CN112098491A - 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 myopic lesion degree. The invention prepares a microelectrode on a transparent polyester film by a photoetching method. And then fixing the nano enzyme containing the sulfur-doped graphene carrier with high electrochemical activity to a microelectrode by an electrochemical deposition method, thereby constructing the nano biosensor based on electrochemical signals. The constructed biosensor is subjected to hot-press molding, can be directly worn in eyes, simply, conveniently and efficiently monitors the myopia degree of a person in real time through tears, and provides a new idea for myopia mechanism discussion and early prevention.
Description
Technical Field
The invention relates to the technical field of research and development of diagnosis and treatment devices for eyes, in particular to an electrochemical sensor for eyes 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, the number of myopia population in China currently exceeds 4.5 hundred million, and one myopia in almost every three people is the myopia. The incidence of myopia in the young years of our country is the first to live in the world. Myopia, an irreversible refractive error, the pathogenesis of which remains unknown, presents significant challenges to the early prevention and monitoring of myopia. Tears are clear liquids secreted by the lacrimal gland with complex compositions. The normal tear secretion has multiple functions of barrier, bacteriostasis, sterilization, immunoregulation and the like. The components in tears are often closely related to certain metabolites in the blood. It has been found that changes in tear composition are often a signal for many eye diseases, even systemic diseases. For example, changes in the glucose content of tears are associated with the development of diabetes, nucleotide concentrations in tears are associated with cataracts, and changes in the dopamine content of tears are also closely associated with the development of myopia. Dopamine is an important hormone and neurotransmission substance to help cells deliver pulsatile chemicals. Previous studies found that dopamine is one of the major neuroactive substances in vertebrate retinas and plays a dominant role in the development of form-deprivation myopia and lens-induced ametropia.
Therefore, the high-sensitivity ocular biosensor designed based on the tears is used for monitoring the change of the concentration of dopamine in the tears in real time, is expected to provide a simple, convenient, 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 tears is very rare relative to that in blood. For example, the content of dopamine in tears is less than one tenth of that in serum, but the content of major interferents such as ascorbic acid, urea, etc. is the same. Therefore, the requirements for sensitivity and selectivity of tear biosensors are necessarily very high, which is also one of the most important factors currently restricting the popularity of ocular biosensors.
Disclosure of Invention
In order to solve the problems that high-efficiency real-time monitoring and early prevention cannot be achieved for major eye diseases such as myopia and the like, the invention provides the sulfur-doped graphene-based electrochemical sensor for eyes, the preparation method and the application thereof and the myopia monitoring device, which can monitor the concentration change of important components such as dopamine in tears in real time and the change situation of the myopia degree of a person obtained from the concentration change, so that the linear relation between the myopia degree of the person and the output current of the sensor for eyes is established, and a new thought is provided for the real-time monitoring and early prevention of myopia.
The technical solution adopted by the invention is as follows: an eye electrochemical sensor based on sulfur-doped graphene comprises nanoenzyme containing a sulfur-doped graphene carrier with high electrochemical activity and a microelectrode, wherein the nanoenzyme of the sulfur-doped graphene carrier covers 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 microns.
A preparation method of an ophthalmic electrochemical sensor based on sulfur-doped graphene comprises the following steps:
(1) preparing a microelectrode: firstly, treating a transparent polymer film by oxygen ions, then spin-coating a layer of photoresist, fixing and forming a photoresist sheet, then using a photoetching machine to irradiate for 30-60 seconds under the shielding of a mask plate by using an ultraviolet lamp, then fixing for 70-120 seconds in a developing solution, removing and cleaning the photoresist on the ultraviolet irradiation part, removing the mask plate to obtain a micro-patterned photoresist, spray-coating a layer of platinum metal by using a magnetron sputtering instrument, then cleaning by using a glue washing solution, and removing the redundant photoresist to obtain a micro-patterned microelectrode;
(2) preparing a sulfur-doped graphene carrier material: blending 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 the rotating speed of 300-;
(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 mixture on a microelectrode by adopting an electrochemical cyclic voltammetry electrochemical method, and circulating for 50-150 times at a voltage scanning speed of 0.05V/s between 0 and 1.0V to prepare an active part enzyme electrode of the sensor;
(4) preparation of the corneal electrode sensor for eyes: and carrying out hot-pressing molding on the prepared nano enzyme electrode for 3-5 h at 200 ℃ in a nitrogen environment 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 microns.
The treatment time of the transparent polymer film subjected to the oxygen ion treatment in the step (1) is 30-100 seconds, and the power is 10-50 watts.
The photoresist in the step (1) is AZ4620 photoresist, the thickness is 10-50 microns, and the photoresist is baked at 80 ℃ for 10-20 minutes and is fixed and molded.
The thickness of the platinum metal layer sprayed on the micro electrode in the step (1) is 100-300 nm.
In the step (2), the sulfur-containing conductive polymer monomer 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 in 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 invention has the beneficial effects that: the invention provides an ophthalmic electrochemical sensor based on sulfur-doped graphene, a preparation method and application thereof and a myopia monitoring device, the specific bioactive enzyme is loaded through the unique ultra-large specific surface area of the two-dimensional graphene nano carrier, the loading capacity of the enzyme on a sensing electrode is improved, the selectivity and the specificity of the biosensor are effectively improved, the graphene is doped through edges such as sulfur-containing macromolecules (such as polythiophene and phenyl sulfide), so that the electron cloud is deviated from the large pi bond of the graphene, thereby obtaining high electrochemical active sites, effectively improving the sensitivity of the biosensor, constructing a wearable high-performance nano biosensor based on electrochemical signals through the independently designed cornea micro-electrode, realizing the concentration of dopamine in tears, and the real-time monitoring of the change condition of the myopia degrees provides a new detection tool and a new technical idea for early clinical ophthalmic monitoring and prevention of serious eye diseases such as myopia.
Drawings
FIG. 1 is a schematic diagram of the preparation of the ocular nano biosensor and a device photo. (a) A schematic diagram of the preparation of the nano microelectrode; (b) the photo of the prepared nanometer microelectrode; (c) after the hot-pressing molding of the nano-electrode for eyes, connecting the three electrodes and then taking a picture; and (c) an inset is the ocular nano-electrode obtained after hot-pressing and shaping.
Fig. 2 shows the result of monitoring the change of dopamine concentration after the prepared ocular nano biosensor is worn in the eye of a new zealand white rabbit. (a) After different concentrations of dopamine/artificial tears were instilled in rabbit eyes, the current signal detected on the ocular sensor changed at a constant voltage of + 0.2V. (b) Calibration curve obtained from the results of (a). (c) Under the same conditions, 5. mu.M dopamine, 1 mM glucose, 1 mM uric acid, 1 mM ascorbic acid, and 5. mu.M dopamine were added dropwise, respectively, and the current signal change detected on the ocular sensor.
FIG. 3 shows the results of tear samples collected from eyes of patients with different degrees of myopia (from 0 to 800 degrees) and tested on the ocular nano-biosensor prepared. (a) Tears of different myopic degrees, at a constant voltage of +0.2V, the current signal detected on the ocular sensor changes. (b) Calibration curve obtained from the results of (a).
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
The invention will be better explained with reference to the drawings and the specific embodiments.
Example 1.
The preparation method of the sulfur-doped graphene-based ophthalmic electrochemical sensor and the application of the sulfur-doped graphene-based ophthalmic electrochemical sensor in myopia monitoring comprises the following steps:
as shown in fig. 1, the constructed biosensor mainly includes the following important parts: (1) micropatterned microelectrodes are fabricated by photolithography on transparent polymer films such as PET, PMMA, and the like. The microelectrode is provided with three joints for connecting a working electrode, a counter electrode and a reference electrode. The active part of the microelectrode was used to deposit graphene nanocarriers and enzymes. The prepared microelectrode is cut according to the shape and size of the cornea (such as the diameter of 1-1.2 cm), and then hot-pressed and shaped (as shown in figure 1 c). And then the electrode is connected through low-temperature soldering tin and used for electrochemically depositing the sulfur-containing graphene nano-carrier and the enzyme.
Example 2.
As shown in fig. 2, after the artificial tears containing different dopamine concentrations were dropped into eyes of new zealand white rabbits, the ocular nano biosensor prepared in example 1 was worn into the rabbit eyes, and the change of dopamine concentration in the tears was monitored in real time. Fig. 2 (a) shows that dopamine in the eye is specifically oxidized by the enzyme on the sensor, thereby outputting an oxidation current, which is detected by the ocular sensor. The current output detected by the eye sensor can be increased along with the increase of the concentration of the dopamine dripped into the eye, and the two are in a good linear relation (R)2=0.9739, fig. 2 b). As can be seen from the results of the selectivity experiment in FIG. 2 (c), the prepared ocular biosensor has good anti-interference performance and can specifically detect dopamine in tears.
Example 3.
The prepared eye sensor can be used for efficiently detecting tears of myopes
Tear samples were taken from the eyes of myopes of varying degrees of myopia and used for the detection by the ocular sensor prepared in example 1. As can be seen from fig. 3 (a), the current intensity detected by the prepared ocular sensor will sensitively vary with the degree of myopia. Fig. 3 (b) further shows that there is a good potential relationship between the change in current detected on the ocular sensor and the increase in myopia power. 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 the myopia degree and the current intensity, the occurrence of myopia is expected to be well predicted and prevented, and a new technical method is provided for prevention and control and early treatment of myopia.
According to the invention, the specific bioactive enzyme is loaded through the super-large specific surface area of the two-dimensional graphene nanosheet, so that the selectivity and specificity of the nano biosensor are effectively improved; furthermore, the graphene is doped on the edge of sulfur-containing macromolecules (such as polythiophene and phenyl sulfide), so that the electron cloud is deviated from a large pi bond of the graphene, a high electrochemical active site is obtained, and the sensitivity of the biosensor is effectively improved. On the basis, the nano enzyme is loaded on the surface of the sulfur-doped graphene carrier by a mild and controllable electrochemical coprecipitation method and is fixed on a cornea microelectrode which is independently designed, so that a wearable high-performance nano biosensor based on electrochemical signals is constructed, the nano biosensor is used for monitoring the dopamine content in tears and the change of myopia degrees in real time, and a new detection tool and a new technical thought are provided for early clinical ophthalmic monitoring and prevention of serious eye diseases such as myopia.
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 embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (10)
1. The sulfur-doped graphene-based ophthalmic electrochemical sensor 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 covers the surface of the microelectrode.
2. The sulfur-doped graphene-based ocular electrochemical sensor according to claim 1, wherein the transparent polymer is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
3. The sulfur-doped graphene-based ocular electrochemical sensor of claim 1, wherein the transparent polymer has a thickness of 50-100 μm.
4. The preparation method of the sulfur-doped graphene-based ophthalmic electrochemical sensor according to claim 1, characterized by comprising the following steps:
(1) preparing a microelectrode: firstly, treating a transparent polymer film by oxygen ions, then spin-coating a layer of photoresist, fixing and forming a photoresist sheet, then using a photoetching machine to irradiate for 30-60 seconds under the shielding of a mask plate by using an ultraviolet lamp, then fixing for 70-120 seconds in a developing solution, removing and cleaning the photoresist on the ultraviolet irradiation part, removing the mask plate to obtain a micro-patterned photoresist, spray-coating a layer of platinum metal by using a magnetron sputtering instrument, then cleaning by using a glue washing solution, and removing the redundant photoresist to obtain a micro-patterned microelectrode;
(2) preparing a sulfur-doped graphene carrier material: blending 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 the rotating speed of 300-;
(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 mixture on a microelectrode by adopting an electrochemical cyclic voltammetry electrochemical method, and circulating for 50-150 times at a voltage scanning speed of 0.05V/s between 0 and 1.0V to prepare an active part enzyme electrode of the sensor;
(4) preparation of the corneal electrode sensor for eyes: and carrying out hot-pressing molding on the prepared nano enzyme electrode for 3-5 h at 200 ℃ in a nitrogen environment to obtain the cornea electrode sensor for eyes.
5. The method for preparing the sulfur-doped graphene-based ophthalmic electrochemical sensor according to claim 1, wherein the transparent polymer is polyethylene terephthalate (PET) or polymethyl methacrylate (PMMA).
6. The method for preparing an ophthalmic electrochemical sensor based on sulfur-doped graphene according to claim 4, wherein the thickness of the transparent polymer is 50-100 μm.
7. The method for preparing the sulfur-doped graphene-based ophthalmic electrochemical sensor according to claim 4, wherein the sulfur-containing conductive polymer monomer in the step (2) is 3, 4-ethylenedioxythiophene, and the sulfur-containing conductive polymer is polyphenylene sulfide.
8. Use of the sulfur-doped graphene-based ophthalmic electrochemical sensor of claim 1 as a device for monitoring the concentration of dopamine in tears in real time.
9. A myopia monitoring device is characterized by comprising a module for monitoring the concentration of dopamine in tears in real time.
10. A myopia monitoring device according to claim 9, wherein the means for monitoring the dopamine concentration in tear fluid in real time comprises the sulfur-doped graphene-based ocular electrochemical sensor of claim 1.
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