CN110865101A - Flexible and elastic biosensor and application thereof - Google Patents
Flexible and elastic biosensor and application thereof Download PDFInfo
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- CN110865101A CN110865101A CN201910926014.9A CN201910926014A CN110865101A CN 110865101 A CN110865101 A CN 110865101A CN 201910926014 A CN201910926014 A CN 201910926014A CN 110865101 A CN110865101 A CN 110865101A
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- 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
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- 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
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- 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
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
Abstract
The invention discloses a biosensor with flexibility, which comprises a working electrode; the working electrode comprises a flexible elastic conductive composite film substrate and an enzyme fixed on the surface of the flexible elastic conductive composite film substrate; the invention also discloses application of the biosensor in preparation of wearable bioelectronic equipment, which can realize high-sensitivity acquisition of bioelectric signals and physiological and biochemical signals and has the advantages of strong anti-interference capability, high sensitivity, no need of an additional medium and the like.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a flexible biosensor and application thereof.
Background
With the improvement of the living standard of modern people and the aggravation of the aging problem of the population, the real-time detection and long-term monitoring technology of physiological and biochemical indexes of the human body is more and more widely concerned; the wearable biomedical sensing device is developed, physiological and biochemical signals of a human body are monitored in real time, and the wearable biomedical sensing device has important value and great demand in multiple fields of daily health management, health care, diagnosis and treatment rehabilitation and the like; although wearable devices have made some progress in monitoring vital signs of human bodies such as heart rate in recent years, generally, the current wearable sensors have single detection indexes, low detection efficiency and insufficient detection flux, and although high-flux detection can be realized by using the existing biochips, there are still many problems, such as high manufacturing cost of the biochips, additional arrangement of signal detection elements in the detection process, poor flexibility of the sensors, difficulty in making good contact with the skin and acquiring signals in real time, and the like.
Disclosure of Invention
The invention mainly aims to overcome the defects and provide a flexible biosensor and application thereof.
The technical scheme of the invention is as follows:
a biosensor having flexibility, comprising a working electrode; the working electrode comprises a flexible elastic conductive composite film substrate and an enzyme fixed on the surface of the flexible elastic conductive composite film substrate; the flexible elastic conductive composite membrane substrate consists of a conductive nano body and a flexible elastic carrier.
The conductive nano-body adopted by the invention has large specific surface area and excellent conductivity, and the flexible elastic carrier has excellent plasticity and can be made into various shapes, thereby effectively improving the conductivity, the ductility and the biocompatibility of the flexible elastic conductive composite film.
Furthermore, the mass ratio of the conductive nano-bodies to the flexible elastic carrier is 1: 9-9: 1, and the flexibility and the conductivity of the flexible elastic conductive composite film can be adjusted according to the mass ratio of the conductive nano-bodies to the flexible elastic carrier.
Further, the enzyme comprises more than one of horseradish peroxidase, glucose oxidase, lactate oxidase, alcohol oxidase and cholesterol oxidase.
Further, the material of the conductive nano-body comprises more than one of gold nano-material, silver nano-material and carbon nano-material.
Further, the flexible carrier comprises ethylene-vinyl acetate copolymer and polydimethylsiloxane.
Further, the working electrode is prepared by adopting the following steps:
s1, respectively and uniformly dispersing the conductive nano-bodies and the flexible elastic carriers in a good solvent, mixing and continuously performing ultrasonic treatment for 0.5-1 h to obtain conductive ink, and performing self-assembly to obtain a flexible elastic conductive composite film substrate;
s2, dripping 25-100 mu L of solution containing enzyme on the surface of the flexible and elastic conductive composite film substrate, standing for 1.5-2 h at 4-10 ℃, and fixing the enzyme on the surface of the flexible and elastic conductive composite film substrate after a crosslinking reaction to obtain the working electrode.
Among them, xylene and cyclohexane are commonly used as good solvents.
The substrate of the flexible elastic conductive composite membrane has good biocompatibility and can effectively keep the bioactivity of immobilized enzyme.
The invention also provides application of the biosensor with flexibility in preparation of wearable bioelectronic equipment.
Further, the wearable bioelectronic device is applied to acquisition of bioelectric signals and physiological and biochemical signals.
Further, the physiological and biochemical signals include glucose concentration, lactic acid concentration, ethanol concentration and cholesterol concentration.
The biosensor with flexibility provided by the invention adopts a three-electrode system (comprising a working electrode, a counter electrode and a reference electrode) or a two-electrode system (comprising the working electrode and the counter electrode). The counter electrode adopts a platinum electrode or a carbon electrode, but not limited to, and the reference electrode adopts an Ag/AgCl electrode or a saturated calomel electrode.
The working principle of the biosensor with flexibility provided by the invention is as follows: the biological enzyme fixed on the surface of the flexible elastic conductive composite membrane substrate and a substrate to be detected are subjected to catalytic reaction, electron transfer exists in the process, so that the current of an electrode system is changed, the higher the concentration of the substrate to be detected is, the higher the current is, and the concentration of the substrate to be detected can be indirectly calculated by detecting the variable quantity of the current.
The biosensor with flexibility provided by the invention can be used for detecting a substrate which can be specifically catalyzed by enzyme fixed on the surface of the substrate of the flexible and elastic conductive composite membrane, for example, horseradish peroxidase is fixed on the surface of the substrate of the flexible and elastic conductive composite membrane, and the substrate which can be catalyzed is hydrogen peroxide, so that the biosensor can be applied to detecting the concentration of the hydrogen peroxide; if the glucose oxidase and the horseradish peroxidase are fixed on the surface of the substrate of the flexible elastic conductive composite membrane together, and the substrate capable of catalyzing is glucose, the biosensor can be applied to detecting the concentration of the glucose; the biosensor with flexibility of the invention can fix enzymes including, but not limited to, horseradish peroxidase, glucose oxidase, lactate oxidase, alcohol oxidase and cholesterol oxidase.
Compared with the prior art, the invention has the following advantages:
1. the flexible elastic conductive composite membrane is prepared by mutually winding and self-assembling the conductive nano-body and the flexible elastic carrier through hydrophobic interaction at room temperature, the surface of the flexible elastic conductive composite membrane is a compact and uniform three-dimensional porous structure and has higher conductivity, the conductivity of the flexible elastic composite membrane is measured through a linear sweep voltammetry, the flexibility of the flexible elastic composite membrane is characterized through bending, folding, twisting, knotting and the like, and the flexible elastic composite membrane is cut into flexible elastic electrodes, so that the high-sensitivity acquisition of biological electric signals (such as myoelectric signals) can be realized, and the flexible elastic composite membrane has the characteristics of low cost, easiness in processing and the like, and is easy for industrial production.
2. The invention adopts the flexible elastic conductive composite membrane prepared by conductive nano-body and flexible elastic carrier, takes the flexible elastic conductive composite membrane as a substrate, adsorbs and fixes enzyme on the surface of the substrate through cross-linking reaction, and generates direct electron transfer, thereby realizing the direct high-sensitivity acquisition of physiological and biochemical signals, having simple detection method and no need of markingStrong recording and anti-interference ability, high sensitivity (for example, in the detection of hydrogen peroxide, its linear sensitivity can be up to 2 mA mM in the concentration range of 0.1 mM-1 mM-1cm-2The response time is 2-3 s), no additional medium is needed, and the like, and the method can be used for preparing wearable bioelectronic equipment and can be widely applied to the fields of biomedical detection, health monitoring, environmental early warning and the like.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) photograph of a flexible and elastic carbon nanotube composite film according to the present invention;
FIG. 2 is an image of the present invention using a flexible carbon nanotube composite membrane for collecting electromyographic signals;
FIG. 3 is a time-current response (A) and a detection curve (B) of the biosensor having flexibility according to the present invention when a 0.1 mM hydrogen peroxide solution is continuously added;
FIG. 4 is a time-current response (A) and a detection curve (B) of the biosensor having flexibility according to the present invention when a 0.1 mM glucose solution is continuously added;
FIG. 5 is a time-current response (A) and a detection curve (B) of the flexible biosensor of the present invention for glucose concentration detection in sweat;
the implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand for those skilled in the art and will therefore make the scope of the invention more clearly defined.
Example 1
The embodiment provides a flexible biosensor, which is a three-electrode or two-electrode system and comprises a working electrode; the working electrode comprises a flexible elastic conductive composite film substrate and an enzyme fixed on the surface of the flexible elastic conductive composite film substrate; the flexible and elastic conductive composite membrane substrate consists of a carbon nano tube and an ethylene-vinyl acetate copolymer.
The biosensor of this example was used for specific detection of different targets using cyclic voltammetry measurements.
In this embodiment, the working electrode is prepared by the following steps:
weighing 300 mg and 450 mg of Carbon Nanotubes (CNTs) and ethylene-vinyl acetate copolymer (EVAc) respectively, dispersing the carbon nanotubes and the EVAc in a xylene solvent at room temperature (25-30 ℃), continuously performing ultrasonic treatment for 1 h to obtain uniform conductive ink, pouring the conductive ink into a flat glass culture dish, and placing the flat glass culture dish in a fume hood at room temperature (25-30 ℃) for overnight volatilization to remove the solvent to obtain a large-area uniform flexible and elastic carbon nanotube composite membrane, wherein as shown in a figure 1, the Scanning Electron Microscope (SEM) picture of the flexible and elastic carbon nanotube composite membrane is shown, and the large-area uniform and elastic carbon nanotube composite membrane has a uniform and compact three-dimensional porous structure.
Cutting the flexible and elastic carbon nanotube composite film into 1 cm2Processing the lead-bonding part of the flexible elastic electrode, uniformly coating conductive gel on 3 flexible elastic electrodes bonded with leads, placing 2 flexible elastic electrodes on the abdominal part of the tested muscle (such as biceps brachii, triceps brachii, flexor radialis, tibialis anterior muscle, etc.), placing the other 1 flexible elastic electrode on the joint of the tail end of the muscle, and tightly attaching with medical adhesive tape; the 3 electrodes are connected with an electromyographic signal acquisition instrument, an electromyographic test program is started, and the electromyographic signals during muscle relaxation and muscle contraction are respectively tested, as shown in fig. 2, in order to adopt the flexible elastic carbon nanotube composite membrane for acquiring images of the electromyographic signals, high-sensitivity acquisition of the electromyographic signals is realized, and the response time is less than 0.5 s.
Example 2
This example is the same as example 1, and further, the immobilization and direct electron transfer reaction of horseradish peroxidase are realized for detecting the hydrogen peroxide concentration in the solution environment.
The method comprises the following steps of (1) taking a flexible and elastic carbon nanotube composite membrane as a substrate, loading horseradish peroxidase by adopting a glutaraldehyde crosslinking method, specifically, dropwise adding 25 mu L of horseradish peroxidase-glutaraldehyde mixed solution on the surface of the composite membrane, wherein the concentration of the horseradish peroxidase in the mixed solution is 5mg/mL, the concentration of the glutaraldehyde is 2.5%, standing at 4 ℃ for 2 hours, washing the surface with deionized water, and removing unadsorbed horseradish peroxidase to prepare a horseradish peroxidase modified electrode;
the horseradish peroxidase modified electrode is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, 0.1M phosphate buffer solution (pH = 7) is used as electrolyte, and the three electrodes are connected to an electrochemical workstation for electrochemical test.
Adding hydrogen peroxide solution into phosphate buffer solution every 20 s under the condition of 0V vs. Ag/AgCl working voltage to enable the concentration of hydrogen peroxide to rise by 0.1 mM each time, and measuring the current response (see figure 3A) and the working standard curve (see figure 3B) of the horseradish peroxidase sensor to the hydrogen peroxide standard solution by utilizing a current-time mode to realize the linear detection of the hydrogen peroxide concentration range of 0.1-1 mM, wherein the linear sensitivity is 2 mA cm-2mM-1The response time is 2-3 s.
Example 3
The embodiment is the same as the embodiment 1, further, glucose oxidase and horseradish peroxidase are jointly fixed on the surface of the flexible and elastic carbon nanotube composite membrane and used for detecting the concentration of glucose in a solution environment, hydrogen peroxide is generated due to the reaction of the glucose and the glucose oxidase, the hydrogen peroxide is specifically reduced on the surface of an electrode through the horseradish peroxidase, and then the change of system current is caused, the larger the concentration of the glucose is, the larger the current is, and the glucose concentration can be indirectly calculated through the change of the detection current.
Taking a flexible and elastic carbon nanotube composite membrane as a substrate, loading glucose oxidase-horseradish peroxidase by adopting a glutaraldehyde crosslinking method, specifically, dropwise adding 100 mu L of glucose oxidase-horseradish peroxidase-glutaraldehyde mixed solution on the surface of the flexible and elastic carbon nanotube composite membrane, wherein the concentration of the glucose oxidase, the concentration of the horseradish peroxidase and the concentration of the glutaraldehyde in the mixed solution are 2.5 mg/mL, 0.25mg/mL and 2.5% respectively, standing the mixed solution at 10 ℃ for 1.5h, washing the surface with deionized water, and removing unadsorbed glucose oxidase and horseradish peroxidase to prepare a glucose oxidase-horseradish peroxidase double-enzyme modified electrode;
the glucose oxidase-horseradish peroxidase double-enzyme modified electrode is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, 0.1M phosphate buffer solution (pH = 7) is used as electrolyte, and the three electrodes are connected to an electrochemical workstation for electrochemical test.
Under the condition of 0V vs. Ag/AgCl working voltage, adding glucose solution into phosphate buffer solution every 20 s to enable the glucose concentration to rise by 0.1 mM every time, and measuring the current response (see figure 4A) and the working standard curve (see figure 4B) of the glucose oxidase-horseradish peroxidase double-enzyme sensor to the glucose standard solution by using a current-time mode to realize the linear detection of the glucose concentration range of 0.1 mM-1 mM and the linear sensitivity of 250 muA cm-2mM-1The response time is 3-5 s.
Example 4
This example is the same as example 1, and further, used for measuring the glucose concentration in artificial sweat.
Taking a flexible and elastic carbon nanotube composite membrane as a substrate, loading glucose oxidase-horseradish peroxidase by adopting a glutaraldehyde crosslinking method, specifically, dropwise adding 60 mu L of glucose oxidase-horseradish peroxidase-glutaraldehyde mixed solution on the surface of the flexible and elastic carbon nanotube composite membrane, wherein the concentration of the glucose oxidase, the concentration of the horseradish peroxidase and the concentration of the glutaraldehyde in the mixed solution are 2.5 mg/mL, 0.25mg/mL and 2.5% respectively, standing the flexible and elastic carbon nanotube composite membrane at 7 ℃ for 1.5h, washing the surface by using deionized water, and removing unadsorbed glucose oxidase and horseradish peroxidase to prepare a glucose oxidase-horseradish peroxidase double-enzyme modified electrode;
the wearable flexible elastic biosensor is prepared by taking the glucose oxidase-horseradish peroxidase double-enzyme modified electrode as a working electrode, an Ag/AgCl conductive slurry printing electrode as a reference electrode and an enzyme-free modified flexible elastic carbon nanotube composite membrane as a counter electrode, so that the reagent-free detection of the concentration of glucose in artificial sweat is realized;
under the condition of 0V vs. Ag/AgCl working voltage, the wearable flexible elastic biosensor is used for measuring the glucose concentration of different pairsTime-current response (see FIG. 5A) and detection curve (see FIG. 5B) of artificial sweat (0.1% urea, 0.5% NaCl, 0.1% lactic acid, pH 6.5) (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.6 mM, 0.8mM, respectively) at a linear sensitivity of 80 μ A cm for glucose concentrations in sweat ranging from 0.1 mM to 0.8mM-2mM-1。
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A flexible biosensor comprising a working electrode; the working electrode comprises a flexible elastic conductive composite film substrate and an enzyme fixed on the surface of the flexible elastic conductive composite film substrate; the flexible elastic conductive composite membrane substrate consists of a conductive nano body and a flexible elastic carrier.
2. The biosensor with flexibility according to claim 1, wherein the mass ratio of the conductive nanobody to the flexible carrier is 1: 9-9: 1.
3. The biosensor of claim 1, wherein the enzyme comprises one or more of horseradish peroxidase, glucose oxidase, lactate oxidase, alcohol oxidase, and cholesterol oxidase.
4. The biosensor having flexibility according to claim 1, wherein the material of the conductive nano-body includes one or more of gold nano-material, silver nano-material, and carbon nano-material.
5. The biosensor having flexibility according to claim 1, wherein the flexible carrier comprises ethylene-vinyl acetate copolymer, polydimethylsiloxane.
6. The flexible biosensor as claimed in claim 1, wherein the working electrode is prepared by the steps of:
s1, respectively and uniformly dispersing the conductive nano-bodies and the flexible elastic carriers in a good solvent, mixing and continuously performing ultrasonic treatment for 0.5-1 h to obtain conductive ink, and performing self-assembly to obtain a flexible elastic conductive composite film substrate;
s2, dripping 25-100 mu L of solution containing enzyme on the surface of the flexible and elastic conductive composite film substrate, standing for 1.5-2 h at 4-10 ℃, and fixing the enzyme on the surface of the flexible and elastic conductive composite film substrate after a crosslinking reaction to obtain the working electrode.
7. Use of a flexible biosensor in the manufacture of a wearable bioelectronic device, wherein the flexible biosensor is a flexible biosensor according to any one of claims 1-6.
8. Use of the flexible biosensor in the manufacture of a wearable device according to claim 7, wherein the wearable bioelectronic device is used for the acquisition of bioelectric signals, physiological and biochemical signals.
9. Use of a flexible biosensor as claimed in claim 8 in the manufacture of a wearable device, wherein the physiological and biochemical signals comprise glucose, lactate, ethanol and cholesterol concentrations.
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| US20160304929A1 (en) * | 2013-12-05 | 2016-10-20 | Suzhou Institute Of Nano-Tech And Nano-Bionics(Sinano), Chinese Academy Of Sciences | Bio-enzyme sensor capable of super hydrophobic solid-liquid-gas three-phase coexistence and method for preparing same |
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