CN111208130B - Test strip for rapidly detecting tyrosinase in serum and preparation method and application thereof - Google Patents
Test strip for rapidly detecting tyrosinase in serum and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a test strip for rapidly detecting tyrosinase in serum and a preparation method and application thereof. The method for detecting tyrosinase by using the test strip comprises the steps of preparing a detection test strip; then preparing a series of tyrosinase standard solutions with different concentrations, mixing the tyrosinase standard solutions with dopamine with the same concentration for a certain time, dripping the mixed solution on a test strip for reaction, and displaying different colors on the test strip to be used as a standard colorimetric card; according to the invention, the standard colorimetric card is designed, the activity of tyrosinase to be detected can be detected in real time and rapidly according to the color of the system, a standard curve of ultraviolet absorbance and tyrosinase activity is designed, and the accurate tyrosinase activity can be obtained by substituting related data into the standard curve through testing the ultraviolet visible light absorption spectrum of the system, so that a valuable instrument is not required, and the detection method is simpler, more convenient and more economic and has higher practical value.
Description
Technical Field
The invention relates to a test strip for rapidly detecting tyrosinase in serum and a preparation method and application thereof, belonging to the technical field of analysis and detection.
Background
Tyrosinase (TYR), also known as polyphenol oxidase, is a copper-containing metalloenzyme widely distributed in microorganisms, animals, plants, and human bodies. Tyrosinase has important physiological functions in organisms, is a key enzyme in a melanin biosynthesis pathway, is related to the occurrence of diseases such as excessive deposition of melanin such as freckles, chloasma and the like of human bodies, and is also greatly related to molting of insects and browning of fruits and vegetables. Therefore, the detection of the activity level of tyrosinase can provide a basis for diagnosis and treatment of clinical diseases such as malignant melanoma and the like, and has important significance in multiple fields such as fruit and vegetable preservation, environmental monitoring and the like in the food industry.
At present, the methods for measuring the activity of tyrosinase reported at home and abroad mainly comprise a radioisotope detection method, a high performance liquid chromatography method, a fluorescence detection method and the like. However, these methods have some limitations, such as complicated operation, long analysis time, high analysis cost, etc., which limits their wide application. Chemical and biological sensor methods have the advantages of simplicity, rapidness and effective detection and have been developed, but the chemical and biological sensor methods for detecting mercury ions cannot achieve the effect of on-site detection. Chinese patent No. CN 108152495 a discloses a colloidal gold test paper for detecting tyrosine, but it needs enzyme-linked reaction, and the price of antigen and antibody is high. Therefore, the method for detecting tyrosinase, which is rapid, free of instrument field instant detection, high in sensitivity and selectivity, has great significance in the fields of food safety and clinical diagnosis.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for detecting tyrosinase in serum by using a test strip based on a functionalized nano gold core-shell structure material, so as to detect the tyrosinase content in serum in real time, rapidly, economically and practically.
The technical scheme of the invention is as follows:
a preparation method of a test strip for rapidly detecting tyrosinase in serum comprises the following steps:
S1、Au PB synthesis of @ Au colloid
Taking 10mL of nano gold colloid solution, adding 10 microliter of Prussian blue solution with the concentration of 0.1mol/L as Raman signal molecules, stirring and mixing uniformly, centrifuging, and resuspending in 10mL of ultrapure water to obtain Au @ PB colloid; to 10mL of the prepared Au @ PB colloid was added 250. mu.L of 6 x 10 -2 magnetic stirring and heating mol/L sodium citrate solution to boiling, adding 2.5mL chloroauric acid solution with the concentration of 1mmol/L, stopping heating, continuing magnetic stirring for 1h, centrifugally washing colloid for 2 times, and re-dispersing in 5mL ultrapure water to obtain Au PB @ Au colloid;
s2, functionalized Au PB Preparation of @ Au NPs
Au prepared in the step S1 PB Mixing @ Au colloid with Fe (NTA) solution at a volume ratio of 1:1, stirring, centrifuging to remove unreacted Fe (NTA), and removing supernatant Re-dispersing the solution for later use to obtain the functionalized Au PB @Au NPs；
S3, fully soaking the test strip substrate in a 3-mercaptopropyl methyl dimethoxy silane ethanol solution for 30min, taking out, respectively rinsing with distilled water and absolute ethyl alcohol, forming a monolayer on the surface of paper fibers through self-assembly of organosilane coupling, and drying for later use to obtain the functionalized test strip; the functionalized test strip substrate is put into a glass sand core funnel device of 100mLG4, and 50mL of prepared functionalized Au is taken PB And (3) pouring the @ Au NPs material into a sand core funnel device, carrying out suction filtration, and finally taking out the filter paper and drying to obtain the detection test strip.
Further, the synthesis method of the Fe (NTA) solution is as follows:
firstly, the concentration is 10 −3 M ferric nitrate nonahydrate solution and concentration of 10 −3 Mixing M nitrilotriacetic acid trisodium solution according to the volume ratio of 1:1, adding a pH regulator into the solution to regulate the pH value of the Fe (NTA) solution to 7.0, and standing the mixed solution to obtain the Fe (NTA) solution.
Further, the preparation method of the nanogold glue solution comprises the following steps:
heating 100mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; then adding 1.5 mL of 1% sodium citrate solution into the boiling chloroauric acid solution, after boiling for 15min, finishing the reaction, and cooling to room temperature to prepare nano gold sol;
Further, the method for detecting tyrosinase by the detection test strip comprises the following steps:
1) manufacturing a standard color comparison card: mixing tyrosinase standard solutions with different concentrations (500U/mL, 400U/mL, 300U/mL, 200U/mL, 100U/mL, 50U/mL, 10U/mL and 0U/mL) with dopamine, sequentially dropwise adding 10 mu L onto a detection test strip, reacting at room temperature for 1.5 minutes, then airing, recording the colors of the detection test strip as dark red, wine red, mauve, light purple, blue purple, purple gray and gray respectively, and finishing according to different colors to obtain a tyrosinase standard colorimetric card;
2) detecting tyrosinase: mixing a sample to be detected with dopamine, dripping the mixture onto a detection test strip, reacting at room temperature for 1.5 minutes, then airing, observing the color of the detection test strip, comparing with the standard colorimetric card obtained in the step 1), and estimating the tyrosinase content in serum to be detected through color comparison.
The invention has the following beneficial effects:
1. the invention provides a detection test strip for simply, conveniently and rapidly detecting tyrosinase in serum. Au prepared by the invention PB The @ Au NPs are more stable than the traditional Au NPs, are not easy to deteriorate and are convenient to store for later use;
2. the test strip provided by the invention has a wide test range and a low detection limit.
3. The invention does not need large instruments, has simple, quick and easy operation, can identify the detection result by observing color comparison with naked eyes, and can carry out in-situ quick detection.
4. The preparation method of the test strip is more stable, and the nano particles are not easy to fall off from the filter paper.
5. The reagent and the operation process used in the invention have no toxic or side effect.
6. According to the invention, the standard colorimetric card is designed, the concentration of tyrosinase to be detected can be detected in real time, quickly, simply and economically according to the color, a standard curve of ultraviolet absorbance and the concentration of tyrosinase is designed, and the accurate concentration of tyrosinase can be obtained by substituting related data into the standard curve through the ultraviolet visible light absorption spectrum of a test system.
7. The invention utilizes Au PB @ AuNPs as SERS enhancing substrate, Fe (NTA) forms Fe (NTA) DA complex with DA, which can amplify the Raman signal of DA. Because the tyrosinase catalyzes and oxidizes dopamine to generate dopaquinone, the content of DA is reduced along with the increase of the concentration of the tyrosinase in the same time, the DA consumed in the same time is increased, the content of the residual DA in the mixed solution is low, and the Raman signal is reduced, the activity of TYR can be indirectly detected through DA. The method greatly improves the detection limit of TYR by amplifying DA signals.
8. Au of the present invention PB @ Au core-shell structure toolThe method has the following advantages: the nano-gold has good biocompatibility and low cytotoxicity; the external gold shell can not only enhance the signal of the internal standard molecule, but also protect the internal standard molecule from the influence of external environment, pH, temperature and the like.
9. Prussian blue is used as a Raman signal molecule in a biological silence region (more than 1800 cm) -1 ) Has a characteristic peak with high signal-to-noise ratio, and the cell or blood is 1800cm -1 Characteristic peaks exist before the wave number, and cannot be overlapped with the spectral peaks of the cells, so that background interference is caused.
Drawings
FIG. 1 is a schematic diagram illustrating a color reaction mechanism between a functionalized nanogold core-shell structure material and tyrosinase according to the present invention;
FIG. 2 is a transmission electron micrograph of AuNPs prepared in example 1;
FIG. 3 is Au prepared in example 1 PB Transmission electron microscopy of @ Au NPs;
FIG. 4 is a flowchart of the manufacturing of the color chart of example 1;
FIG. 5 is a color picture of the test paper of the solution to be tested in example 1;
FIG. 6 is a UV spectrum of tyrosinase at various concentrations in example 1;
FIG. 7 is a graph of the linear relationship between the concentration of tyrosinase and the absorbance in example 1;
FIG. 8 is a graph showing color changes corresponding to different concentrations of tyrosinase in example 1.
Detailed Description
The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. But are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1
(1) Preparation of nano gold
The preparation method of the nano gold colloid solution comprises the following steps: heating 100mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then adding 1.5 mL of 1% sodium citrate solution into the boiling chloroauric acid solution, after boiling for 15min, finishing the reaction, cooling to room temperature to prepare nano gold sol, and observing the nano gold by using a transmission electron microscope. As shown in FIG. 2, the nanoparticles are distributed under a transmission electron microscope, and the particle size is 20-30 nm.
（2）Au PB Synthesis of @ Au NPs
Taking 10mL of nano gold colloid solution, adding 10 microliter of Prussian blue solution with the concentration of 0.1mol/L as Raman signal molecules, stirring and mixing uniformly, centrifuging, and resuspending in 10mL of ultrapure water to obtain Au @ PB colloid; to 10mL of the prepared Au @ PB colloid was added 250. mu.L of 6 x 10 -2 magnetic stirring and heating mol/L sodium citrate solution to boiling, adding 2.5mL chloroauric acid solution with the concentration of 1mmol/L, stopping heating, continuing magnetic stirring for 1h, centrifugally washing colloid for 2 times, and re-dispersing in 5mL ultrapure water to obtain Au PB @ Au colloid. Observation of Au Using Transmission Electron microscope PB And @ AuNPs, as shown in FIG. 3, are in nanoparticle distribution under a transmission electron microscope, and have particle sizes of 30-40 nm.
(3) Synthesis of Fe (NTA) solution
Firstly, the concentration is 10 −3 M ferric nitrate nonahydrate solution and concentration of 10 −3 Mixing M nitrilotriacetic acid trisodium solution according to the volume of 1:1, adding a pH regulator into the solution to regulate the pH value of the Fe (NTA) solution to 7.0, and standing the mixed solution to obtain an Fe (NTA) solution;
(4) functionalized Au PB Preparation of @ Au NPs
Au prepared in the step (2) PB Mixing the @ Au colloid with the Fe (NTA) solution prepared in the step (3) according to the volume ratio of 1:1, stirring and mixing, centrifugally washing to remove unreacted Fe (NTA), removing supernatant, and re-dispersing for later use to obtain the functionalized Au PB @Au NPs；
(5) Manufacturing test paper strip
Fully soaking filter paper in 3-mercaptopropyl-methyldimethoxysilaneTaking out the test paper from the test paper, rinsing the test paper in an alcohol solution for 30min, respectively rinsing the test paper with distilled water and absolute ethyl alcohol, forming a monolayer on the surface of the paper fiber through self-assembly of organosilane coupling, and drying the test paper for later use to obtain a functionalized test paper strip; putting the functionalized test strip substrate into a glass sand core funnel device of 100mLG4, and taking 50mL of prepared functionalized Au PB The @ Au NPs material is poured into a sand core funnel device, is subjected to suction filtration, and is finally taken out and dried to obtain the detection test strip, wherein the experimental process is shown in figure 4;
(6) making standard color comparison card
Sequentially dropwise adding the mixed solution (tyrosinase solutions with different concentrations and dopamine with the same concentration) into the functional nano gold core-shell structure material test strips, reacting the tyrosinase with different concentrations and the test strips to generate different colors, photographing each test strip, and finishing to obtain a tyrosinase standard colorimetric card as shown in fig. 5, wherein the colors are dark red, wine red, mauve, light purple, blue purple, purple gray and gray;
(7) detection of tyrosinase
And (3) mixing dopamine of a sample to be detected, dripping the mixed dopamine on the detection test strip obtained in the step 1), reacting at room temperature for 1.5 minutes, drying in the air, observing the color of the detection test strip, comparing the color with the standard colorimetric card obtained in the step 6), and estimating tyrosinase in serum to be detected through color comparison.
(8) Detection of actual samples
Randomly drawing 5 human serum samples, dripping 10 mu L of the samples on a test strip, wherein the test strip is purple gray, and comparing the test strip with a standard colorimetric card, the tyrosinase content in the human serum can be estimated to be about 10U/mL approximately.
(9) Ultraviolet detection of tyrosinase
Preparing tyrosinase standard solutions with different concentrations, adding dopamine for co-culture, and then adding Au PB The @ Au-Fe (NTA) solution was mixed well and subjected to UV detection as shown in FIG. 6. FIG. 7 is a linear relationship graph of absorbance at 523nm and tyrosinase concentration, wherein the absorbance at 523nm gradually increases with the increase of tyrosinase concentration, and the tyrosinase concentration is detectedThe limit of measurement was 10U/mL. FIG. 8 is a graph of the corresponding color change, with the colors dark red, wine red, purplish red, light purple, blue purple, purple gray, and gray, respectively, as tyrosinase concentration increases.
Example 2
(1) Preparation of nano gold
The preparation method of the nano gold colloid solution comprises the following steps: heating 100mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then adding 1.5 mL of sodium citrate solution with the mass fraction of 1% into the boiling chloroauric acid solution, after boiling for 15min, finishing the reaction, cooling to room temperature to prepare nano gold sol, and observing the nano gold by using a transmission electron microscope. As shown in FIG. 2, the nanoparticles are distributed under a transmission electron microscope, and the particle size is 20-30 nm.
（2）Au PB Synthesis of @ Au NPs
Taking 10mL of nano gold colloid solution, adding 10 microliter of Prussian blue solution with the concentration of 0.1mol/L as Raman signal molecules, stirring and mixing uniformly, centrifuging, and resuspending in 10mL of ultrapure water to obtain Au @ PB colloid; to 10mL of the prepared Au @ PB colloid was added 250. mu.L of 6 x 10 -2 magnetic stirring and heating mol/L sodium citrate solution to boiling, adding 2.5mL chloroauric acid solution with the concentration of 1mmol/L, stopping heating, continuing magnetic stirring for 1h, centrifugally washing colloid for 2 times, and dispersing in 5mL ultrapure water again to obtain Au PB @ Au colloid. Observation of Au Using Transmission Electron microscope PB And @ AuNPs, as shown in FIG. 3, are in nanoparticle distribution under a transmission electron microscope, and have particle sizes of 30-40 nm.
(3) Synthesis of Fe (NTA) solution
Firstly, the concentration is 10 −3 M ferric nitrate nonahydrate solution and concentration of 10 −3 Mixing M nitrilotriacetic acid trisodium solution according to the volume of 1:1, adding a pH regulator into the solution to regulate the pH value of the Fe (NTA) solution to 7.0, and standing the mixed solution to obtain an Fe (NTA) solution;
(4) functionalized Au PB Preparation of @ Au NPs
Au prepared in the step (2) PB @ Au colloid and procedure(3) Mixing the prepared Fe (NTA) solution according to the volume of 1:1, stirring and mixing, centrifugally washing to remove unreacted Fe (NTA), removing supernatant, and re-dispersing for later use to obtain the functionalized Au PB @Au NPs；
(5) Manufacturing test paper strip
Fully soaking filter paper in an ethanol solution of 3-mercaptopropyl-methyl-dimethoxysilane for 60min, taking out, respectively rinsing with distilled water and absolute ethanol, forming a monolayer on the surface of paper fibers through self-assembly of organosilane coupling, and drying for later use to obtain a functionalized test strip; putting the functionalized test strip substrate into a glass sand core funnel device of 100mLG4, and taking 100mL of prepared functionalized Au PB The @ Au NPs material is poured into a sand core funnel device, suction filtration is carried out, finally, the filter paper is taken out and dried, and the test strip is obtained, wherein the experimental process is shown in figure 4;
(6) making standard color comparison card
Sequentially dropwise adding the mixed solution (tyrosinase solutions with different concentrations and dopamine with the same concentration) into the functional nano gold core-shell structure material test strips, reacting the tyrosinase with different concentrations and the test strips to generate different colors, photographing each test strip, and finishing to obtain a tyrosinase standard colorimetric card as shown in fig. 5, wherein the colors are dark red, wine red, mauve, light purple, blue purple, purple gray and gray;
(7) detection of tyrosinase
And (3) mixing dopamine of a sample to be detected, dripping the mixed dopamine on the detection test strip obtained in the step 1), reacting at room temperature for 0.5 minute, airing, observing the color of the detection test strip, comparing with the standard colorimetric card obtained in the step 6), and estimating tyrosinase in serum to be detected through color comparison.
(9) Detection of actual samples
Randomly drawing 5 human serum samples, dripping 10 mu L of the samples on a test strip, wherein the test strip is purple gray, and comparing the test strip with a standard colorimetric card, the tyrosinase content in the human serum can be estimated to be about 10U/mL approximately.
Claims (9)
1. A preparation method of a detection test strip for rapidly detecting tyrosinase in serum is characterized by comprising the following steps:
S1、Au PB synthesis of @ Au colloid
Taking 10mL of nano gold colloid solution, adding 10-20 mu L of Prussian blue solution with the concentration of 0.1mol/L as Raman signal molecules, stirring, mixing uniformly, centrifuging, and resuspending in 10mL of ultrapure water to obtain Au @ PB colloid; to 10mL of the prepared Au @ PB colloid was added 250. mu.L of 6 x 10 -2 magnetic stirring and heating mol/L sodium citrate solution to boiling, adding 2.5mL chloroauric acid solution with the concentration of 1mmol/L, stopping heating, continuing magnetic stirring for 1h, centrifugally washing colloid for 2 times, and re-dispersing in 5mL ultrapure water to obtain Au PB @ Au colloid;
s2, functionalized Au PB Preparation of @ Au NPs
Au prepared in the step S1 PB Mixing the @ Au colloid with the Fe (NTA) solution according to the volume of 1:1, stirring and mixing, centrifugally washing to remove unreacted Fe (NTA), removing supernatant, and re-dispersing for later use to obtain the functionalized Au PB @Au NPs；
S3, fully soaking the test strip substrate in a 3-mercaptopropyl methyl dimethoxy silane ethanol solution, taking out, respectively rinsing with distilled water and absolute ethyl alcohol, forming a monolayer on the surface of paper fibers through self-assembly of organosilane coupling, and drying for later use to obtain the functionalized test strip; putting the functionalized test strip substrate into a glass sand core funnel device of 100mLG4, and taking 50mL-100mL of prepared functionalized Au PB And (3) pouring the @ Au NPs material into an even sand core funnel device, carrying out suction filtration, and finally taking out and drying the filter paper to obtain the detection test strip.
2. The method for preparing a test strip for rapidly detecting tyrosinase in serum according to claim 1, wherein the fe (nta) solution is synthesized by the following steps: firstly, the concentration is 10 −3 M ferric nitrate nonahydrate solution and concentration of 10 −3 M nitrilotriacetic acid trisodium solution is mixed according to the volume ratio of 1:1, and then the mixture is added into the solutionAdding pH regulator to regulate pH of Fe (NTA) solution to 7.0, and standing the mixed solution to obtain Fe (NTA) solution.
3. The preparation method of the test strip for rapidly detecting tyrosinase in serum according to claim 1, wherein the preparation method of the nanogold solution comprises the following steps: heating 100mL of chloroauric acid solution with the mass fraction of 0.01% to boil under magnetic stirring; and then 1-1.5 mL of 1% sodium citrate solution by mass fraction is added into the boiling chloroauric acid solution, after boiling for 15min, the reaction is finished, and the solution is cooled to room temperature to prepare the nano gold sol.
4. The preparation method of the test strip for rapidly detecting tyrosinase in serum according to claim 1, wherein the preparation method comprises the following steps: the test strip substrate in step S3 is filter paper, cellophane, non-woven fabric, cellulose membrane or starch membrane.
5. The preparation method of the test strip for rapidly detecting tyrosinase in serum according to claim 1, wherein the preparation method comprises the following steps: and the soaking time of the filter paper and the ethanol solution of the 3-mercaptopropyl methyl dimethoxy silane in the step S3 is 20-60 min.
6. A test strip for detecting tyrosinase in serum prepared according to any one of claims 1-5.
7. A method for detecting tyrosinase by using the test strip of claim 6, comprising the following steps:
1) manufacturing a standard color comparison card: mixing tyrosinase standard solutions with different concentrations (500U/mL, 400U/mL, 300U/mL, 200U/mL, 100U/mL, 50U/mL, 10U/mL and 0U/mL) with dopamine, sequentially dropwise adding 10 mu L onto a detection test strip, reacting at room temperature for 0.5-1.5 min, then airing, recording the color of the detection test strip, and finishing according to different colors to obtain a tyrosinase standard colorimetric card;
2) detecting tyrosinase: and (2) mixing a sample to be detected with dopamine, dripping the mixture onto a detection test strip, reacting at room temperature for 0.5-1.5 minutes, drying in the air, observing the color of the detection test strip, comparing with the standard colorimetric card obtained in the step 1), and estimating the tyrosinase content in the serum to be detected through color comparison.
8. The method of detecting tyrosinase according to claim 7, wherein: the concentration of the tyrosinase standard solution is 500U/mL, 400U/mL, 300U/mL, 200U/mL, 100U/mL, 50U/mL, 10U/mL and 0U/mL; the colors of the corresponding color comparison cards are dark red, wine red, purple red, light purple, blue purple, purple gray and gray.
9. The method of detecting tyrosinase according to claim 7, wherein: the concentration of dopamine in step 1) is 10 -3 M。
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| CN109342391B (en) * | 2018-11-03 | 2021-03-30 | 华东理工大学 | Tyrosinase activity detection method based on recyclable SERS sensor |
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