CN113533469B - Lactate ion sensor based on graphene/polypyrrole and preparation method and application thereof - Google Patents
Lactate ion sensor based on graphene/polypyrrole and preparation method and application thereof Download PDFInfo
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 156
- 229910021389 graphene Inorganic materials 0.000 title claims abstract description 131
- 229920000128 polypyrrole Polymers 0.000 title claims abstract description 83
- -1 Lactate ion Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- VCJMYUPGQJHHFU-UHFFFAOYSA-N iron(III) nitrate Inorganic materials [Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O VCJMYUPGQJHHFU-UHFFFAOYSA-N 0.000 description 1
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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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/307—Disposable laminated or multilayered electrodes
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/333—Ion-selective electrodes or membranes
- G01N27/3335—Ion-selective electrodes or membranes the membrane containing at least one organic component
-
- 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
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses a graphene/polypyrrole-based lactate ion sensor and a preparation method and application thereof. The method has the advantages of simple process, composite of graphene and polypyrrole two sensitive materials in a layered/layered mode, no fixation and inactivation of biological enzymes, high ion responsiveness, wide detection range, high surface flatness, adjustable material thickness and performance, stable chemical properties and the like, and has long service life and good material durability.
Description
Technical Field
The invention relates to the field of ion sensors, in particular to a lactate ion sensor and a preparation method thereof.
Background
In recent years, wearable electronic sensors for monitoring endocrine metabolic products in human sweat in real time are increasingly significant in health detection. For example, the content of lactic acid in sweat, which is a key product of carbohydrate anaerobic metabolism in humans, is an important parameter in sports medicine and clinical analysis. At present, most of the lactic acid sensors are based on catalytic reaction of lactic acid oxidase, and the activity of the enzyme is easily affected by environment although the selectivity is high, so that the use condition of the sensor is limited and the service life is low. Although enzyme-free sensors represent an important research direction, their related research is still limited and immature. It is urgent to explore an enzyme-free sensor having a wide detection range and high sensitivity characteristics.
Disclosure of Invention
The invention aims to: the invention aims to provide an enzyme-free lactate ion sensor based on a two-dimensional material graphene/polypyrrole composite film, and meanwhile, the enzyme-free lactate ion sensor has high ion responsiveness, a wide detection range and a long service life.
The technical scheme is as follows: the invention discloses a graphene/polypyrrole-based lactate ion sensor, which comprises a substrate, spiral interdigital electrodes and a composite film, wherein the composite film comprises layered graphene and a polypyrrole film coated on the upper surface of the graphene. Namely: unlike most of enzyme-containing lactic acid sensors, the graphene/polypyrrole composite lactate ion-sensitive functional material is arranged on a substrate, the graphene/polypyrrole composite functional material comprises layered graphene, and the surface of the layered graphene is electrochemically polymerized with layered polypyrrole in situ; the lactate ion sensor is provided with spiral interdigital electrodes, and the electrodes are connected with the graphene/polypyrrole composite ion sensitive material.
Wherein, the substrate adopts at least one of silicon slice with oxide layer, polyimide slice and polydimethylsiloxane slice, and the thickness of the substrate is 0.3-0.5 mm.
The transparency of the graphene is greater than 95%, so that the graphene is pure and has no covering; the number of layers of the graphene is smaller than three, and the thickness of the polypyrrole layer is 20-200 nm so as to ensure enough surface area and adsorption sites.
Wherein, the shape of the spiral interdigital electrode is double spiral lines, the width of the electrode finger is 10-30 mu m, the interdigital distance is 20-60 mu m, and the number of turns of a single electrode is 2-10 turns.
The graphene/polypyrrole composite film is only filled in the interdigital electrode channel and is in contact with the electrodes at the two sides. The spiral interdigital electrode is composed of a Ti film and an Au film covered on the Ti film.
The invention also provides a preparation method of the lactate ion sensor based on graphene/polypyrrole, which comprises the following steps:
(1) Patterning the surface of the pretreated substrate through a patterning process;
(2) Ti and Au are sequentially deposited on the patterned substrate by a physical vapor deposition method, and redundant Ti/Au is stripped from the substrate by a stripping process, so that the interdigital electrode with the spiral shape is obtained.
(3) Graphene is prepared by a chemical vapor deposition method and transferred to a substrate surface with electrodes using wet transfer.
(4) In-situ polymerizing polypyrrole on the graphene film by an electrochemical polymerization method to obtain a graphene/polypyrrole composite film;
(5) And carrying out etching channeling treatment on the graphene/polypyrrole composite film, and connecting two ends of the composite film with electrodes to obtain the lactate ion sensor.
In the step (1), the patterning method includes: at least one of electron beam lithography, ultraviolet lithography, extreme ultraviolet lithography, and nanoimprint techniques;
in the step (2), the physical vapor deposition method includes: at least one of electron beam evaporation, magnetron sputtering or thermal evaporation; the titanium electrode comprises the following components: the thickness of titanium is 5-20 nm, and the thickness of gold is 40-60 nm.
In the step (2), the wet transfer is an electrochemical stripping method.
In the step (3), the preparation method of the monolayer graphene film is chemical vapor deposition with copper foil as a substrate. The prepared graphene has the transparency more than 95 percent and the number of layers less than 3.
In the step (3), the wet transfer method is an electrochemical stripping method, so that the transfer speed is improved, the impurity pollution is reduced, and the sensing performance is improved;
in the step (3), graphene is transferred to the surface of the substrate with the electrode by using wet transfer; the method comprises the following specific steps:
(a) Coating polymethyl methacrylate (PMMA) on the surface of a substrate (copper foil) on which single-layer graphene grows, and heating and drying; the coating may be at least one of spin coating, spray coating, knife coating, or dip coating;
(b) The peripheral edge of the copper foil is cut off, and a straight line penetrating through the edge is drawn by a sharp object at a position close to the edge to be measured. The copper foil is held by a wire with alligator clips at the narrower side of the line.
(c) Copper foil is used as a cathode, a platinum electrode or a carbon electrode is used as an anode, and NaOH solution or Na with the concentration of 0.1-5 mol/L is used 2 S 2 O 8 The solution is electrolyte, and a voltage of 1-5V is applied. And placing the anode in electrolyte, slowly extending the cathode copper foil into the electrolyte, and keeping the separation interface of PMMA and the copper foil at the liquid level position all the time. And finally, stripping the PMMA from the copper foil to finish stripping.
(d) Transferring the P MMA/graphene onto a substrate, washing with deionized water and drying before transferring, heating to soften the P MMA at 150-180 ℃, removing PMMA by using an organic solvent, and drying.
The electrochemical polymerization method of the step (4) comprises the following steps:
(a) Preparing sodium paratoluenesulfonate containing 0.01-0.10 mol/L, adding pyrrole liquid to the concentration of 0.1-0.3 mol/L, adding hydrochloric acid to adjust the pH to 1.5-2.5, and carrying out ultrasonic treatment for 5-20 min to completely emulsify and uniformly disperse;
(b) An electrochemical workstation is used, a saturated calomel electrode with an electrolyte being a saturated potassium chloride solution is used as a reference electrode, a platinum sheet electrode is used as an auxiliary electrode, and a working electrode is connected to an electrode contact on a substrate.
(c) In-situ polymerizing polypyrrole on the surface of the graphene film by using a constant pressure polymerization method: the polarization voltage is set to be 0.7-0.9V. The low-pressure low-speed polymerization is adopted, so that the order of polypyrrole is improved, the electrons on the outer layer of the polypyrrole can be better delocalized, and the sensing sensitivity can be remarkably improved. The polymerization determines the thickness of polypyrrole, and is generally set to 20-60 min, and when the polymerization is too short, the sensitivity and selectivity of the sensor are reduced, and when the polymerization is too long, the response speed is lowered.
And (3) adopting plasma etching in the step (5), wherein high-purity oxygen or high-purity argon is used as a gas source, the pressure in the gas cavity is 1-50 Pa, and the flow is 1-2 sccm. Because the film is thinner, the etching time is not required to be too long, and the time is 5-10 min.
The invention also provides an application of the lactate ion sensor based on graphene/polypyrrole as a sensor for detecting the lactate concentration in a liquid environment.
The principle of the invention: although the traditional detection method of oxidase and electrochemical electrode has the advantages of high selectivity and molecular recognition in detection, the problems of difficult maintenance of biological enzyme activity, narrow detection range, low detection sensitivity and the like in continuous operation generate difficulties for the development of lactate ion sensors. Graphene is expected to solve the above-mentioned difficulties because it has various excellent electrical, mechanical, physical and chemical properties. In addition, the electrical property and the surface chemical potential of the graphene can be regulated and controlled through doping, so that the sensitivity and the selectivity are further improved. The invention utilizes the high carrier concentration and mobility of graphene and combines the specificity correspondence of polypyrrole to lactic acid groups, and adopts a physical adsorption and charge transfer method to realize the high sensitivity, wide detection range and simple device manufacture of the lactate ion sensor, and the concentration detection of lactate ions can be realized without the participation of lactate biological oxidase. Meanwhile, the use of the interdigital electrode further increases the detection area and anti-interference performance of sensing, and compared with the traditional double-electrode (or three-electrode) system, the liquid quantity required by detection is less, and micro detection can be realized.
Compared with the common parallel double electrodes, the sensor adopts the spiral interdigital electrode pattern, and the shape electrode has the advantages of high detection sensitivity, strong anti-interference performance, large coverage area and the like. The lactate example sensor of the invention has the detection range of 10-110mM at maximum and the sensitivity of 0.309% mM -1 (2.609μA mM -1 ) The method comprises the steps of carrying out a first treatment on the surface of the In a narrow detection range (10-40 mM) the sensitivity is as high as 0.936% mM -1 (10.923μA mM -1 ) And the response time (55 s) and the recovery time (43 s) are shorter, which is obviously superior to the sensor in the prior invention.
The beneficial effects are that:
(1) The lactate ion sensing device of the graphene/polypyrrole layered composite ion sensitive material provided by the invention has a sensing area of only 3mm 2 Compared with the prior artThe large electrochemical detection instrument can realize high sensitivity under a small volume, and can meet the requirements of wearable equipment and microelectronic devices.
The method has the advantages of simple process, high ion responsiveness, low detection limit, high surface flatness, adjustable material thickness, stable chemical property and the like, and has long service life and good material durability by compounding two sensitive materials of graphene and polypyrrole in a layered/layered mode without involving the activity and fixation of biological enzymes.
(2) Due to the high chemical stability of graphene, the graphene/polypyrrole layered composite material disclosed by the invention can maintain the stability of sensing performance for a long time in a room temperature and atmospheric environment.
(3) Compared with the common parallel double electrodes, the spiral interdigital electrode has the advantages of high detection sensitivity, strong anti-interference performance, large coverage area and the like.
(4) The method adopts the electrochemical bubbling method to transfer the graphene, has high transfer speed, avoids copper residues caused by insufficient etching in the etching process, and increases the purity and flatness of large-area graphene.
(5) According to the invention, due to the clustered surface morphology of the graphene/polypyrrole composite material ball, the graphene/polypyrrole composite material ball has a large specific surface area, so that lactate adsorption sites are greatly increased, the sensor has a higher linear interval compared with most commercial sensors, has good response linearity within 10mM to 100mM, and omits the step of diluting most sensors.
Drawings
Fig. 1 is a schematic diagram of a preparation flow of a lactate ion sensor of graphene/polypyrrole.
Fig. 2 is a schematic diagram of an electrochemical exfoliated graphene transfer flow scheme.
FIG. 3 is an AFM micrograph of a graphene/polypyrrole layered composite ion sensitive material; fig. (a) is example 1, and fig. (b) is comparative example 1.
Fig. 4 is a raman characterization graph of a graphene/polypyrrole layered composite ion sensitive material.
FIG. 5 is a reticle design (top: primary lithography; bottom: secondary lithography).
FIG. 6 is a detailed view of a reticle sensing region; FIG. (a) is a single lithography; fig. (b) shows a secondary photolithography.
Fig. 7 is a microscopic image of a graphene/polypyrrole lactate ion sensing device of the present invention with channel material.
FIG. 8 is a graph showing the current response of the ion sensor prepared in example 1 at an operating voltage of 0.1V.
Fig. 9 is a sensor structure diagram.
Detailed Description
The present invention will be described in further detail with reference to examples.
The raw materials and reagents referred to in the following examples and comparative examples are commercially available.
Example 1:
the preparation method of the lactate ion sensor based on graphene/polypyrrole comprises the following steps, and a specific preparation flow chart is shown in fig. 1:
(1) Preparing a single-layer graphene film by using a chemical vapor deposition method, wherein the prepared graphene is a single layer.
(2) Constructing an electrode pattern on a silicon wafer with a 300nm oxide layer by using ultraviolet lithography, wherein the size parameters of the electrode pattern are as follows: the finger width is 30 μm, the finger spacing is 60 μm, and the single finger turns are 5 turns. A titanium gold electrode layer was then deposited on the silicon substrate using an electron beam evaporation method, wherein the titanium thickness was 5nm and the gold thickness was 45nm. Finally, a silicon wafer with spiral interdigital electrodes on the surface is prepared through a stripping process, and the specific electrode shape design is shown in figures 5 and 6.
(3) Electrochemical wet transfer of graphene thin films onto silicon substrates with electrodes: and (3) heating and drying the surface PMMA with the single-layer graphene on the copper foil, shearing off the peripheral edge part of the copper foil, and drawing a penetrating straight line at a position close to a measured edge by using a sharp object. The copper foil is held by a wire with alligator clips at the narrower side of the line. A copper foil is used as a cathode, a platinum electrode or a carbon electrode is used as an anode, a NaOH solution with the concentration of 1mol/L is used as an electrolyte, and a voltage of 3.5V is applied. And placing the anode in electrolyte, slowly extending the cathode copper foil into the electrolyte, and keeping the separation line of PMMA and the copper foil at the liquid level position all the time. And finally, stripping the PMMA from the copper foil to finish stripping. And obtaining the PMMA film with the single-layer graphene on the lower surface. The P MMA sheet with the single-layer graphene was washed with deionized water and dried, then the single-layer graphene was transferred to a silicon substrate with electrodes, after heating and softening PMMA at 160 ℃, PMMA was removed using acetone, and then residual PMMA and acetone were washed with isopropyl alcohol and dried (as shown in fig. 2).
(4) Using an electrochemical polymerization method, in-situ polymerizing a polypyrrole thin layer on the single-layer graphene film: preparing an electrolytic cell solution: 0.971g of sodium p-toluenesulfonate was dissolved in 49mL of deionized water, 1mL of 1mol/L hydrochloric acid and 350. Mu.L of pyrrole were added, and after stirring uniformly, the mixture was sonicated for 10min. And connecting a working electrode to an electrode contact on the silicon wafer, and constructing an electrolytic cell by using a platinum sheet electrode as an auxiliary electrode. The electrochemical workstation working mode is set to be a constant voltage test mode, the voltage is set to be 0.9V, and the polymerization time is 20min. And taking down the silicon wafer after polymerization, and cleaning the silicon wafer by acetone, isopropanol and deionized water in sequence. And naturally air-drying to avoid damaging the composite film. The raman spectrum characterization of the prepared graphene polypyrrole composite film is shown in fig. 4.
(5) The device channel region protection layer is prepared using ultraviolet lithography. Setting the air cavity pressure at 50Pa by using an argon plasma etching technology, introducing 2sccm of argon, etching for 5min to remove the unprotected graphene/polypyrrole composite film, and finally obtaining the lactate ion sensing device (shown in figure 7) with a single-layer graphene/polypyrrole composite film as a channel material;
in the embodiment, the lactate ion sensing device of the single-layer graphene/polypyrrole layered composite ion sensitive material prepared by the method has the average polypyrrole sphere cluster size of 50nm and uniform distribution (shown in the left graph of fig. 3). As shown in FIG. 8, the detection range is 10-110mM at maximum, and the sensitivity can reach 0.309% mM -1 (2.609μA mM -1 ) The method comprises the steps of carrying out a first treatment on the surface of the In a narrow detection range (10-40 mM) the sensitivity is as high as 0.936% mM -1 (10.923μA mM -1 ) And the response time (55 s) and the recovery time (43 s) are relatively longShort.
Example 2:
a method for preparing a lactate ion sensor based on graphene/polypyrrole, comprising the following steps:
(1) Preparing a single-layer graphene film by using a chemical vapor deposition method, wherein the prepared graphene is a single layer.
(2) Constructing an electrode pattern on a silicon wafer with a 300nm oxide layer by using ultraviolet lithography, wherein the size parameters of the electrode pattern are as follows: the finger width is 30 μm, the finger spacing is 60 μm, and the single finger turns are 5 turns. A titanium gold electrode layer was then deposited on the silicon substrate using an electron beam evaporation method, wherein the titanium thickness was 5nm and the gold thickness was 45nm. And finally, preparing the silicon wafer with the spiral interdigital electrode on the surface through a stripping process.
(3) Electrochemical wet transfer of graphene thin films onto silicon substrates with electrodes: and (3) heating and drying the surface PMMA with the single-layer graphene on the copper foil, shearing off the peripheral edge part of the copper foil, and drawing a penetrating straight line at a position close to a measured edge by using a sharp object. The copper foil is held by a wire with alligator clips at the narrower side of the line. A copper foil is used as a cathode, a platinum electrode or a carbon electrode is used as an anode, a NaOH solution with the concentration of 1mol/L is used as an electrolyte, and a voltage of 3.5V is applied. And placing the anode in electrolyte, slowly extending the cathode copper foil into the electrolyte, and keeping the separation line of PMMA and the copper foil at the liquid level position all the time. And finally, stripping the PMMA from the copper foil to finish stripping. And obtaining the PMMA film with the single-layer graphene on the lower surface. And (3) cleaning and drying the PMMA sheet with the single-layer graphene by using deionized water, transferring the single-layer graphene onto a silicon substrate with an electrode, heating and softening the PMMA at 160 ℃, removing the PMMA by using acetone, cleaning residual PMMA and acetone by using isopropanol, and drying.
(4) Using an electrochemical polymerization method, in-situ polymerizing a polypyrrole thin layer on the single-layer graphene film: preparing an electrolytic cell solution: 0.971g of sodium p-toluenesulfonate was dissolved in 49mL of deionized water, 1mL of 1mol/L hydrochloric acid and 350. Mu.L of pyrrole were added, and after stirring uniformly, the mixture was sonicated for 10min. And connecting a working electrode to an electrode contact on the silicon wafer, and constructing an electrolytic cell by using a platinum sheet electrode as an auxiliary electrode. The electrochemical workstation working mode is set to be a constant voltage test mode, the voltage is set to be 0.7V, and the polymerization time is 30min. And taking down the silicon wafer after polymerization, and cleaning the silicon wafer by acetone, isopropanol and deionized water in sequence. And naturally air-drying to avoid damaging the composite film.
(5) The device channel region protection layer is prepared using ultraviolet lithography. Setting the air cavity pressure at 50Pa by using an argon plasma etching technology, introducing argon with the pressure of 2sccm, etching for 5min to remove the unprotected graphene/polypyrrole composite film, and finally obtaining the lactate ion sensing device with the channel material of a single-layer graphene/polypyrrole composite film;
in this embodiment, the polymerization voltage is reduced and the polymerization time is increased. The lactate ion sensing device of the single-layer graphene/polypyrrole layered composite ion sensitive material prepared by the method has the average polypyrrole sphere cluster size of 50nm, is uniformly distributed, and has the sensitivity and detection range basically consistent with those of the embodiment 1.
Comparative example 1:
a method for preparing a lactate ion sensor based on graphene/polypyrrole, comprising the following steps:
(1) Preparing a single-layer graphene film by using a chemical vapor deposition method, wherein the prepared graphene is a single layer.
(2) Constructing an electrode pattern on a silicon wafer with a 300nm oxide layer by using ultraviolet lithography, wherein the size parameters of the electrode pattern are as follows: the finger width is 30 μm, the finger spacing is 60 μm, and the single finger turns are 5 turns. A titanium gold electrode layer was then deposited on the silicon substrate using an electron beam evaporation method, wherein the titanium thickness was 5nm and the gold thickness was 45nm. And finally, preparing the silicon wafer with the spiral interdigital electrode on the surface through a stripping process.
(3) Electrochemical wet transfer of graphene thin films onto silicon substrates with electrodes: and heating and drying the surface P MMA with the single-layer graphene on the copper foil, shearing off the peripheral edge part of the copper foil, and drawing a penetrating straight line at a position close to a measured edge by using a sharp object. The copper foil is held by a wire with alligator clips at the narrower side of the line. A copper foil is used as a cathode, a platinum electrode or a carbon electrode is used as an anode, a NaOH solution with the concentration of 1mol/L is used as an electrolyte, and a voltage of 3.5V is applied. And placing the anode in electrolyte, slowly extending the cathode copper foil into the electrolyte, and keeping the separation line of PMMA and the copper foil at the liquid level position all the time. And finally, stripping the PMMA from the copper foil to finish stripping. And obtaining the PMMA film with the single-layer graphene on the lower surface. And (3) cleaning and drying the PMMA sheet with the single-layer graphene by using deionized water, transferring the single-layer graphene onto a silicon substrate with an electrode, heating and softening the PMMA at 160 ℃, removing the PMMA by using acetone, cleaning residual PMMA and acetone by using isopropanol, and drying.
(4) Using an electrochemical polymerization method, in-situ polymerizing a polypyrrole thin layer on the single-layer graphene film: preparing an electrolytic cell solution: 50mL of 1mol/L sodium lactate solution is prepared, 1mL of 1mol/L hydrochloric acid and 350 mu L of pyrrole are added, and after uniform stirring, ultrasonic treatment is performed for 10min. And connecting a working electrode to an electrode contact on the silicon wafer, and constructing an electrolytic cell by using a platinum sheet electrode as an auxiliary electrode. The electrochemical workstation working mode is set to be a constant voltage test mode, the voltage is set to be 0.9V, and the polymerization time is 20min. And taking down the silicon wafer after polymerization, and cleaning the silicon wafer by acetone, isopropanol and deionized water in sequence. And naturally air-drying to avoid damaging the composite film.
(5) The device channel region protection layer is prepared using ultraviolet lithography. Setting the air cavity pressure at 50Pa by using an argon plasma etching technology, introducing argon with the pressure of 2sccm, etching for 5min to remove the unprotected graphene/polypyrrole composite film, and finally obtaining the lactate ion sensing device with the channel material of a single-layer graphene/polypyrrole composite film;
in the comparative example, the lactate ion sensing device of the single-layer graphene/polypyrrole layered composite ion sensitive material prepared by the method has the average size of polypyrrole clusters of 90nm, and clusters are aggregated (as shown in the right graph of the attached figure 3).
Conclusions can be drawn by comparing the test results: the response degree of the sensor prepared under the electrolyte environment with the sodium paratoluenesulfonate as the emulsifier is about three times that of the sensor prepared by the invention with the sodium lactate as the emulsifier in the comparative example, and the sensitivity is the first in the prior reported sensor. And under the condition of multiple liquid environment changes, current changes can be rapidly generated, and the sensor has strong stability and repeatability. And as can be illustrated by the figure 3, the sodium paratoluenesulfonate can lead the distribution of the polypyrrole clusters to be more uniform, and the sensing sensitivity is obviously improved.
Comparative example 2:
a method for preparing a lactate ion sensor based on graphene/polypyrrole, comprising the following steps:
(1) Preparing a single-layer graphene film by using a chemical vapor deposition method, wherein the prepared graphene is a single layer.
(2) Constructing an electrode pattern on a silicon wafer with a 300nm oxide layer by using ultraviolet lithography, wherein the size parameters of the electrode pattern are as follows: the finger width is 30 μm, the finger spacing is 60 μm, and the single finger turns are 5 turns. A titanium gold electrode layer was then deposited on the silicon substrate using an electron beam evaporation method, wherein the titanium thickness was 5nm and the gold thickness was 45nm. And finally, preparing the silicon wafer with the spiral interdigital electrode on the surface through a stripping process.
(3) Transferring the graphene film onto a silicon substrate with an electrode by a substrate etching method: spin-coating PMMA on the surface of the copper foil with the single-layer graphene, heating and drying, and then immersing the PMMA-free surface of the copper foil into 0.5mol/L ammonium persulfate solution for corrosion to obtain the PMMA film with the single-layer graphene on the lower surface. And (3) cleaning and drying the PMMA sheet with the single-layer graphene by using deionized water, transferring the single-layer graphene onto a silicon substrate with an electrode, heating and softening the PMMA at 165 ℃, removing the PMMA by using acetone, cleaning residual PMMA and acetone by using isopropanol, and drying.
(4) Using an electrochemical polymerization method, in-situ polymerizing a polypyrrole thin layer on the single-layer graphene film: preparing an electrolytic cell solution: 0.971g of sodium p-toluenesulfonate was dissolved in 49mL of deionized water, 1mL of 1mol/L hydrochloric acid and 350. Mu.L of pyrrole were added, and after stirring uniformly, the mixture was sonicated for 10min. And connecting a working electrode to an electrode contact on the silicon wafer, and constructing an electrolytic cell by using a platinum sheet electrode as an auxiliary electrode. The electrochemical workstation working mode is set to be a constant voltage test mode, the voltage is set to be 0.9V, and the polymerization time is 20min. And taking down the silicon wafer after polymerization, and cleaning the silicon wafer by acetone, isopropanol and deionized water in sequence. And naturally air-drying to avoid damaging the composite film.
(5) The device channel region protection layer is prepared using ultraviolet lithography. Setting the air cavity pressure at 50Pa by using an argon plasma etching technology, introducing argon with the pressure of 2sccm, etching for 5min to remove the unprotected graphene/polypyrrole composite film, and finally obtaining the lactate ion sensing device with the channel material of a single-layer graphene/polypyrrole composite film;
in the comparative example, the single-layer graphene obtained by the method in (3) has copper residues, and is particularly obvious at the edge positions. The residue reduces the purity and flatness of the graphene, influences the subsequent polymerization process in (4), reduces the thickness controllability of polypyrrole polymerization, and reduces the performance of the sensor. Meanwhile, the residue causes the impurity of the whole sensing material and also has adverse effect on the performance of the sensor. Therefore, the stability of the manufacturing quality of the sensor and the performance thereof can be ensured by adopting an electrochemical stripping method in wet transfer.
Comparative example 3:
a method for preparing a lactate ion sensor based on graphene/polypyrrole, comprising the following steps:
(1) Preparing a single-layer graphene film by using a chemical vapor deposition method, wherein the prepared graphene is a single layer.
(2) Constructing an electrode pattern on a silicon wafer with a 300nm oxide layer by using ultraviolet lithography, wherein the size parameters of the electrode pattern are as follows: the finger width is 30 μm, the finger spacing is 60 μm, and the single finger turns are 5 turns. A titanium gold electrode layer was then deposited on the silicon substrate using an electron beam evaporation method, wherein the titanium thickness was 5nm and the gold thickness was 45nm. And finally, preparing the silicon wafer with the spiral interdigital electrode on the surface through a stripping process.
(3) Electrochemical wet transfer of graphene thin films onto silicon substrates with electrodes: and (3) heating and drying the surface PMMA with the single-layer graphene on the copper foil, shearing off the peripheral edge part of the copper foil, and drawing a penetrating straight line at a position close to a measured edge by using a sharp object. The copper foil is held by a wire with alligator clips at the narrower side of the line. A copper foil is used as a cathode, a platinum electrode or a carbon electrode is used as an anode, a NaOH solution with the concentration of 1mol/L is used as an electrolyte, and a voltage of 3.5V is applied. And placing the anode in electrolyte, slowly extending the cathode copper foil into the electrolyte, and keeping the separation line of PMMA and the copper foil at the liquid level position all the time. And finally, stripping the PMMA from the copper foil to finish stripping. And obtaining the PMMA film with the single-layer graphene on the lower surface. And (3) cleaning and drying the PMMA sheet with the single-layer graphene by using deionized water, transferring the single-layer graphene onto a silicon substrate with an electrode, heating and softening the PMMA at 160 ℃, removing the PMMA by using acetone, cleaning residual PMMA and acetone by using isopropanol, and drying.
(4) In-situ polymerizing a polypyrrole thin layer on a monolayer graphene film using cyclic voltammetry mode polymerization: preparing an electrolytic cell solution: 0.971g of sodium p-toluenesulfonate was dissolved in 49mL of deionized water, 1mL of 1mol/L hydrochloric acid and 350. Mu.L of pyrrole were added, and after stirring uniformly, the mixture was sonicated for 10min. And connecting a working electrode to an electrode contact on the silicon wafer, and constructing an electrolytic cell by using a platinum sheet electrode as an auxiliary electrode. Setting an electrochemical workstation working mode as a cyclic voltammetry mode, setting a first potential as 0V, setting a second potential as 0.65V, setting a scanning step length as 1mV, setting a scanning speed as 5mV, and setting the number of cycles as 5. And taking down the silicon wafer after polymerization, and cleaning the silicon wafer by acetone, isopropanol and deionized water in sequence. And naturally air-drying to avoid damaging the composite film.
(5) The device channel region protection layer is prepared using ultraviolet lithography. Setting the air cavity pressure at 50Pa by using an argon plasma etching technology, introducing argon with the pressure of 2sccm, etching for 5min to remove the unprotected graphene/polypyrrole composite film, and finally obtaining the lactate ion sensing device with the channel material of a single-layer graphene/polypyrrole composite film;
in this comparative example, the uniformity of the polypyrrole film obtained by the cyclic voltammetry polymerization in (4) was significantly worse (compared with the use of example 1), and particularly the thickness of the portion of the polypyrrole film near the electrode was significantly increased. Such non-uniformity results in non-uniformity of the sensing material as a whole, which adversely affects the stability of the sensor manufacturing quality and its performance.
Comparative example 4:
the present comparative example is based on a carbon nanotube/polypyrrole composite fiber-based transistor sensor:
(1) Preparing a carbon nanotube dispersion liquid: taking 5g of carbon nano tube, 10g of sodium dodecyl sulfate and 100g of deionized water respectively, mixing, and performing ultrasonic treatment for 1 hour to obtain a carbon nano tube dispersion liquid;
(2) Preparing flexible fiber with surface coated with carbon nano tube layer: repeatedly soaking acrylic fiber with the surface subjected to ultrasonic cleaning by ethanol in the carbon nano tube dispersion liquid in the step 1) for 2 hours, taking out, washing with water, and drying to obtain flexible fiber with the surface coated with the carbon nano tube layer;
(3) Preparing a mixed solution A: 2.5g of anthraquinone-2, 7-disulfonic acid disodium salt and 8.0g of pyrrole monomer are dissolved in deionized water to obtain 250mL of mixed solution A;
(4) Preparing a mixed solution B: 45g of 9-ferric nitrate hydrate and 30g of 5-sulfosalicylic acid are dissolved in deionized water to obtain 150mL of mixed solution B;
(5) Preparing a carbon nano tube/polypyrrole composite fiber: taking the flexible fiber coated with the carbon nano tube layer on the surface of the step (2) with the length and the width of 20cm, placing the flexible fiber in 250mL of mixed solution A, stirring for 10 minutes at room temperature, then dripping 150mL of mixed solution B, controlling the stirring speed to be 800r/min under the water bath condition of 0 ℃, stirring and reacting for 4 hours, taking out, adopting ethanol and deionized water for cleaning, and then air-drying at normal temperature to obtain the carbon nano tube/polypyrrole composite fiber;
(6) Preparing electrolyte: mixing 1g of polyvinyl alcohol, 1g of phosphoric acid and 10mL of deionized water, and stirring and dissolving at the temperature of 60 ℃ to obtain electrolyte;
(7) Preparing a carbon nano tube/polypyrrole composite fiber-based transistor sensor:
(8) Preparing source and drain electrodes of a transistor sensor: the carbon nano tube/polypyrrole composite fiber prepared in the step (5) with the length of 2cm is taken, conductive silver paste is smeared at two ends along the length, the length of 2mm is reserved in the middle and is not smeared, and then the fiber smeared with the conductive silver paste is fixed on a PVC plastic plate to be used as a source electrode and a drain electrode of a transistor sensor;
(9) Preparing a gate electrode of a transistor sensor: taking the carbon nano tube/polypyrrole composite fiber prepared in the step (5) with the length of 2cm, soaking the carbon nano tube/polypyrrole composite fiber in a lactic acid oxidase solution with the concentration of 10mg/mL for 24 hours under the ice bath condition, continuously soaking the carbon nano tube/polypyrrole composite fiber in a perfluorosulfonic acid solution with the mass percent concentration of 2% for 24 hours under the ice bath condition after taking out, taking out the carbon nano tube/polypyrrole composite fiber, and air-drying the carbon nano tube/polypyrrole composite fiber under the ice bath condition to obtain the gate electrode of the transistor sensor;
(10) Assembly of the transistor sensor: and (3) arranging the source electrode and the drain electrode in the step (7) in a crisscross manner, and smearing the electrolyte configured in the step (6) for blocking the direct contact of the two electrodes at the crossing point of the crisscross manner, so that the carbon nano tube/polypyrrole composite fiber-based transistor sensor is obtained.
This comparative example requires the immobilization of oxidase and is relatively complicated to manufacture. Meanwhile, the detection range of the lactic acid sensor prepared by the comparative example method is as follows: 1nM to 1mM, the detection range is much smaller than 10mM to 110mM of the present invention. The invention has obvious improvement on the detection range.
Comparative example 5:
in this comparative example, lactic acid in artificial sweat was detected and the annular wearing model was printed out with a 3D printing technique as a cavity ring of epoxy resin having an outer radius of 15mm, an inner radius of 10mm and a height of 5mm. Sputtering a graphite electrode with the length and the width of 15mm and the thickness of 2mm and the thickness of 1mm on the inner surface of the annular wearing model by utilizing a photoetching technology, wherein the graphite electrode is used as a working electrode; then a silver metal electrode with the same size characteristic as the working electrode is evaporated at the position 2mm away from the working electrode, and the electrode is used for carrying out electrolytic treatment to form a silver/silver chloride electrode which is used as a counter electrode. The AD conversion circuit, the current collection and microprocessor and the button battery with the voltage of 1.5V are installed and fixed in a cavity of the ring of the wearing model, and a 4-bit LED display nixie tube with the width of 1cm and the length of 2cm is fixed on the outer surface of the annular wearing model to be used as a display screen and connected with the microprocessor through the display circuit. The positive electrode of the battery is connected with the working electrode, and the counter electrode is sequentially connected with the current acquisition circuit, the A/D conversion circuit and the microprocessor in series and is connected to the negative electrode of the battery, so that the homeotropic voltage of the electrochemical reaction is ensured. Thereafter, 1. Mu.l of 1% glutaraldehyde and 1. Mu.l of a 10U/. Mu.l of a lactic acid enzyme solution were mixed with a shaker, and then added dropwise to the working electrode until dry and coagulated.
The detection limit of the sensor prepared by the method of the comparative example is 0.00771mM, the concentration range of the sensor can be accurately detected to be 0.00771-4 mM, the relative average error is about 2%, and the detection time is about 2 minutes. The detection range is far smaller than 10 mM-110 mM of the invention, and the detection time is longer than 64s of the invention, so that the invention has obvious improvement on the detection range and the detection time compared with the comparative example.
Comparative example 6:
the comparative example is based on a high-efficiency sensing electrode of a lactic acid sensor, and the preparation method comprises the preparation of LOX protease and the preparation process of electrode materials;
(1) The preparation process of the electrode material comprises the preparation process of graphene oxide and graphene quantum dot composite material and the treatment process of the glassy carbon electrode;
(2) The preparation method of the graphene oxide and graphene quantum dot composite material comprises the steps of dissolving graphene oxide and graphene quantum dot in ultrapure water, wherein the concentration is 1.3mg/mL, and performing ultrasonic treatment for 2 hours; then the obtained solution is reacted for 14 hours at 180 ℃; centrifuging the solution obtained by the reaction at 8000rpm for 6 hours; drying the mixture in a vacuum oven at 60 ℃ for 14 hours;
(3) The particle size of the graphene quantum dots is 5nm, the sheet size of the graphene oxide is 250nm, and the weight ratio of the graphene oxide to the graphene quantum dots is 1:1, a step of;
(4) The treatment process of the glassy carbon electrode comprises the following steps:
A. polishing the glassy carbon electrode by using alumina to obtain a glassy carbon electrode with a mirror surface, carrying out ultrasonic treatment on the glassy carbon electrode for 3 hours by using a treatment liquid, and drying the glassy carbon electrode under the condition of nitrogen;
B. c, dissolving graphene and graphene quantum dot composite material in ultrapure water, wherein the concentration is 1.0mg/mL, performing ultrasonic treatment for 3 hours, pouring 25 mu L of the graphene and graphene quantum dot composite material on the surface of the material obtained in the step A, and drying at room temperature;
C. dripping 5 mu LLOX protease on the surface of the material obtained in the step B, standing and drying at 4 ℃ for 2.5 hours;
the treatment solution comprises 50% (v/v) nitric acid solution, absolute ethyl alcohol and water, wherein the volume ratio of the nitric acid solution to the absolute ethyl alcohol to the water is 1:1:1, a step of;
(5) The diameter of the glassy carbon electrode is 2mm, and the diameter of the alumina is 0.25 mu m;
(6) The preparation of the LOX protease comprises the following steps:
A. extracting the whole gene of the balloon fungus, designing a pair of primers 5' -GCGCGGCAGCCATATGATGAATA
The target gene LOX is amplified by the ACAATGACATT-3' and 5'-GGTGGTGGTGCTCGAGCTAGTATTCATAACCG-3' through PCR to obtain the target gene sequence;
C. using homologous recombination method, using one-step cloning kit, and water-bathing at 37deg.C for 30min for the purpose obtained in step (1)
The gene of (2) is connected with a starting vector to construct an expression vector;
D. constructing genetically engineered bacteria containing LOX genes by using host bacteria, inoculating the constructed LOX strains into an LB shake flask, culturing for 3.5 hours at 37 ℃, adding 50mM lactose solution 25ul, and culturing for 24 hours at 20 ℃. Expressing a lactate oxidase gene;
E. purifying the recombinant expression lactate oxidase by using a nickel ion column, and concentrating to obtain LOX protease;
the starting vector is pET-28a (+), and the expression vector is pET-LOX; the host bacterium is E.coliBL21 (DE 3), and the genetically engineered bacterium is E.coliBL21 (DE 3-pET-LOX).
The detection range of the comparative example is 0.1 mM-0.8 mM, and the sensitivity is 0.98667 mu AmM -1 The method is significantly lower than the method of the invention, and still utilizes oxidase, and lacks the stability of long-time use and the simplicity of manufacture. The invention has remarkable improvement on the sensitivity and the detection range.
Claims (9)
1. The application of the lactate ion sensor based on graphene/polypyrrole as the detection of the lactate concentration in a liquid environment comprises a substrate, a spiral interdigital electrode and a composite film, wherein the composite film comprises layered graphene and a polypyrrole film coated on the upper surface of the graphene.
2. The use according to claim 1, characterized in that: the transparency of the graphene is greater than 95%, the number of layers of the graphene is smaller than three, and the thickness of the polypyrrole layer is 20-200 nm.
3. The use according to claim 1, characterized in that: the spiral interdigital electrode is in a double spiral line shape, the width of electrode fingers is 10-30 mu m, the interdigital distance is 20-60 mu m, and the number of turns of a single electrode is 2-10 turns.
4. The use according to claim 1, characterized in that: the spiral interdigital electrode is composed of a Ti film and an Au film covered on the Ti film.
5. The use according to claim 1, characterized in that: the graphene/polypyrrole composite film is filled in the interdigital electrode channel and is contacted with the electrodes at the two sides.
6. The use according to claim 1, characterized in that the preparation method of the graphene/polypyrrole-based lactate ion sensor comprises the following steps:
(1) Patterning the pretreated substrate surface through a photoetching process;
(2) Depositing Ti and Au on the patterned substrate by a physical vapor deposition method, and stripping the redundant Ti/Au from the substrate by a stripping process to obtain an interdigital electrode with a spiral shape;
(3) Preparing graphene by a chemical vapor deposition method, and transferring the graphene to the surface of a substrate with an electrode by using wet transfer;
(4) In-situ polymerizing polypyrrole on the graphene film by an electrochemical polymerization method to obtain a graphene/polypyrrole composite film;
(5) And carrying out etching channeling treatment on the graphene/polypyrrole composite film, and connecting two ends of the composite film with electrodes to obtain the lactate ion sensor.
7. The use according to claim 6, characterized in that step (4) comprises the steps of:
(a) Preparing sodium p-toluenesulfonate containing 0.01-0.10 mol/L, adding pyrrole liquid to the concentration of 0.1-0.3 mol/L, adding hydrochloric acid to adjust the pH to 1.5-2.5, and carrying out ultrasonic treatment for 5-20 min to completely emulsify and uniformly disperse;
(b) Connecting a working electrode to an electrode contact on a substrate by an electrochemical workstation, wherein the electrode is a saturated calomel electrode of saturated potassium chloride solution and is used as a reference electrode, and the electrode is a platinum sheet electrode and is used as an auxiliary electrode;
(c) In-situ polymerizing polypyrrole on the surface of the graphene film by using a constant pressure polymerization method: setting the polarization voltage to be 0.7-0.9V, and setting the polarization time to be 20-60 min.
8. The use according to claim 6, characterized in that: in the step (1), the substrate is one of polyethylene terephthalate, polyimide and a silicon wafer with an oxide layer.
9. The use according to claim 6, characterized in that: in step (3), the wet transfer is an electrochemical stripping process.
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