CN112098491B - Sulfur-doped graphene-based ophthalmic electrochemical sensor, preparation method and application thereof, and myopia monitoring device - Google Patents
Sulfur-doped graphene-based ophthalmic electrochemical sensor, preparation method and application thereof, and myopia monitoring device Download PDFInfo
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Abstract
An eye electrochemical sensor based on sulfur doped graphene, a preparation method and application thereof and a myopia monitoring device can be used for detecting the pathological change degree of myopia. The invention prepares microelectrodes on transparent polyester films by a photoetching method. And then fixing the nano enzyme containing the sulfur doped graphene carrier with high electrochemical activity to a microelectrode through an electrochemical deposition method, so as to construct the nano biosensor based on electrochemical signals. The constructed biosensor is formed by hot pressing, can be worn in eyes directly, is simple and efficient, monitors the myopia degree of a person in real time by tears, and provides a new thought for mechanism discussion and early prevention of myopia.
Description
Technical Field
The invention relates to the technical field of research and development of ophthalmic diagnosis and treatment devices, in particular to an ophthalmic electrochemical sensor based on sulfur-doped graphene, a preparation method and application thereof and a myopia monitoring device.
Background
Myopia is regarded as a national disease in China, and the current myopia population in China exceeds 4.5 hundred million, and almost every three people are near-sighted. The incidence rate of myopia in the young of China is the first in the world. Myopia is an irreversible refractive error, the pathogenesis of which is currently unknown, which presents a great challenge for early prevention and monitoring of myopia. Tear fluid is a transparent liquid secreted by the lacrimal gland and has a complex composition. The normal tear secretion has a plurality of functions such as barrier, bacteriostasis, sterilization, immunoregulation and the like. The components of tears often have a close relationship with certain metabolites in the blood. It has been found that changes in tear composition are often a sign of many ocular diseases, even systemic diseases. For example, changes in glucose levels in tears are associated with the development of diabetes, nucleotide levels in tears are associated with cataracts, and changes in dopamine levels in tears are also closely associated with the development of myopia. Dopamine is an important hormonal and neurotransmission substance that helps cells deliver pulsed chemicals. Previous studies have found that dopamine is one of the major neuroactive substances in the vertebrate retina, and plays a dominant role in the formation of form deprived myopia and lens-induced refractive error.
Therefore, the ocular biosensor with high sensitivity is designed based on tears and is used for monitoring the change of the concentration of dopamine in tears in real time, is hopeful to provide a simple, efficient and noninvasive clinical thought and technical means for early prevention and monitoring of myopia and provides a possible mechanism explanation for the occurrence of myopia. On the other hand, the content of the active ingredient in the tear fluid is very rare relative to that in the blood. For example, the content of dopamine in tears is less than one tenth that in serum, but the content of major interferents such as ascorbic acid, urea, etc. is the same. Thus, the sensitivity and selectivity requirements for tear biosensors are necessarily very high, which is also currently one of the most important factors restricting the popularity of ophthalmic biosensors.
Disclosure of Invention
Aiming at the problems that high-efficiency real-time monitoring, early prevention and the like cannot be realized for serious eye diseases such as myopia and the like, the invention provides the sulfur-doped graphene-based eye electrochemical sensor, the preparation method and the application thereof and the myopia monitoring device, which can monitor the concentration change of important components in tears such as dopamine and the like and the obtained change condition of the degree of myopia of a person in real time, thereby establishing the linear relation between the degree of myopia of the person and the output current of the eye sensor and providing a new thought for real-time monitoring and early prevention of myopia.
The technical scheme adopted by the invention is as follows: the eye electrochemical sensor based on the sulfur-doped graphene comprises a nanoenzyme containing a sulfur-doped graphene carrier with high electrochemical activity and a microelectrode, wherein the nanoenzyme of the sulfur-doped graphene carrier is covered on the surface of the microelectrode.
The transparent polymer is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
The thickness of the transparent polymer is 50-100 micrometers.
A preparation method of an ophthalmic electrochemical sensor based on sulfur-doped graphene comprises the following steps:
(1) Preparation of microelectrodes: firstly, carrying out oxygen ion treatment on a transparent polymer film, then spin-coating a layer of photoresist, fixing and forming a photoresist sheet, using a photoetching machine, irradiating for 30-60 seconds by using an ultraviolet lamp under the shielding of a mask, then fixing for 70-120 seconds in a developing solution, removing the photoresist of which the ultraviolet irradiation part is washed away, removing the mask to obtain a micro-patterned photoresist, spraying a layer of platinum metal by using a magnetron sputtering instrument, then washing by using a glue washing solution, and removing redundant photoresist to obtain a micro-patterned microelectrode;
(2) Preparing a sulfur-doped graphene carrier material: mixing and ball milling solid graphite powder and a sulfur-containing conductive high molecular monomer or a sulfur-containing conductive polymer, continuously ball milling for 3-6 hours at a rotating speed of 300-500 rpm, and then performing low-speed centrifugation at 500-1,000 rpm and high-speed centrifugation at 8,000-12,000 rpm on a sample to remove impurities to prepare a sulfur-doped graphene nano carrier;
(3) Preparation of enzyme electrode: dispersing a sulfur-doped graphene carrier material into an aqueous solution of sodium dodecyl sulfate, then adding tyrosinase, magnetically stirring for 30 minutes at 4 ℃, fully and uniformly mixing, depositing the material onto a microelectrode by an electrochemical cyclic voltammetry electrochemical method, and circulating for 50-150 times at a voltage scanning speed of 0.05V/s to prepare an active part enzyme electrode of the sensor;
(4) Preparation of an ophthalmic corneal electrode sensor: and performing thermal compression molding on the prepared nano enzyme electrode at 200 ℃ in a nitrogen environment to form 3-5 h, so as to obtain the cornea electrode sensor for eyes.
The transparent polymer is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
The thickness of the transparent polymer is 50-100 micrometers.
The treatment time of the transparent polymer film treated by oxygen ions in the step (1) is 30-100 seconds, and the power is 10-50 watts.
The photoresist in the step (1) is AZ4620 photoresist with the thickness of 10-50 micrometers, and is baked for 10-20 minutes at the temperature of 80 ℃ for fixed forming.
The thickness of the platinum metal layer sprayed on the microelectrode in the step (1) is 100-300 nanometers.
The sulfur-containing conductive high molecular monomer in the step (2) is 3, 4-ethylenedioxythiophene, and the sulfur-containing conductive polymer is polyphenylene sulfide.
An application of an ophthalmic electrochemical sensor based on sulfur doped graphene as a device for monitoring the concentration of dopamine in tears in real time.
A myopia monitoring device comprises a module for monitoring the concentration of dopamine in tears in real time.
The module for monitoring the concentration of dopamine in tears in real time comprises the sulfur doped graphene-based ophthalmic electrochemical sensor.
The beneficial effects of the invention are as follows: the invention provides an eye electrochemical sensor based on sulfur doped graphene, a preparation method and application thereof and a myopia monitoring device, wherein a specific bioactive enzyme is loaded on a unique ultra-large specific surface area of a two-dimensional graphene nano carrier, so that the loading capacity of the enzyme on a sensing electrode is improved, the selectivity and the specificity of the biosensor are effectively improved, and an electron cloud is deviated from a large pi bond of the graphene by doping graphene on edges such as sulfur-containing macromolecules (such as polythiophene and phenyl sulfide) and the like, so that a high electrochemical active site is obtained, the sensitivity of the biosensor is effectively improved, and a wearable, high-performance and electrochemical signal-based nano biosensor is constructed on an autonomously designed cornea microelectrode, so that real-time monitoring of dopamine concentration in tears and myopia degree change conditions is realized, and a novel detection tool and a novel technical idea are provided for clinical early monitoring and preventing serious eye diseases such as myopia.
Drawings
Fig. 1 is a schematic diagram of the preparation of an ophthalmic nano-biosensor and a photograph of the device. (a) schematic preparation of nano microelectrodes; (b) a photograph of the prepared nano microelectrode; (c) After hot press molding, the ocular nano-electrode is connected with a photo after three electrodes; the illustration (c) is an ophthalmic nano-electrode obtained after hot press molding.
Fig. 2 is a graph showing the result of monitoring the change of the concentration of dopamine after the prepared ocular nano-biosensor is worn in eyes of New Zealand white rabbits. (a) After instilling different concentrations of dopamine/artificial tear in rabbit eyes, the current signal detected on the ocular sensor changes at a constant voltage of +0.2v. (b) a calibration curve derived from the results of (a). (c) Under the same conditions, 5 μm dopamine, 1 mM glucose, 1 mM uric acid, 1 mM ascorbic acid, and 5 μm dopamine were added dropwise, respectively, and the current signal detected on the ocular sensor was changed.
Fig. 3 is a result of collecting tear samples from the eyes of patients with different myopia degrees (from 0 to 800 degrees) and testing on prepared ocular nano-biosensors. (a) Tears of different myopic degrees change in current signal detected on the ocular sensor at a constant voltage of +0.2v. (b) a calibration curve derived from the results of (a).
Detailed Description
The technical scheme of the invention is a conventional scheme in the field unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available.
The invention will be better illustrated with reference to the following drawings and specific examples.
Example 1.
Preparation of an ophthalmic electrochemical sensor based on sulfur-doped graphene and application of the ophthalmic electrochemical sensor in myopia monitoring, the preparation method comprises the following steps:
as shown in fig. 1, the constructed biosensor mainly includes the following important parts: (1) Micropatterned microelectrodes are prepared by photolithography on transparent polymer films (such as PET, PMMA, etc.). The microelectrode is provided with three connectors for connecting a working electrode, a counter electrode and a reference electrode. The active portion of the microelectrode is used to deposit graphene nanocarriers and enzymes. The prepared microelectrodes were cut according to the morphology and size of the cornea (e.g. 1-1.2 cm diameter) and then thermoformed (e.g. FIG. 1 c). And connecting electrodes through low-temperature soldering tin for electrochemical deposition of the sulfur-containing graphene nano-carrier and the enzyme.
Example 2.
As shown in fig. 2, after artificial tears containing different dopamine concentrations are instilled into eyes of new zealand white rabbits, the ocular nano-biosensor prepared in example 1 is worn in the eyes of the rabbits, and the change of the dopamine concentration in the tears can be monitored in real time. Fig. 2 (a) shows that dopamine in the eye will be specifically oxidized by enzymes on the sensor, thereby outputting an oxidation current that is detected by the ocular sensor. The current output detected by the ocular sensor increases with increasing concentration of dopamine added dropwise in the eye, and the two are in good linear relationship (R 2 = 0.9739, fig. 2 b). As can be seen from the selective experimental results of FIG. 2 (c), the prepared ophthalmic biosensor has good anti-interference performance and can specifically detect dopamine in tear.
Example 3.
The prepared ophthalmic sensor can be used for efficiently detecting tears of myopic patients
Tear samples were taken from the eyes of myopes of different myopes for detection by the ophthalmic sensors prepared in example 1. As can be seen from fig. 3 (a), the current intensity detected by the prepared ophthalmic sensor varies sensitively with the variation of myopia degree. Fig. 3 (b) further shows that there is a good potential relationship between the current change detected on the ocular sensor and the increase in myopic degree. These results fully indicate that the prepared wearable ocular nano-biosensor is expected to be used for early monitoring of myopia. And by constructing big data comparison between myopia degree and current intensity, the occurrence of myopia is expected to be well predicted and prevented, and a new technical method is provided for myopia prevention, control and early treatment.
According to the invention, the specific bioactive enzyme is loaded through the ultra-large specific surface area of the two-dimensional graphene nano-sheet, so that the selectivity and the specificity of the nano-biosensor are effectively improved; the edges of the sulfur-containing polymer (such as polythiophene and phenyl sulfide) are doped with graphene, so that electron cloud is offset from the large pi bond of the graphene, high electrochemical active sites are obtained, and the sensitivity of the biosensor is effectively improved. On the basis, a nanometer enzyme is loaded on the surface of the sulfur-doped graphene carrier by a mild and controllable electrochemical coprecipitation method, and is fixed on an autonomously designed cornea microelectrode, so that a wearable, high-performance and electrochemical signal-based nanometer biosensor is constructed, and the nanometer biosensor is used for monitoring the content of dopamine in tears and the change of myopia degree in real time, and provides a new detection tool and a new technical idea for ophthalmic clinical early monitoring and myopia prevention and other important eye diseases.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (8)
1. The ophthalmic electrochemical sensor based on the sulfur-doped graphene is characterized by comprising a nanoenzyme containing a sulfur-doped graphene carrier with high electrochemical activity and a microelectrode, wherein the nanoenzyme of the sulfur-doped graphene carrier is covered on the surface of the microelectrode, and the ophthalmic electrochemical sensor is prepared by the following steps: (1) preparation of microelectrodes: firstly, carrying out oxygen ion treatment on a transparent polymer film, then spin-coating a layer of photoresist, fixing and forming a photoresist sheet, using a photoetching machine, irradiating for 30-60 seconds by using an ultraviolet lamp under the shielding of a mask, then fixing for 70-120 seconds in a developing solution, removing the photoresist of which the ultraviolet irradiation part is washed away, removing the mask to obtain a micro-patterned photoresist, spraying a layer of platinum metal by using a magnetron sputtering instrument, then washing by using a glue washing solution, and removing redundant photoresist to obtain a micro-patterned microelectrode;
(2) Preparing a sulfur-doped graphene carrier material: mixing and ball milling solid graphite powder and a sulfur-containing conductive high molecular monomer or a sulfur-containing conductive polymer, continuously ball milling for 3-6 hours at a rotating speed of 300-500 rpm, and then performing low-speed centrifugation at 500-1,000 rpm and high-speed centrifugation at 8,000-12,000 rpm on a sample to remove impurities to prepare a sulfur-doped graphene nano carrier;
(3) Preparation of enzyme electrode: dispersing a sulfur-doped graphene carrier material into an aqueous solution of sodium dodecyl sulfate, then adding tyrosinase, magnetically stirring for 30 minutes at 4 ℃, fully and uniformly mixing, depositing the material onto a microelectrode by an electrochemical cyclic voltammetry electrochemical method, and circulating for 50-150 times at a voltage scanning speed of 0.05V/s to prepare an active part enzyme electrode of the sensor;
(4) Preparation of an ophthalmic corneal electrode sensor: and performing thermal compression molding on the prepared nano enzyme electrode at 200 ℃ in a nitrogen environment to form 3-5 h, so as to obtain the cornea electrode sensor for eyes.
2. The sulfur-doped graphene-based ocular electrochemical sensor of claim 1, wherein the microelectrode is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
3. The sulfur-doped graphene-based ocular electrochemical sensor of claim 1, wherein the microelectrode has a thickness of 50-100 microns.
4. The sulfur-doped graphene-based ocular electrochemical sensor of claim 1, wherein the transparent polymer is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
5. The sulfur-doped graphene-based ocular electrochemical sensor of claim 1, wherein the transparent polymer has a thickness of 50-100 microns.
6. The sulfur-doped graphene-based ophthalmic electrochemical sensor according to claim 1, wherein the sulfur-containing conductive polymer monomer in the step (2) is 3, 4-ethylenedioxythiophene, and the sulfur-containing conductive polymer is polyphenylene sulfide.
7. Use of the sulfur-doped graphene-based ocular electrochemical sensor of claim 1 as a device for monitoring the concentration of dopamine in tears in real time.
8. A myopia monitoring device, which is characterized by comprising a module for monitoring the concentration of dopamine in tear in real time, wherein the module for monitoring the concentration of dopamine in tear in real time comprises the sulfur-doped graphene-based ophthalmic electrochemical sensor as claimed in claim 1.
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| CN106793943A (en) * | 2014-04-15 | 2017-05-31 | 明眸科技股份有限公司 | The system and method for feature contact lenses and correlation |
| CN107037093A (en) * | 2017-03-02 | 2017-08-11 | 辽宁科技大学 | A kind of tyrosinase electrochemica biological sensor and its preparation, application process |
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