CN111426841A - Preparation method of anti-fouling microgel chip, chip and sensor - Google Patents
Preparation method of anti-fouling microgel chip, chip and sensor Download PDFInfo
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- CN111426841A CN111426841A CN202010241576.2A CN202010241576A CN111426841A CN 111426841 A CN111426841 A CN 111426841A CN 202010241576 A CN202010241576 A CN 202010241576A CN 111426841 A CN111426841 A CN 111426841A
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- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims abstract description 14
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- SWXQHJMEARSCRK-UHFFFAOYSA-N 1-(6,6-dimethoxycyclohexa-2,4-dien-1-yl)propan-1-one Chemical compound C(C)C(=O)C1C(C=CC=C1)(OC)OC SWXQHJMEARSCRK-UHFFFAOYSA-N 0.000 claims abstract description 7
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims abstract description 7
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- ULVXDHIJOKEBMW-UHFFFAOYSA-N [3-(prop-2-enoylamino)phenyl]boronic acid Chemical compound OB(O)C1=CC=CC(NC(=O)C=C)=C1 ULVXDHIJOKEBMW-UHFFFAOYSA-N 0.000 claims abstract description 7
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims abstract description 7
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- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims abstract description 7
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- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 238000006116 polymerization reaction Methods 0.000 claims description 6
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 4
- 238000012360 testing method Methods 0.000 abstract description 9
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 30
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
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- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
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Abstract
The invention provides a preparation method of a chip with antifouling microgel, which comprises the following steps: synthesizing microgel: adding 3-acrylamidophenylboronic acid, acrylamide, glycidyl methacrylate, N, N' -methylene bisacrylamide and azobisisobutyronitrile into acetonitrile, polymerizing at 90 ℃, and repeatedly cleaning with distilled water; synthesis of microgel antifouling layer: threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value, dispersing the microgel into the mixed solution, washing the product with distilled water, adding ethanol, and centrifuging; fixing on a crystal oscillator: dispersing the microgel antifouling layer, N-vinyl pyrrolidone, ethylene glycol dimethacrylate and 2, 2-dimethoxy-phenyl ethyl ketone in dimethyl sulfoxide to prepare a prepolymerization solution, then placing the quartz plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the quartz plate, irradiating for 1 hour by an ultraviolet lamp, washing with distilled water and drying with nitrogen. The invention can eliminate the inaccurate test caused by the high non-specificity of the protein.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a preparation method of an antifouling microgel chip, the chip and a sensor.
Background
Diagnosis and treatment of diabetes requires periodic or (ideally) continuous control of glucose levels in the blood in order to optimize treatment and improve the quality of life of a diabetic. Currently, blood glucose level measurement relies mainly on two methods, one is to pierce a finger to measure blood glucose, and the other is to insert a microprobe into the skin to continuously measure glucose in the intercellular fluid. However, both of these methods are invasive. In order to meet the medical requirements of patients with diabetes and improve the quality of life of the patients, a non-invasive technology has become a research hotspot of clinical and scientific research. Particularly, the correlation between the glucose concentration in saliva and the blood glucose level is high, and saliva has natural advantages of safety, convenience for real-time collection and the like, so that the saliva-based noninvasive blood glucose monitoring becomes a research hotspot.
The QCM has the characteristics of high sensitivity, high response speed, low production cost, strong real-time measurement capability, simple integration and the like, and is a suitable tool for detecting various molecular recognition events such as liquid DNA, nucleic acid, protein, bacteria and the like on line.
Therefore, in order to solve the problems of the prior art, there is a need for a method of preparing a chip having an antifouling microgel, a chip, and a sensor.
Disclosure of Invention
One aspect of the present invention is to provide a method for preparing a chip with an antifouling microgel, the method comprising:
synthesizing microgel, namely adding 516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile into 40m L acetonitrile, polymerizing for 1 hour at 90 ℃, and repeatedly washing by using distilled water to obtain the microgel;
synthesis of microgel antifouling layer: threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of the microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing a product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance;
fixing on a crystal oscillator, preparing a prepolymerization solution by taking 30mg of the white pasty substance, 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone to be dispersed in 50 mu L dimethyl sulfoxide,
and then placing 25 mu L of the prepolymerization solution on a quartz plate, placing a crystal oscillator plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator plate by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp, washing with distilled water and drying with nitrogen.
Preferably, the method further comprises fixing the microgel antifouling layer before the crystal oscillator plate, and treating the crystal oscillator plate by:
ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece with distilled water and drying the crystal oscillation piece with nitrogen;
then, the mixture is put into ethanol containing 100 mu L gamma-aminopropyltriethoxysilane to react for 12 hours at room temperature;
washing the crystal oscillation piece with distilled water, drying the crystal oscillation piece with nitrogen, adding N, N-dimethylformamide of maleic anhydride for reacting for 12 hours, washing with distilled water again, and drying with nitrogen.
Preferably, the piranha solution is: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
Preferably, threonine and serine are present in a molar ratio of 1: 1, to an ethanol solution having a volume ratio of ethanol to water of 1: 3.
another aspect of the present invention is to provide a chip, which includes a crystal oscillator plate, wherein the crystal oscillator plate fixes the antifouling microgel through chemical crosslinking;
the anti-fouling microgel is grafted with amino acid to form a microgel anti-fouling layer, so that the non-specific adsorption of protein is reduced for the chip.
Preferably, the crystal oscillator plate fixes the antifouling microgel through chemical crosslinking as follows:
the following treatment is carried out on the crystal oscillation piece:
ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece with distilled water and drying the crystal oscillation piece with nitrogen;
then, the mixture is put into ethanol containing 100 mu L gamma-aminopropyltriethoxysilane to react for 12 hours at room temperature;
washing the crystal oscillation sheet with distilled water, drying the crystal oscillation sheet with nitrogen, adding N, N-dimethylformamide of maleic anhydride for reacting for 12 hours, washing with distilled water again, and drying the crystal oscillation sheet with nitrogen;
fixing the antifouling microgel on the crystal oscillator through chemical crosslinking:
preparing a pre-polymerization solution by dispersing 30mg of the microgel anti-fouling layer, 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone in 50 mu L dimethyl sulfoxide,
and then placing 25 mu L of the prepolymerization solution on a quartz plate, placing a crystal oscillator plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator plate by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp, washing with distilled water and drying with nitrogen.
Preferably, the piranha solution is: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
Preferably, the microgel is synthesized by:
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile are added into 40m L of acetonitrile, polymerization is carried out for 1 hour at 90 ℃, and distilled water is repeatedly washed to obtain the microgel.
Preferably, the microgel antifouling layer is synthesized by:
threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing a product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
Yet another aspect of the present invention is to provide a sensor comprising the chip of claim, the chip comprising a crystal oscillator plate, the crystal oscillator plate fixing the anti-fouling microgel through chemical crosslinking;
the anti-fouling microgel is grafted with amino acid to form a microgel anti-fouling layer, so that the non-specific adsorption of protein is reduced for the chip.
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention synthesize the antifouling microgel on the QCM chip, and the non-specific adsorption of protein is overcome when the saliva glucose is detected.
According to the preparation method of the chip with the antifouling microgel, the chip and the sensor, the microgel containing boric acid is used as a binding site of glucose, the amino acid grafted to the microgel is used as a protein resistant component, and then the protein resistant component is fixed on the chip through chemical crosslinking, so that the glucose sensor has glucose responsiveness and anti-pollution performance, and can detect 0-40 mg/L saliva glucose under physiological conditions.
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention have excellent protein and organic molecule tolerance, and realize detection (V) of glucose in saliva solutionSaliva＝VPBS＝1:1)。
According to the preparation method of the chip with the antifouling microgel, the chip and the sensor, provided by the invention, the problem of inaccurate test caused by high non-specificity of protein is solved under the conditions that the content of glucose molecules in saliva is extremely low (3.6-36 mg/L) and the frequency change is very small.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Drawings
Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:
FIG. 1 schematically shows a flow chart for preparing chips with anti-fouling microgels in one embodiment of the present invention.
Fig. 2a shows the microstructure of the synthesized microgel.
Fig. 2b shows the microscopic appearance of the synthetic microgel anti-fouling layer.
Fig. 2c shows the microstructure after the microgel anti-fouling layer was immobilized on the chip.
FIG. 3 is a graph showing stability test of chips having the antifouling microgel of the present invention.
FIG. 4 is a schematic view showing a detection limit test of chips having an antifouling microgel of the present invention.
FIG. 5 is a graph showing the effect of different pH on the test with the anti-fouling microgel chips of the present invention.
FIG. 6 is a schematic view showing a recovery test of chips having the antifouling microgel of the present invention.
FIG. 7 shows a schematic diagram of a real saliva test with an anti-fouling microgel chip of the present invention.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numerals denote the same or similar parts, or the same or similar steps.
In order to solve the problem of inaccuracy in detection of salivary sugar caused by huge background signals generated by high non-specificity of protein in the prior art, a preparation method of an antifouling microgel chip is provided.
The first embodiment.
Referring to fig. 1, a flow chart for preparing chips with anti-fouling microgels in one embodiment of the present invention, according to one embodiment of the present invention, a method for preparing chips with anti-fouling microgels comprises:
fixing the microgel antifouling layer in front of the crystal oscillator plate, and treating the crystal oscillator plate
And ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece 1 with distilled water and drying the crystal oscillation piece with nitrogen.
Then, the mixture was put into ethanol (50m L) containing 100. mu. L. gamma. -aminopropyltriethoxysilane, and reacted at room temperature for 12 hours.
The crystal oscillator plate is washed by distilled water and dried by nitrogen, then the crystal oscillator plate is put into N, N-dimethylformamide (50m L) of maleic anhydride (1g) for reaction for 12 hours, and then the crystal oscillator plate is washed by distilled water again and dried by nitrogen.
piranha solution was: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
Synthetic microgels
Adding 516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile into 40m L of acetonitrile, polymerizing at 90 ℃ for 1 hour, and repeatedly cleaning with distilled water to obtain microgel;
composite microgel antifouling layer
Threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of the microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing the product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
In some preferred embodiments, threonine and serine are present in a molar ratio of 1: 1, to an ethanol solution having a volume ratio of ethanol to water of 1: 3.
fixed on a crystal oscillator
Preparing a prepolymerization solution by taking 30mg of the white pasty substance (microgel antifouling layer), 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone to be dispersed in 50 mu L dimethyl sulfoxide,
and then placing 25 mu L of the pre-polymerization solution on a quartz plate (10 × 10cm), placing the crystal oscillator piece 1 on the pre-polymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator piece by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp (365nm), washing by using distilled water, drying by using nitrogen, and finishing the cross-linking and fixing of the microgel antifouling layer 2 on the crystal oscillator piece 1.
And II, implementing.
The embodiment provides a chip, which includes a crystal oscillator plate, wherein the crystal oscillator plate fixes an antifouling microgel through chemical crosslinking, and the antifouling microgel is formed by grafting amino acid on the microgel to form a microgel antifouling layer, so that the nonspecific adsorption of protein is reduced for the chip.
The anti-fouling microgel is fixed by the crystal oscillator through chemical crosslinking as follows:
the following treatment is carried out on the crystal oscillation piece:
and ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece with distilled water and drying the crystal oscillation piece with nitrogen. piranha solution was: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
Then, the mixture was put into ethanol (50m L) containing 100. mu. L. gamma. -aminopropyltriethoxysilane, and reacted at room temperature for 12 hours.
The crystal oscillator plate is washed by distilled water and dried by nitrogen, then the crystal oscillator plate is put into N, N-dimethylformamide (50m L) of maleic anhydride (1g) for reaction for 12 hours, and then the crystal oscillator plate is washed by distilled water again and dried by nitrogen.
Fixing the antifouling microgel on the crystal oscillator through chemical crosslinking:
30mg of the microgel antifouling layer, 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone are dispersed in 50 mu L dimethyl sulfoxide to prepare a prepolymerization solution.
Then placing 25 mu L of the prepolymerization solution on a quartz plate (10 × 10cm), placing a crystal oscillator plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator plate by using a film pressing machine, irradiating for 1 hour by using an ultraviolet (365nm) lamp, washing with distilled water and drying by using nitrogen.
The microgel is synthesized by the following method:
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile are added into 40m L of acetonitrile, polymerization is carried out for 1 hour at 90 ℃, and distilled water is repeatedly washed to obtain the microgel.
The microgel antifouling layer is synthesized by the following method:
threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing a product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
Example three.
The embodiment provides a sensor, which comprises a chip, wherein the chip comprises a crystal oscillation sheet, the crystal oscillation sheet fixes an antifouling microgel through chemical crosslinking, and the antifouling microgel is used for reducing the nonspecific adsorption of protein for the chip by grafting amino acid on the microgel to form a microgel antifouling layer.
The process of fixing the anti-fouling microgel by cross-linking the quartz plate is the same as that of the first embodiment and the second embodiment, and the details are not repeated here.
The following experiments verify that the preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention are carried out.
1. And (3) characterization:
scanning electron microscopy was used to evaluate the morphology and particle size of the prepared antifouling microgel.
The microgel microstructure synthesized as shown in fig. 2a has smooth surface, uniform size (about 600nm), and spherical morphology.
As shown in FIG. 2b, the micro-gel anti-fouling layer synthesized has a micro-structure, and the micro-gel after the amino acid is grafted to the surface has non-uniform surface.
The microstructure after the microgel anti-fouling layer was immobilized on the chip, a crosslinked layer in a package of many beads, as shown in fig. 2c, demonstrates that the microgel anti-fouling layer was successfully immobilized on the surface of the chip quartz plate.
2. Stability:
the stability of the sensor was evaluated in PBS (phosphate buffered saline).
The stability test chart with the anti-fouling microgel chips of the present invention, as shown in fig. 3, shows that the fluctuation value of △ F (frequency shift) was only △ F ═ 2.4Hz when the polymer-coated chips were exposed to PBS for about 2 hours.
3. Detection limit:
in order to obtain the detection limit of the glucose sensor, the glucose concentration is gradually increased in the example.
As shown in fig. 4, a schematic diagram of a detection limit test of the chip with the anti-fouling microgel according to the present invention shows that the detection limit of the preparation method with the anti-fouling microgel chip according to the present invention is 5mg per liter.
4. Detection range:
the glucose detection in different ranges is achieved by adjusting different pH values.
As shown in FIG. 5, the effect of different pH on the test of the anti-fouling microgel chip of the present invention is shown, and the absolute value of Δ F gradually increases with the increase of pH. The glucose concentration is from 0 to 40 mg/L, the sensor shows good linear relation, and the linear correlation coefficients are 0.9523(pH6.8), 0.9274(pH 7.2) and 0.9482(pH 7.5)
5. Recovery property:
as shown in FIG. 6, the recovery test of the anti-fouling microgel chip of the invention is schematically shown, and the frequency change of the chip is basically unchanged when the chip is exposed to 200 mg/L glucose and 0 mg/L glucose alternately for a plurality of times.
6. Real saliva test:
to further demonstrate the anti-contamination performance of the glucose chip (sensor) of the present invention, real saliva test was used in the examples.
The actual saliva test with the anti-fouling microgel chip of the present invention, as shown in fig. 7, shows an absolute value of △ F increasing with increasing glucose concentration (a in fig. 7), the glucose concentration has a good linear relationship with △ F with a linear correlation coefficient of 0.9671 (b in fig. 7).
The preparation method of the chip with the antifouling microgel, the chip and the sensor provided by the invention synthesize the antifouling microgel on the QCM chip, and the non-specific adsorption of protein is overcome when the saliva glucose is detected.
According to the preparation method of the chip with the antifouling microgel, the chip and the sensor, the microgel containing boric acid is used as a binding site of glucose, the amino acid grafted to the microgel is used as a protein resistant component, and then the protein resistant component is fixed on the chip through chemical crosslinking, so that the glucose sensor has glucose responsiveness and anti-pollution performance, and can detect 0-40 mg/L saliva glucose under physiological conditions.
The preparation method of the anti-fouling microgel chip, the chip and the sensor provided by the invention have excellent protein and organic molecule tolerance, and realize detection (V) of glucose in saliva solutionSaliva＝VPBS＝1:1)。
According to the preparation method of the chip with the antifouling microgel, the chip and the sensor, provided by the invention, the problem of inaccurate test caused by high non-specificity of protein is solved under the conditions that the content of glucose molecules in saliva is extremely low (3.6-36 mg/L) and the frequency change is very small.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (10)
1. A method for preparing a chip with an antifouling microgel, which is characterized by comprising the following steps:
synthesizing microgel, namely adding 516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile into 40m L acetonitrile, polymerizing for 1 hour at 90 ℃, and repeatedly washing by using distilled water to obtain the microgel;
synthesis of microgel antifouling layer: threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of the microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing a product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance;
fixing on a crystal oscillator, preparing a prepolymerization solution by taking 30mg of the white pasty substance, 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone to be dispersed in 50 mu L dimethyl sulfoxide,
and then placing 25 mu L of the prepolymerization solution on a quartz plate, placing a crystal oscillator plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator plate by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp, washing with distilled water and drying with nitrogen.
2. The method as set forth in claim 1, further comprising fixing the microgel anti-fouling layer on the quartz plate by:
ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece with distilled water and drying the crystal oscillation piece with nitrogen;
then, the mixture is put into ethanol containing 100 mu L gamma-aminopropyltriethoxysilane to react for 12 hours at room temperature;
washing the crystal oscillation piece with distilled water, drying the crystal oscillation piece with nitrogen, adding N, N-dimethylformamide of maleic anhydride for reacting for 12 hours, washing with distilled water again, and drying with nitrogen.
3. The method according to claim 2, characterized in that said piranha solution is: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
4. The method according to claim 1, wherein threonine and serine are present in a molar ratio of 1: 1, to an ethanol solution having a volume ratio of ethanol to water of 1: 3.
5. a chip, which comprises a crystal oscillation sheet, wherein the crystal oscillation sheet fixes the antifouling microgel through chemical crosslinking;
the anti-fouling microgel is grafted with amino acid to form a microgel anti-fouling layer, so that the non-specific adsorption of protein is reduced for the chip.
6. The chip of claim 5, wherein the quartz plate fixes the anti-fouling microgel by chemical crosslinking:
the following treatment is carried out on the crystal oscillation piece:
ultrasonically treating the crystal oscillation piece in piranha solution for 10 minutes, washing the crystal oscillation piece with distilled water and drying the crystal oscillation piece with nitrogen;
then, the mixture is put into ethanol containing 100 mu L gamma-aminopropyltriethoxysilane to react for 12 hours at room temperature;
washing the crystal oscillation sheet with distilled water, drying the crystal oscillation sheet with nitrogen, adding N, N-dimethylformamide of maleic anhydride for reacting for 12 hours, washing with distilled water again, and drying the crystal oscillation sheet with nitrogen;
fixing the antifouling microgel on the crystal oscillator through chemical crosslinking:
preparing a pre-polymerization solution by dispersing 30mg of the microgel anti-fouling layer, 50 mu L N-vinyl pyrrolidone, 5 mu L ethylene glycol dimethacrylate and 3mg of 2, 2-dimethoxy-phenyl ethyl ketone in 50 mu L dimethyl sulfoxide,
and then placing 25 mu L of the prepolymerization solution on a quartz plate, placing a crystal oscillator plate on the prepolymerization solution with the front side facing downwards, pressing the back side of the crystal oscillator plate by using a film pressing machine, irradiating for 1 hour by using an ultraviolet lamp, washing with distilled water and drying with nitrogen.
7. The chip of claim 6, wherein the piranha solution is: h with mass percentage concentration of 96% w/w2SO4And H with the mass percentage concentration of 30% w/w2O2According to the volume ratio of 7: 3, and (3) obtaining a mixed solution.
8. The chip of claim 5, wherein the microgel is synthesized by:
516mg of 3-acrylamidophenylboronic acid, 831mg of acrylamide, 350u L of glycidyl methacrylate, 46.2mg of N, N' -methylenebisacrylamide and 5mg of azobisisobutyronitrile are added into 40m L of acetonitrile, and the mixture is polymerized for 1 hour at 90 ℃, and repeatedly washed with distilled water to obtain the microgel.
9. The chip of claim 5, wherein the microgel stainproof layer is synthesized by:
threonine and serine in a molar ratio of 1: 1, adding the mixture into an ethanol solution, adjusting the pH value to 10.5 to obtain a mixed solution, dispersing 500mg of microgel into the mixed solution, reacting at 50 ℃ for 24 hours, washing a product to be neutral by distilled water, adding ethanol, and centrifuging at a high speed to obtain a white pasty substance.
10. A sensor comprising a chip according to any one of claims 5 to 9.
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| CN202110338792.3A CN113156132B (en) | 2020-03-31 | 2021-03-30 | Preparation method of chip with antifouling microgel, chip and sensor |
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| CN113156132A (en) * | 2020-03-31 | 2021-07-23 | 中科康磁医疗科技（苏州）有限公司 | Preparation method of anti-fouling microgel chip, chip and sensor |
| WO2022111613A1 (en) * | 2020-11-30 | 2022-06-02 | 深圳市真迈生物科技有限公司 | Substrate, and preparation method therefor and use of substrate |
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| WO2003025627A2 (en) * | 2001-06-01 | 2003-03-27 | Colorado State University Research Foundation | Optical biosensor with enhanced activity retention for detection of halogenated organic compounds |
| US20110008404A1 (en) * | 2007-12-19 | 2011-01-13 | Georgia Tech Research Corporation | Modification Of Biomaterials With Microgel Films |
| KR100921237B1 (en) * | 2007-12-31 | 2009-10-12 | 한국식품연구원 | Quartz crystal microbalance immunosensor Apparatus for C-reactive protein detection and High-sensitivity detection method for C-reactive protein using quartz crystal microbalance immunosensor |
| JP6160862B2 (en) * | 2013-07-25 | 2017-07-12 | 国立大学法人神戸大学 | Amino acid ionic liquid-containing polymer gel and method for producing the same |
| CN104148038B (en) * | 2014-03-18 | 2016-02-10 | 苏州康磁医疗科技有限公司 | A kind of based on gold surface boric acid class glucose-sensitive type trace hydrogel and preparation method thereof |
| CN104014323B (en) * | 2014-04-29 | 2016-02-10 | 苏州康磁医疗科技有限公司 | A kind of preparation method based on gold surface boric acid class glucose-sensitive type hydrogel and application thereof |
| CN104434131A (en) * | 2014-10-16 | 2015-03-25 | 苏州康磁医疗科技有限公司 | Quartz-crystal-vibration-piece-based method for preparing glucose sensitive film with excellent biocompatibility and application of method |
| KR102255158B1 (en) * | 2014-10-21 | 2021-05-27 | 엘지전자 주식회사 | Sensor for detecting saccharide and manufacturing method thereof and detection method of glycated hemoglobin using the same |
| CN111426841A (en) * | 2020-03-31 | 2020-07-17 | 中科康磁医疗科技（苏州）有限公司 | Preparation method of anti-fouling microgel chip, chip and sensor |
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| CN113156132A (en) * | 2020-03-31 | 2021-07-23 | 中科康磁医疗科技（苏州）有限公司 | Preparation method of anti-fouling microgel chip, chip and sensor |
| CN113156132B (en) * | 2020-03-31 | 2024-03-01 | 中科康磁医疗科技（苏州）有限公司 | Preparation method of chip with antifouling microgel, chip and sensor |
| WO2022111613A1 (en) * | 2020-11-30 | 2022-06-02 | 深圳市真迈生物科技有限公司 | Substrate, and preparation method therefor and use of substrate |
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