano conductive layer 2 by means of complementary pairing of a heavy metal ion probe chain and 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, so that 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 a current signal, wherein the ceria-gold nanocomposite catalyzes the reaction of hydrogen peroxide and o-phenylenediamine to obtain the strongest current signal, when the heavy metal ion specific reaction enzyme is activated by heavy metal ions to catalyze the corresponding heavy metal ion probe chain to break, the ceria-gold nanocomposite falls off from the surface of the working electrode to reduce the current signal, and detection of heavy metal ions is realized.
The heavy metal ion specific reaction enzyme is any one of lead ion reaction enzyme, zinc ion reaction enzyme, copper ion reaction enzyme, cadmium ion enzyme or magnesium ion enzyme, and the corresponding probe chain is one of lead ion probe chain, zinc ion probe chain, copper ion probe chain, cadmium ion probe chain or magnesium ion probe chain. Wherein, the liquid crystal display device comprises a liquid crystal display device,
lead ion reaction enzyme: 5' -SH- (T) 7 CAT 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 -(CH 2 ) 6 -SH-3′
Zinc ion probe chain: 5'-ACT CAC TAT rA GGA AGA GAT G-SH-3'
Copper ion reaction enzyme: 5' -GGT AAG CCT GGG CCT CTT TCT TTT TAA GAA AGA AC (A) 9 -(CH 2 ) 6 -SH-3′
Copper ion probe chain: 5'-AGC TTC TTT CTA ATA CGG CTT ACC-SH-3'
Cadmium ion reaction 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.
The preparation method of the electrochemical microfluidic sensing chip for heavy metal ion detection comprises the following specific preparation steps:
s1, selecting a glass slide with the size of 76 multiplied by 26mm as a basal layer 7, immersing the glass slide in a Micro90 solution, boiling and washing the glass slide, ultrasonically cleaning the glass slide with deionized water, and finally drying the glass slide with nitrogen for later use;
s2, designing a mask pattern by utilizing Ledit software on a computer, manufacturing a mask 1, immersing the mask 1 pattern in a Micro90 solution with the concentration of 1-3%, boiling and washing for 2-3 hours, ultrasonically cleaning with deionized water for 5-10 minutes, and finally drying with nitrogen, wherein the manufactured mask 1 is covered on one side of the substrate layer 7;
s3, sputtering 2-5nm of titanium on the side covered with the mask plate 1 by using an electron beam evaporation coating machine, and then sputtering 40-60nm of gold to obtain a gold nano conductive layer 2, wherein the shape of the gold nano conductive layer is shown in the figure 2, and a scanning electron microscope image is shown in the figure 3; the gold nano-conductive layer 2 of each detection unit has a length of 11mm and a width of 4mm.
S4, mixing PDMS and a curing agent according to a proportion of 10:1, vacuumizing until no obvious bubbles are generated in the colloid, continuously vacuumizing for 1-2 hours, taking out, pouring into a mold, heating for 1-3 hours at 60-80 ℃, cooling, taking out PDMS from the mold to obtain a cuboid PDMS layer 9 with the diameter of 76 multiplied by 18 multiplied by 3mm, punching the cuboid PDMS layer 9 by using a round puncher with the square shape of 4 multiplied by 4mm and the diameter of 1mm, 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-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 mu L.
S5, bonding the bonding surface of the PDMS layer 9 without the micro-channel grooves with the bonding surface of the substrate of the gold nano conductive layer 2;
s6, dripping 20 mu L of heavy metal ion specific reaction enzyme solution into the working electrode pool 5, incubating for 12 hours at room temperature, and then cleaning the working electrode pool 5 by using a buffer solution and drying; dripping 20 mu L of mercapto hexanol into the working electrode pool 5, incubating for 2 hours at room temperature, and then cleaning the working electrode pool 5 by using a buffer solution and drying; and (3) dripping 20 mu L of the ceria-gold nanocomposite modified probe chain solution onto the surface of the working electrode 5, incubating for 2 hours at 37 ℃, cleaning the working electrode pool 5 by using a buffer solution, drying, and preparing 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 purple red, heating is stopped after the mixed solution is continuously heated and stirred for 8-15min, the reaction is stopped after the mixed solution is stirred for 3-5min, the mixture is centrifuged for 10min at a rotating speed of 8000rpm, and the obtained solid is washed by deionized water, so that gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone in a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180-220 mu L of ammonia water, continuously stirring for 15min to form white colloid, adding 100 mu L of 30% hydrogen peroxide, continuously stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating for 4-12h in a 120-170 ℃ oven, removing the reaction kettle from the oven, naturally cooling to room temperature, flushing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium oxide nanomaterial;
s63, redispersing gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium oxide dispersion liquid into 0.5-1.5mL of 1% bovine serum albumin, stirring for 4 hours at the temperature of 4 ℃, redispersing the mixture into 2mL of gold nanoparticles, incubating for 8-14 hours, centrifuging at 8000-16000rpm, and washing with deionized water to obtain the cerium oxide-gold nanoparticle composite material.
The specific synthesis steps of the heavy metal ion probe chain marked by the ceria-gold nanocomposite are as follows: 4-8. Mu.L of 5. Mu.M probe chains and 100-200. Mu.L of cerium oxide-gold nanomaterial are added into a buffer solution to be uniformly dispersed, hatching reaction is carried out for 10-18 hours under magnetic stirring, and the obtained complex is centrifuged and redispersed to remove free probe chains, and then stored at 4 ℃ for further use. The buffer solution includes PBS buffer solution, acetic acid buffer solution and Tris-HCl buffer solution.
The heavy metal ion detection method of electrochemical microfluidic sensing chip includes the following steps,
p1. adding 10-30 μl of Tris-HCl solution containing lead ions at a certain concentration onto the surface of gold electrode, incubating at 37deg.C, washing with 10mM Tris-HCl with pH=7.4, and drying;
p2. weighing 0.011g of o-phenylenediamine, dissolving in 10mL of 10mM pH=7.4 Tris-HCl, adding 8 mu L of 30% hydrogen peroxide, uniformly mixing, dripping 40-60 mu L of the mixture onto the surface of a gold electrode, and flowing to a reference electrode pool and a counter electrode pool through a micro-channel;
p3. inserting a counter electrode, a silver/silver chloride reference electrode and a working electrode of an electrochemical workstation 8 into a counter electrode cell 4, a reference electrode cell 6 and a working electrode cell 5 of the chip respectively, and recording current intensity I, wherein the schematic diagram of the whole structure is shown in figure 11;
p4. the relationship curve of current intensity and different lead ion concentration is drawn, the relationship of current intensity value and logarithmic value of concentration is in direct proportion, the relationship of current intensity value and logarithmic value of concentration is shown in figure 7, the regression equation is I= 17.8975lgc-89.0982, the detection result is as low as 3.1pM, and the sensitivity of the chip is good;
p5. adding 20 mu L of Tris-HCl solution of a heavy metal ion sample to be detected to the surface of a gold electrode, incubating at 37 ℃, and washing and drying with 10mM of Tris-HCl with pH=7.4;
p6. repeating the steps p2 and p3 in turn, and recording the current intensity value I of the sample Sample Substituting into a regression equation established in p4 to calculate the lead ion concentration c Sample The sample detection is completed;
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 steps: zn (zinc) 2+ 、Cr 2+ 、Cu 2 + 、Mn 2+ 、Co 2+ 、Fe 3+ 、K + 、Mg 2+ The measured change in current intensity of 1 μm of lead ions and the above-mentioned mixed solution was plotted as an interference contrast bar graph. In fig. 10, the current intensity variation value of the interfering ions is lower, and has a larger difference compared with the lead ions and the mixed liquid, and the current intensity value of the lead ions and the mixed liquid is similar, which indicates that the chip has better specificity, is less affected by the interference, and has wider practical application prospect.
As shown in fig. 12, when lead ions are contained in the liquid to be detected, the surface reaction enzyme of the working electrode is activated by the lead ions, and the chain of the catalytic probe breaks, so that the cerium oxide/gold nanocomposite material falls off from the surface of the electrode, and the signal is reduced, thereby realizing detection;
when no lead ions exist in the liquid to be detected, the surface of the working electrode is unchanged, and the electrochemical workstation detects a stronger current signal due to the good catalysis of the cerium oxide/gold nanocomposite.
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, incubating the specific reaction enzyme corresponding to the heavy metal ions in an electrode pool, complementarily pairing a probe chain modified and matched by the ceria-gold nanocomposite material with the base of the probe chain, reacting the probe, and then detecting.
Of course, the above description is not intended to limit the present technology, and the present technology is not limited to the above examples, but rather, changes, modifications, additions or substitutions made by those skilled in the art within the spirit and scope of the present invention are also within the scope of the present technology.
Claims (9)
1. The preparation method of the electrochemical microfluidic sensing chip for detecting heavy metal ions 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 with deionized water, and finally drying with nitrogen for later use;
s2, designing and manufacturing a mask, and covering the manufactured mask on one side of the basal layer;
s3, preparing a gold nano conductive layer;
s4, mixing PDMS and a curing agent according to a proportion of 10:1, vacuumizing until no obvious bubbles are generated in the colloid, continuously vacuumizing for 1-2 hours, taking out, pouring into a mold, heating for 1-3 hours at 60-80 ℃, cooling, taking out PDMS from the mold to obtain cuboid PDMS with the thickness of 76 multiplied by 18 multiplied by 3mm, punching the cuboid 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-channel grooves;
s5, bonding the bonding surface of the PDMS non-micro flow channel groove with the bonding surface of the substrate of the gold nano conductive layer;
s6, taking 15-25 mu L of heavy metal ion specific reaction enzyme solution, dripping the solution into a working electrode pool, incubating the solution for 10-15 hours at room temperature, and then cleaning the working electrode pool by using a buffer solution and drying the working electrode pool; dripping 15-25 mu L of mercapto hexanol into a working electrode pool, incubating for 1.5-2.5h at room temperature, washing the working electrode pool with a buffer solution, and drying; and (3) dripping 15-25 mu L of the ceria-gold nanocomposite modified probe chain solution onto the surface of the working electrode, incubating for 1.5-2.5 hours at 30-40 ℃, cleaning the working electrode pool with a buffer solution, drying, and preparing the chip.
2. The method for preparing the electrochemical microfluidic sensing chip for detecting heavy metal ions according to claim 1, wherein the synthesis steps of the ceria-gold nanocomposite 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 purple red, heating is stopped after the mixed solution is continuously heated and stirred for 8-15min, the reaction is stopped after the mixed solution is stirred for 3-5min, the mixture is centrifuged for 10min at a rotating speed of 8000rpm, and the obtained solid is washed by deionized water, so that gold nanoparticles are obtained;
s62, weighing 0.08-0.12g of cerium chloride heptahydrate and 0.4-0.6g of polyvinylpyrrolidone in a beaker with 18-20mL of absolute ethyl alcohol, carrying out ultrasonic dissolution, then adding 80-120 mu L of formic acid and 180-220 mu L of ammonia water, continuously stirring for 15min to form white colloid, adding 100 mu L of 30% hydrogen peroxide, continuously stirring, gradually changing the color of the solution from white to yellow, transferring the yellow mixed solution into a 25mL stainless steel autoclave, heating for 4-12h in a 120-170 ℃ oven, removing the reaction kettle from the oven, naturally cooling to room temperature, flushing with absolute ethyl alcohol and deionized water, and drying in the 70-80 ℃ oven for 8-12h to obtain the cerium oxide nanomaterial;
s63, redispersing gold nanoparticles into 18-20mL of ultrapure water, adding 1-3mL of cerium oxide dispersion liquid into 0.5-1.5mL of 1% bovine serum albumin, stirring for 3-5h at the temperature of 0-8 ℃, redispersing the mixture into 2-5mL of gold nanoparticles, incubating for 8-14h, centrifuging at the speed of 8000-16000rpm, and washing with deionized water to obtain the cerium oxide-gold nanocomposite.
3. The method for preparing the electrochemical microfluidic sensing chip for detecting heavy metal ions according to claim 1, 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 chains and 100-200. Mu.L of cerium oxide-gold nanomaterial are added into a buffer solution to be uniformly dispersed, hatching reaction is carried out for 10-18 hours under magnetic stirring, and the obtained complex is centrifuged and redispersed to remove free probe chains, and then stored at 4 ℃ for further use.
4. The method for preparing the electrochemical microfluidic sensing chip for detecting heavy metal ions according to claim 1, wherein the liquid conducted by the working electrode pool, the counter electrode pool and the reference electrode pool in the detection unit ranges from 36 to 70 mu L.
5. The method for preparing the electrochemical microfluidic sensing chip for heavy metal ion detection according to claim 1, wherein the heavy metal ion specific reaction enzyme is any one of a lead ion reaction enzyme chain, a zinc ion reaction enzyme chain, a copper ion reaction enzyme chain, a cadmium ion enzyme chain and a magnesium ion enzyme 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 and a magnesium ion probe chain.
6. A microfluidic sensing chip prepared by any one of the preparation methods of claims 1-5, comprising a substrate layer and a PDMS layer, wherein the PDMS layer is fixed above the substrate layer by physical adsorption, and a plurality of symmetrically distributed detection units are planned; each detection unit comprises a heavy metal ion probe chain marked by heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite, and a working electrode, a reference electrode, a counter electrode, a working electrode pool, a counter electrode pool, a reference electrode pool and a micro-channel groove which are provided with conducting layers; 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-channel groove, and the counter electrode and the reference electrode are respectively inserted into the counter electrode pool and the reference electrode pool.
7. The microfluidic sensing chip according to claim 6, wherein the conductive layer is a gold nano-conductive layer, and the gold nano-conductive layer has a length of 6-16mm and a width of 2-6mm.
8. The microfluidic sensing chip according to claim 6, wherein heavy metal ion probe chains marked by heavy metal ion specific reaction enzyme/mercapto hexanol/ceria-gold nanocomposite are fixed on the gold nano conductive layer through gold-sulfur bonds respectively; the ceria-gold nanocomposite is fixed on the surface of the gold nano conductive layer in a way of complementary pairing of a heavy metal ion probe chain and heavy metal ion specific reaction enzyme.
9. The microfluidic sensing chip according to claim 8, 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 ceria-gold nanocomposite catalyzes the reaction of hydrogen peroxide and o-phenylenediamine to obtain the strongest current signal, and when the heavy metal ion specific reaction enzyme is activated by heavy metal ions to catalyze the corresponding heavy metal ion probe chain to break, the current signal is reduced due to the fact that the ceria-gold nanocomposite falls off from the surface of the working electrode, and the detection of heavy metal ions is realized.
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