A method for detecting trace proteins

Abstract

The application is suitable for the field of protein detection, and provides a microprotein detection method, comprising the following steps: step one, preparing a protein solution, and adsorbing the protein solution through a polystyrene hole plate, and then washing; step two, synthesizing titanium dioxide nanoparticles by a solvothermal method, and adding the titanium dioxide into the protein solution, so that the titanium dioxide is combined with the hole plate protein, and washing after the combination; step three, adding a chromogenic solution into the hole plate, and irradiating the chromogenic solution through ultraviolet light, so that the resazurin in the chromogenic solution is converted into resorcinol; and step four, reading and recording the fluorescence value of the hole plate through an enzyme label instrument. The research proposes a new microprotein detection method, realizes signal amplification through a catalytic reaction, has the characteristics of convenient operation, high speed and low cost, and takes urine protein detection as an example to verify the feasibility, sensitivity and stability of the method, has high practical application value, and provides a new solution for clinical urine protein detection.

Description

Technical Field

[0001] This invention belongs to the field of protein detection, and in particular relates to a method for detecting trace amounts of protein. Background Technology

[0002] Currently, common methods for detecting total protein include: Kjeldahl method, biuret method, phenol reagent method, ultraviolet spectrophotometry, dye binding method, turbidimetric method, and BCA quantification method. Among these, the BCA quantification method has high sensitivity and good stability, detecting protein concentrations ranging from 20 to 200 μg / mL, while micro-BCA quantification methods can detect concentrations of 0.5 to 10 μg / mL. Clinically, the pyrogallol-molybdenum method is used to detect total protein content in urine; however, existing methods have low sensitivity and can only detect protein at the microgram level. Summary of the Invention

[0003] The purpose of this invention is to provide a method for detecting trace proteins, which aims to solve the problem that the detection method has low sensitivity and can only detect proteins at the microgram level.

[0004] The present invention is implemented as follows: a method for detecting trace proteins, the method comprising the following steps:

[0005] Step 1: Prepare a protein solution and adsorb it through a polystyrene well plate, followed by washing;

[0006] Step 2: Titanium dioxide nanoparticles were synthesized using a solvothermal method, and titanium dioxide was added to the well plate to bind with the well plate protein. After binding, the plate was washed.

[0007] Step 3: Add the colorimetric solution to the well plate and irradiate it with ultraviolet light to develop the colorimetric solution (C). 12 H6NNaO4) is converted into halogenated (C) 12 H7NO2);

[0008] Step 4: Read and record the fluorescence values ​​in the protein solution using an ELISA reader.

[0009] As a further embodiment of the present invention, the preparation of the protein solution in step one, and its adsorption through a polystyrene plate, specifically includes the following steps:

[0010] S1. Weigh 10 mg of human serum albumin using a balance and prepare a standard with a concentration of 10 mg / mL using 1 ml of phosphate buffer.

[0011] S2. Dilute human serum albumin with phosphate buffer to solutions with concentrations of 1, 2, 3, 4, and 5 ng / mL, respectively.

[0012] S3. Add the four concentrations of standard to the microplate, adding 200 μL of sample to each well. Set up three parallel wells for each concentration and incubate at 37 ℃ for 5 min. Then wash with 10 mM phosphate buffer (pH 7.4), adding 360 μL to each well and washing for 1 min each time. Shake vigorously to dry, and repeat the washing process three times in total.

[0013] As a further embodiment of the present invention, the synthesis of titanium dioxide nanoparticles by solvothermal method in step two specifically includes the following steps:

[0014] S1. Add 5 mL of tetrabutyl titanate to 10 mL of methanol solution under constant stirring at room temperature, then add 0.8 mL of hydrofluoric acid dropwise. After stirring for 0.5 h, transfer the mixed solution to a 40 mL polytetrafluoroethylene high-pressure reactor.

[0015] S2. React at 180 °C for 24 h in a constant temperature drying oven, then cool at room temperature, centrifuge the obtained product, and wash it 5 times with ethanol solution and 2 times with water.

[0016] S3. Take 100 mg of the obtained solid white precipitate and disperse it in 20 mL of 0.1 M sodium hydroxide solution and stir overnight.

[0017] S4. Centrifuge the solution at 5000 rpm for 5 min to remove the supernatant. Then disperse the precipitate in 20 mL of ultrapure water and dispense it into 10 tubes using 2 mL centrifuge tubes. Centrifuge the tubes at 13000 rpm for 15 min to remove the supernatant. Take one tube, dry it at 60 °C, and weigh it. Store the other samples at 4 °C for later use.

[0018] As a further embodiment of the present invention, the addition of titanium dioxide to the perforated plate in step two specifically includes the following steps:

[0019] S1. Disperse titanium dioxide nanoparticles in a 40 mM, pH 5.0 phosphate-Tween buffer to achieve a colloidal concentration of 20 μg / mL, wherein the final concentration of Tween is 1 / 10000 (v / v).

[0020] S2. Add the solution prepared in S1 to each well of the microplate and incubate at 37 °C for 5 min.

[0021] S3. Wash the plate by adding 360 μL of 40 mM phosphate-Tween buffer (pH 5.0) to each well for 1 min each time, then shake vigorously to dry. Repeat this washing process three times.

[0022] As a further embodiment of the present invention, step three, which involves adding a color-developing solution to the well plate and irradiating it with ultraviolet light, specifically includes the following steps:

[0023] S1. Weigh 1.28 g of Tris and dissolve it in 1 L of ultrapure water to obtain a concentration of 10 mM. Adjust the pH to 6.8 with 2 M hydrogen chloride and add 1 mL of 5 mM resazurin aqueous solution.

[0024] S2. Add 220 μL of colorimetric solution to each well of the polystyrene plate and irradiate under UV-254 nm ultraviolet light for 5 min.

[0025] As a further embodiment of the present invention, step four, which involves reading and recording the fluorescence values ​​in the protein solution using an ELISA reader, specifically includes the following steps:

[0026] S1. Use a pipette to take out 200 μL of the reaction product and transfer it to a black ELISA plate. Use the fluorescence module of the ELISA reader to read the value.

[0027] S2. Detect the fluorescence intensity F of each well and establish a standard curve between F and the mass concentration c of albumin standard;

[0028] S3. Dilute the actual urine sample 10,000 times with phosphate buffer, and perform the same operation as the standard sample to detect the fluorescence intensity F. Substitute the result into the standard curve to calculate the protein content in the actual sample.

[0029] The method for detecting trace proteins provided in this invention has the following beneficial effects:

[0030] This study proposes a novel method for detecting trace proteins, which amplifies the signal through a catalytic reaction. This method is characterized by its ease of operation, high speed, and low cost. The feasibility, sensitivity, and stability of the method are verified using urine protein detection as an example. It has high practical application value and provides a new solution for clinical urine protein detection. Attached Figure Description

[0031] Figure 1 Transmission electron microscopy (TEM) image of titanium dioxide nanoparticles provided in an embodiment of the present invention;

[0032] Figure 2 The standard curve for human serum albumin detection is provided for embodiments of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0035] In an embodiment of the present invention, a method for detecting trace proteins is characterized by comprising the following steps:

[0036] Step 1: Prepare a protein solution and adsorb it through a polystyrene well plate, followed by washing;

[0037] Step 2: Titanium dioxide nanoparticles were synthesized using a solvothermal method, and titanium dioxide was added to the protein solution to bind with the pore plate protein. The mixture was then washed after binding.

[0038] Step 3: Add the colorimetric solution to the well plate and irradiate it with ultraviolet light to develop the colorimetric solution (C). 12 H6NNaO4) is converted into halogenated (C) 12 H7NO2);

[0039] Step 4: Read and record the fluorescence values ​​in the protein solution using an ELISA reader.

[0040] Example 1: Titanium dioxide nanoparticles of approximately 20 nm were synthesized in methanol via a solvothermal method using tetrabutyl titanate and hydrofluoric acid as raw materials. (See attached image) Figure 1 )

[0041] Synthesis method: Under stirring at room temperature, 5 mL of tetrabutyl titanate was added dropwise to 10 mL of methanol, followed by 0.8 mL of hydrofluoric acid. After stirring for 0.5 h, the mixture was transferred to a 40 mL polytetrafluoroethylene high-pressure reactor. The reaction was carried out at 180 °C for 24 h in a constant temperature drying oven.

[0042] Washing method: After cooling to room temperature, the obtained product was centrifuged and washed 5 times with ethanol and twice with water. 100 mg of the resulting white solid precipitate was dispersed in 20 mL of 0.1 M sodium hydroxide solution and stirred overnight. The supernatant was removed by centrifugation at 5000 rpm for 5 min. The precipitate was dispersed in 20 mL of ultrapure water and dispensed into 10 2 mL centrifuge tubes. The tubes were centrifuged at 13000 rpm for 15 min to remove the supernatant. One tube was dried at 60 ℃ and weighed. The other samples were stored at 4 ℃ for later use.

[0043] Example 2: Preparation of standard solutions of different concentrations and incubation with polystyrene well plates:

[0044] Specific method: Weigh 10 mg of human serum albumin (HSA) using an analytical balance and prepare a 10 mg / mL standard with 1 ml of phosphate-buffered saline (PBS). Then, dilute the HSA to concentrations of 1, 2, 3, 4, and 5 ng / mL with PBS. Add 200 μL of the five concentrations of standard to each well of an ELISA plate, with three parallel wells for each concentration. Incubate at 37 ℃ for 5 min in a constant temperature oven. Then, vigorously shake off the liquid from the plate and pat dry on absorbent paper. Using a multichannel pipette, add 360 μL of the prepared phosphate buffer PB (10 mM, pH 7.4) to each well, let stand for 1 min, shake dry, and wash three times in total.

[0045] Example 3: Binding of titanium dioxide nanoparticles with pore plate proteins:

[0046] Specific method: Preparation of titanium dioxide binding solution (PBT): 1 L phosphate buffer PB (40 mM, pH 5.0) with 100 μL Tween-20 added. Take 100 ml of PBT and add 2 mg of titanium dioxide. Sonicate the solution for 10 min to obtain the working solution. Add 200 μL of the working solution to each well of the microplate and incubate at 37 ℃ for 5 min. Then, vigorously shake out the liquid from the wells, pat dry on absorbent paper, and wash with PBT. Add 360 μL to each well, let stand for 1 min, shake dry vigorously, and repeat the washing process three times.

[0047] Example 4: Adding a colorimetric solution to a well plate and achieving photocatalysis under ultraviolet light irradiation.

[0048] Specific method: Preparation of colorimetric solution: Weigh 1.28 g of Tris and dissolve it in 1 L of ultrapure water to obtain a concentration of 10 mM. Adjust the pH to 6.8 with 2 M HCl. The final concentration of resazurin (RZ) is 5 μM. Add 220 μL of colorimetric solution to each well of a polystyrene plate, irradiate under UV-254 nm ultraviolet light (two 8 W lamps) for 5 min, and then mix well on a vortex mixer.

[0049] Example 5: Microplate reader readings:

[0050] Specific method: Use a pipette to take 200 μL of the above reaction product and transfer it to a black ELISA plate. Use the fluorescence module of an ELISA reader to read the fluorescence intensity F of each well, using the Ex: 590 nm (20 nm) and Em: 620 nm (20 nm) modules. Establish a standard curve between F and the mass concentration c of the albumin standard. (Appendix) Figure 2 )

[0051] Example 6: Actual Sample Detection:

[0052] Specific method: Dilute the actual urine sample 10,000 times with PBS, and perform the same operation as the standard sample as above to detect the fluorescence intensity F. Substitute the result into the standard curve to calculate the protein content, and then multiply by 10,000 to obtain the actual protein content in the urine sample.

[0053] This study proposes a novel method for detecting trace proteins, which amplifies the signal through a catalytic reaction. This method is characterized by its ease of operation, high speed, and low cost. The feasibility, sensitivity, and stability of the method are verified using urine protein detection as an example. It has high practical application value and provides a new solution for clinical urine protein detection.

[0054] Titanium dioxide possesses excellent chemical stability and redox properties, making it a low-toxicity, low-cost photocatalyst widely used in solar cells and the purification of toxic organic matter in wastewater. When the surface of titanium dioxide nanoparticles is exposed to ultraviolet light (λ<384 nm), charge separation occurs on the particle surface, generating positively charged holes and electrons. Electrons on the particle surface can reduce molecules in the surrounding medium, while holes on the particle surface can be removed by corresponding hole scavengers, thus enabling the continuous photocatalytic process.

[0055] In this study, a photocatalytic fluorescence activation method based on the interaction between titanium dioxide and protein was established for the analysis of total protein content in samples.

[0056] The amount of protein coated on a polystyrene microplate is directly related to the protein concentration in the solution. Titanium dioxide nanoparticles can be non-specifically adsorbed onto the protein-coated microplate via phosphate. In this experiment, resamarium was used as the substrate for titanium dioxide photocatalysis, and tris was used for hole scavenging. Under illumination, resamarium was reduced to the fluorescent product halogen, leading to the activation of the fluorescence signal. The increase in fluorescence intensity was directly related to the protein concentration added to the microwells. By detecting the fluorescence intensity of the product, a linear relationship between fluorescence intensity and the concentration of human serum albumin standard was established, and the concentration of the test sample could be calculated using this linear relationship.

[0057] Tween significantly enhances the adsorption capacity of titanium dioxide for proteins in the pores. With the enhancement effect of Tween, the detection method is highly sensitive, capable of non-specifically detecting 1 ng / mL human serum albumin within half an hour. Finally, using urine as the actual sample, the total protein content was quantitatively analyzed. The results indicate that our method can be used for the determination of total protein in urine, showing great application potential for kidney disease screening.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting trace proteins, characterized in that, The method for detecting trace proteins includes the following steps: Step 1: Prepare a protein solution and adsorb it through a polystyrene well plate, followed by washing; Step 2: Titanium dioxide nanoparticles were synthesized using a solvothermal method, and titanium dioxide was added to the protein solution to bind with the pore plate protein, followed by washing. Step 3: Add the colorimetric solution to the protein solution and irradiate it with ultraviolet light to develop the chromogenic reagent. 12 H6NNaO4 is converted to halogen C. 12 H7NO2; Step 4: Read and record the fluorescence values ​​in the protein solution using an ELISA reader; The preparation of the protein solution in step one, and its adsorption through a polystyrene plate, specifically includes the following steps: S1. Weigh 10 mg of human serum albumin using a balance and prepare a standard with a concentration of 10 mg / mL using 1 mL of phosphate buffer. S2. Dilute human serum albumin with phosphate buffer to solutions with concentrations of 2, 4, 6, and 8 ng / mL, respectively. S3. Add the four concentrations of standard to the microplate, adding 200 μL of sample to each well. Set up three parallel wells for each concentration and incubate at 37°C for 5 min. Then wash with 10 mM phosphate buffer (pH 7.4), adding 360 μL to each well and washing for 1 min each time. Shake vigorously to dry, and repeat the washing process three times in total.

2. The method for detecting trace proteins according to claim 1, characterized in that, The solvothermal synthesis of titanium dioxide nanoparticles described in step two specifically includes the following steps: S1. Add 5 mL of tetrabutyl titanate to 10 mL of methanol solution under constant stirring at room temperature, then add 0.8 mL of hydrofluoric acid dropwise. Continue stirring for 0.5 h, then transfer the mixed solution to a 40 mL polytetrafluoroethylene high-pressure reactor. S2. React at 180℃ for 24 hours in a constant temperature drying oven, then cool at room temperature, centrifuge the obtained product, and wash it 5 times with ethanol solution and 2 times with water. S3. Take 100 mg of the obtained solid white precipitate and disperse it in 20 mL of 0.1 M sodium hydroxide solution and stir overnight. S4. Centrifuge the solution at 5000 rpm for 5 min to remove the supernatant. Then disperse the precipitate in 20 mL of ultrapure water and dispense it into 10 tubes with 2 mL of ethylene glycol monopropyl ether solution. Centrifuge at 13000 rpm for 15 min to remove the supernatant. Take one tube, dry it at 60 °C and weigh it. Store the other samples at 4 °C for later use.

3. The method for detecting trace proteins according to claim 1, characterized in that, Step two, which involves adding titanium dioxide to the protein solution, specifically includes the following steps: S1. Disperse titanium dioxide nanoparticles with a 40mM, pH 5.0 phosphate slow-Tween solution to achieve a colloidal concentration of 20μg / mL, wherein the final volume ratio of Tween is 1 / 10000. S2. Add 200 μL of phosphate buffer to each well of the microplate and incubate at 37°C for 5 min. S3. Wash the plate with phosphate buffer. Add 360 μL of phosphate buffer to each well, wash for 1 minute each time, shake vigorously to dry, and repeat three times in total.

4. The method for detecting trace proteins according to claim 1, characterized in that, Step three, which involves adding a colorimetric solution to the protein solution and irradiating it with ultraviolet light, specifically includes the following steps: S1. Weigh 1.28g of Tris and dissolve it in 1L of ultrapure water to obtain a concentration of 10mM. Adjust the pH to 6.8 with 2M hydrogen chloride and prepare a colorimetric solution with a final concentration of 5μM resazurin. S2. Add 220 μL of colorimetric solution to each well of the polystyrene plate and irradiate under UV-254nm ultraviolet light for 5 min.

5. The method for detecting trace proteins according to claim 1, characterized in that, Step four, which involves reading and recording the fluorescence values ​​in the protein solution using an ELISA reader, specifically includes the following steps: S1. Use a pipette to take out 200 μL of the reaction product and transfer it to a black ELISA plate. Use the fluorescence module of the ELISA reader to read the value. S2. Under the conditions of excitation wavelength 590nm and bandwidth 20nm, emission wavelength 620nm and bandwidth 20nm, the fluorescence intensity F of each well was detected, and a standard curve was established between F and the mass concentration c of albumin standard. S3. Dilute the actual urine sample 10,000 times with phosphate buffer, and perform the same operation as the standard sample to detect the fluorescence intensity F. Substitute the result into the standard curve to calculate the protein content in the actual sample.

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