Method for improving protein detection accuracy by utilizing coomassie brilliant blue ratio type probe and application

Abstract

The invention provides a method for improving protein detection accuracy by using a coomassie brilliant blue ratio-type probe and application of the method. Absorbance at two peaks of 465nm and 595nm of bovine serum albumin-coomassie brilliant blue solutions with different concentrations are detected, and the protein detection accuracy is improved by using the ratio of absorbance values from two wavelengths and different absorption peaks. Self-calibration ratio signals are obtained, a linear relation between the peak value ratio of the two peaks and the BSA standard concentration is constructed, and quantitative detection is achieved. The detection method has the advantages of simplicity, rapidness, low cost and less interference, and is suitable for modernization measurement. The method can be used for protein detection, especially for protein detection in milk powder.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of protein detection, and particularly relates to a method for improving the accuracy of protein detection by using a Coomassie brilliant blue ratio probe and application thereof. BACKGROUND

[0002] Proteins are important components of cells and play a crucial role in life phenomena and life processes. Quantitative analysis and detection of proteins have very important practical significance in the fields of pharmacy, biochemistry, biotechnology, clinical medicine and food nutrition. The accuracy of protein quantitative detection results has guiding significance for explaining the relationship between protein structure and function and the mechanism of action of proteins and substances or drugs.

[0003] Bovine serum albumin (BSA) is the highest protein component in bovine plasma, and has the effects of scavenging free radicals, anticoagulation, maintaining plasma osmotic pressure and body fluid balance, etc. In actual production and life, the quantitative detection of bovine serum albumin is particularly important. For example, at present, viral vaccine products (such as measles, mumps, rubella, and varicella vaccines) produced by cell culture method still contain residual bovine serum albumin in the vaccine although a certain amount of bovine serum is added in the culture process to provide necessary nutrients for cell growth, and subsequent washing, purification and other steps. Bovine serum albumin as a heterologous protein may cause serious allergic reactions after inoculation into the human body, so the detection of residual bovine serum albumin (BSA) content is an important quality control index for vaccine products, and is directly related to the safety of vaccine use.

[0004] Traditional bovine serum albumin analysis and detection methods mainly include enzyme-linked immunosorbent assay (ELISA), indirect competitive enzyme-linked immunodetection method (IC-ELISA), Kjeldahl nitrogen determination method, Coomassie brilliant blue method, spectrophotometry, fluorophotometry, chemiluminescence method, near-infrared reflectance spectroscopy, surface plasmon resonance technology (SPR technology), biuret method, etc. The Coomassie brilliant blue G-250 colorimetric method is being more and more widely used due to its outstanding advantages, such as simple method, only one color developing solution is needed; rapid reaction, less interference, etc.

[0005] Coomassie brilliant blue G-250 determination of protein content belongs to a dye binding method, but the traditional Coomassie brilliant blue G-250 colorimetric method is a single absorption probe, which is easily disturbed by the concentration of the probe, the environment and the instrument, so it is difficult to detect slight absorbance value fluctuations, affecting the accuracy of the results, and there are problems such as poor linear relationship, low accuracy, etc. in actual application, which limits the actual application.

[0006] Therefore, it is particularly important to develop a new method for using Coomassie brilliant blue G-250 for high-sensitivity and high-accuracy quantitative detection of proteins. SUMMARY

[0007] The present application aims to provide a method for improving the accuracy of protein detection using a Coomassie brilliant blue ratio probe, which obtains a self-calibrated ratio signal by detecting the absorbance at 465 nm and 595 nm of different concentrations of bovine serum albumin-Coomassie brilliant blue solution, using the absorbance value ratio of two wavelengths and different absorption peaks, and constructing a linear relationship between the two peak value ratios and the standard concentration of BSA to achieve quantitative detection. This detection method is simple, fast, low-cost and less interfered, and is suitable for modern chemical measurement.

[0008] The present application provides a method for improving the accuracy of protein detection using a Coomassie brilliant blue ratio probe, which is used for protein detection, especially for protein detection in milk powder, and has the characteristics of high sensitivity and high accuracy. The present application provides a more sensitive and accurate method for quantitative detection of proteins.

[0009] The specific technical solutions of the present application are as follows:

[0010] A method for improving the accuracy of protein detection using a Coomassie brilliant blue ratio probe, the specific method is as follows:

[0011] Mix different concentrations of bovine serum albumin standard solution with Coomassie brilliant blue solution for staining, and mix the same volume of good solvent PBS with Coomassie brilliant blue solution as a blank sample, after incubation and staining, use an enzyme label instrument to scan the absorbance of the mixed solution at 300 to 800 nm, after successful staining, detect the two peak value data at 465 nm and 595 nm of different concentrations of solution, deduct the blank, and use the linear relationship between the two peak value ratios and the concentration of bovine serum albumin in the mixed staining system to construct the method for improving the accuracy of protein detection using a Coomassie brilliant blue ratio probe.

[0012] The preparation method of the bovine serum albumin standard solution is as follows: dissolve bovine serum albumin BSA in good solvent PBS, and dilute to prepare bovine serum albumin solutions with different concentrations. Preferably, dissolve bovine serum albumin BSA in good solvent PBS to prepare a 4 mg / ml solution. The preparation method of the good solvent PBS used is as follows: weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.29 g of sodium dihydrogen phosphate dihydrate, and 2.96 g of disodium hydrogen phosphate dodecahydrate, dissolve them in distilled water, and make up to 1000 ml. Dilute the 4 mg / ml bovine serum albumin solution to prepare BSA standard solutions with different concentrations, and the concentrations are 10, 20, 40, 80, 160, 320, 640, 960, and 1280 μg / ml.

[0013] The preparation method of the Coomassie brilliant blue solution is: take 5 mg of Coomassie brilliant blue, add 2.5 ml of volume concentration 95% ethanol, completely dissolve, slowly stir and add 5 ml of 85% phosphoric acid, and dilute to 50 ml with deionized water. The obtained solution is 0.1 mg / ml, which is stored in dark and low temperature. Filtration is needed before mixed staining. If too much water is added to ethanol, it will fail to stain successfully.

[0014] The volume ratio of Coomassie brilliant blue solution and different concentrations of bovine serum albumin solution is 19:1. After mixed staining, the BSA concentration in the mixed staining system is 0.5, 1, 2, 4, 8, 16, 32, 48, 64 μg / ml. If the BSA concentration in the mixed staining system is too low, it cannot be stained successfully.

[0015] The blank sample is mixed with Coomassie brilliant blue solution and good solvent PBS at a volume ratio of 19:1, and three groups in parallel.

[0016] The incubation staining refers to incubation at 37℃ for 5-40 min, preferably 20 min at 37℃. If the temperature is too low, such as about 4℃, it cannot be stained successfully. The staining effect at room temperature is not as good as that at 37℃. The linear relationship is better and better from 5 min to 20 min to 40 min. However, the linear relationship is good and stable when incubated for 20 min, and the time is relatively short, which is suitable for application in practice.

[0017] The sign of successful staining is that the absorption spectrum decreases at 465 nm and increases at 595 nm with the increase of the concentration of BSA standard solution.

[0018] The above two peak ratios are the peak ratios after deducting the blank, i.e. 595 nm absorbance value / 465 nm absorbance value, and the experimental verification shows that the linear relationship between the peak ratio and the BSA concentration is stable and has strong correlation. The experimental results and practical value are obviously better than those detected by 465 nm absorbance value / 595 nm absorbance value.

[0019] In the present application, after the Coomassie brilliant blue G-250 is combined with the protein, the dye solution changes from red to blue, and the absorption peak at 595 nm is increased. In the method of the present application, when the protein concentration in the solution is increased under the condition that the Coomassie brilliant blue G-250 is excessive and the concentration is constant, a certain equivalent of the Coomassie brilliant blue G-250 is combined with the protein, the absorbance value of the Coomassie brilliant blue G-250 at 465 nm in the ultraviolet-visible absorption spectrum of the solution is reduced, the absorbance value of the Coomassie brilliant blue G-250 and the protein combination at 595 nm is increased, and the change has a certain quantitative relationship. Therefore, in the method of the present application, the concentration of bovine serum albumin and the ratio of the corresponding 595 nm and 465 nm absorption peak values can be used to obtain an accurate linear relationship for quantifying the protein.

[0020] Preferably, the method of the present application is:

[0021] 10 μl of BSA standard solution with concentrations of 10, 20, 40, 80, 160, 320, 640, 960, 1280 μg / ml, respectively, and 190 μl of G-250 solution with a concentration of 0.1 mg / ml are mixed to obtain a BSA-G-250 mixed solution; the good solvent PBS and the G-250 solution are mixed as a blank sample in the same volume ratio, the mixed staining is incubated at 37°C for 20 min, the absorbance values in the range of 300 nm-800 nm are scanned, and the absorbance values at 465 nm and 595 nm are detected to confirm whether the staining is successful; after confirming that the staining is successful, the blank is deducted, and the 595 nm absorbance value / 465 nm absorbance value is used to plot the BSA concentration in the BSA-G-250 mixed solution to obtain a linear relationship.

[0022] The obtained linear relationship is y=0.04466x+0.65412, and the correlation coefficient R 2 =0.991; wherein, x is the BSA concentration, and the unit is μg / ml; y is the 595 nm absorbance value / 465 nm absorbance value.

[0023] The application of the method for improving the accuracy of protein detection by using the Coomassie brilliant blue ratio probe provided by the present application is used for protein detection, especially for protein detection in milk powder.

[0024] The specific detection method is as follows: the sample to be detected is prepared into a solution, 10 μl of the sample to be detected is mixed with 190 μl of G-250 solution with a concentration of 0.1 mg / ml for staining, the good solvent PBS and the G-250 solution are mixed as a blank sample in the same volume ratio, the absorbance values at 465 nm and 595 nm of the mixed solution are detected, and after the blank is also deducted, the sample protein concentration in the sample solution to be detected is obtained by substituting into the obtained linear relationship.

[0025] The design idea of the present application: the inventor found that G-250 is red in free state, and the maximum light absorption wavelength is at 465 nm; after combining with protein under acidic conditions, the solution changes from red to blue, which increases the absorption peak of Coomassie brilliant blue G-250 and protein combination at 595 nm, and the peak value of Coomassie brilliant blue G-250 at 465 nm is synchronously reduced. The color change is caused by the change of dye molecular structure after the dye combines with protein. The combination of Coomassie brilliant blue G-250 and protein mainly depends on electrostatic interaction (sulfonic acid group of dye combines with basic amino acid residues such as Arg, Lys and His of protein), and the combination stability is enhanced through hydrophobic interaction (aromatic ring of dye combines with non-polar region of aromatic amino acid such as Phe, Tyr and Trp of protein). Therefore, when it combines with protein and becomes blue, the absorption of visible light at 465 nm is increased at 595 nm, and the original peak value is synchronously reduced. Under the condition that Coomassie brilliant blue G-250 is excessive and the concentration is constant, the protein-dye combination has the maximum light absorption at 595 nm, and the light absorption value is proportional to the protein content. Based on this principle, the method can be used for quantitative determination of protein. The combination of protein and Coomassie brilliant blue G-250 reaches equilibrium in about 2 min, the color development is completed within 5 min, and the reaction is very rapid; the combination remains stable within 1 h at room temperature. The reagent preparation of the method is simple, the operation is simple and fast, the reaction is very sensitive, the sensitivity is 4 times higher than that of Lowry method, the microgram level protein content can be determined, the protein concentration range to be determined is 0-1000 μg / mL, and the minimum measurable protein is 2.5 μg / mL. It is a rapid determination method of trace protein.

[0026] The inventor also found that the traditional Coomassie brilliant blue color development method has many disadvantages in practical application. As a single absorption probe, it is easily disturbed by probe concentration, environment and instrument, so it is difficult to detect slight absorbance value fluctuation, and the most prominent is that the linear relationship is not very good. Therefore, the ratio type probe new method of the present application uses the absorbance value ratio from two wavelengths, which is not easily affected by the above factors, can minimize the deviation, greatly improves the linear correlation of the obtained standard curve, and the determination result is more accurate.

[0027] According to the principle of Coomassie brilliant blue method, under the condition that Coomassie brilliant blue G-250 is excessive and the concentration is constant, when the protein concentration in the solution increases, a certain equivalent of Coomassie brilliant blue G-250 will combine with protein, the absorbance value of Coomassie brilliant blue G-250 at 465 nm in the ultraviolet-visible absorption spectrum of the solution is reduced, and the absorbance value of Coomassie brilliant blue G-250 and protein combination at 595 nm is increased (see Figure 3), and the change has a certain quantitative relationship. Specifically, when the protein concentration in the solution increases, the absorbance at 595 nm can basically maintain a linear increase, while the absorbance at 465 nm maintains a linear downward trend. Therefore, this characteristic can be used to construct a ratio probe. The ratio probe has two different absorption peaks, and depending on the absorbance value ratio from the two wavelengths, a self-calibrated ratio signal can be obtained, effectively reducing interference and improving detection accuracy.

[0028] Compared with the prior art, the present application uses bovine serum albumin BSA as a protein model, uses the characteristics of the ratio probe that is sensitive to environmental changes and can self-correct, and uses the absorbance values at two wavelengths to construct a ratio spectrophotometric analysis method with concentration changes, to establish a detection method that is selective, strong in anti-interference, fast and stable. The method has been successfully applied to the detection of protein in milk powder. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the ratio standard working curve of the Coomassie brilliant blue method obtained by the method of the present application (595 nm absorbance value / 465 nm absorbance value);

[0030] Figure 2 is the standard working curve at 595 nm obtained by the traditional Coomassie brilliant blue method;

[0031] Figure 3 is the ultraviolet absorption spectrum of Coomassie brilliant blue staining BSA;

[0032] Figure 4 is the ratio standard working curve of the Coomassie brilliant blue method (465 nm absorbance value / 595 nm absorbance value). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0034] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially.

[0035] Unless otherwise specified, the specific techniques or conditions in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0036] Bovine serum albumin, Coomassie brilliant blue, and sodium dihydrogen phosphate dodecahydrate used in the present invention were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., 85% phosphoric acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd., anhydrous ethanol and sodium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd., and potassium chloride and sodium dihydrogen phosphate dihydrate were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The good solvent PBS was prepared as follows: 8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.29 g of sodium dihydrogen phosphate dihydrate, and 2.96 g of sodium dihydrogen phosphate dodecahydrate were weighed and dissolved in distilled water, and the volume was adjusted to 1000 ml.

[0037] Example 1

[0038] A method for improving protein detection accuracy using a Coomassie brilliant blue ratiometric probe comprises the following steps:

[0039] 1) Prepare BSA standard solutions of different concentrations:

[0040] 1-1) Weigh 4 mg of BSA and dissolve it in 1 ml of PBS to prepare a 4 mg / ml BSA standard solution.

[0041] 1-2) Take 48 μl of the 4 mg / ml BSA solution and dilute it with 102 μl of PBS to obtain a 1280 μg / ml BSA solution. Dilute the solution in half to obtain a 640 μg / ml BSA standard solution, a 320 μg / ml BSA standard solution, a 160 μg / ml BSA standard solution, an 80 μg / ml BSA standard solution, a 40 μg / ml BSA standard solution, a 20 μg / ml BSA standard solution, and a 10 μg / ml BSA standard solution. Then, take 37.5 μl of the 1280 μg / ml BSA standard solution and dilute it with 12.5 μl of PBS to obtain a 960 μg / ml BSA standard solution.

[0042] 2) Prepare G-250 solution

[0043] Weigh 5 mg of G-250 and add 2.5 ml of 95% anhydrous ethanol. Once completely dissolved, add 5 ml of 85% phosphoric acid dropwise while slowly stirring. Transfer to a volumetric flask and dilute to 50 ml with deionized water. Store at low temperature and away from light. Filter the solution before use.

[0044] 3) Mix and dye the standard solution

[0045] Inject 190 μl of filtered G-250 solution into a 96-well plate. Inject 10 μl of each of the nine BSA standard solutions into the wells containing G-250 solution. Perform three replicates for each concentration. Mix thoroughly and incubate at 37°C for 20 minutes.

[0046] 4) Blank sample preparation

[0047] Mix 10ul good solvent PBS with 190ul filtered G-250 solution in a small hole, three groups in parallel, incubate at 37℃ for 20min.

[0048] 5) Absorbance value scanning of standard solution mixture

[0049] Place the 96-well plate in the microplate reader, scan the absorbance value of the standard solution mixture from 300nm to 800nm, make the absorption spectrum of different concentrations to determine whether the staining is successful. Scan the absorbance value of the standard solution mixture at 465nm and 595nm. Plot the absorbance value of 595nm and 595nm / 465nm (subtracting blank) against the concentration of the mixed solution BSA, and obtain the working curve with linear relationship. The standard curve obtained by plotting the absorbance value of 595nm / 465nm (subtracting blank) against the concentration of the mixed solution BSA is y=0.04466x+0.65412, y is the absorbance value of 595nm / 465nm, and x is the concentration of BSA. The correlation coefficient R 2 =0.991;

[0050] By comparing the working curves obtained by plotting the absorbance value of 595nm / 465nm and 595nm, 465nm / 595nm against the concentration (see Figure 1 , Figure 2 , Figure 4 ), Figure 1 The linear relationship is good, and Figure 2 , Figure 4 The linear relationship is poor, and most of the data points are outside the standard curve. It can be seen that the present inventors use the ratio type probe of 595nm / 465nm absorbance value, which has good linear relationship and greatly improves the linear correlation of the working curve.

[0051] Example 2

[0052] A method for improving the accuracy of protein detection using Coomassie brilliant blue ratio type probe, comprising the following steps:

[0053] 1) Preparation of BSA standard solution with different concentrations:

[0054] 1-1) Weigh 4mg BSA, dissolve in 1ml PBS to prepare 4mg / ml BSA standard solution.

[0055] 1-2) Take 160 μl of 4 mg / ml BSA solution, add 340 μl of PBS to dilute to 1280 μg / ml BSA standard solution, and sequentially dilute to 640 μg / ml BSA standard solution, 320 μg / ml BSA standard solution, 160 μg / ml BSA standard solution, 80 μg / ml BSA standard solution, 40 μg / ml BSA standard solution, and 20 μg / ml BSA standard solution. Take another 60 μl of 4 mg / ml BSA solution, add 190 μl of PBS to dilute to 960 μg / ml BSA standard solution. Take another 100 μl of 4 mg / ml BSA solution, add 150 μl of PBS to dilute to 1600 μg / ml BSA solution; take another 75 μl of 4 mg / ml BSA solution, add 675 μl of PBS to dilute to 400 μg / ml BSA solution.

[0056] 2) Preparation of G-250 solution

[0057] The same as Example 1;

[0058] 3) Standard solution mixed staining

[0059] The concentration and operation are the same as Example 1;

[0060] 4) Sample addition recovery

[0061] 4-1) Test sample preparation: mix 400 μg / ml BSA solution with PBS solution at a volume ratio of 1:1 to obtain unspiked sample a.

[0062] 4-2) Spiked sample preparation: mix 320 μg / ml BSA solution, 640 μg / ml BSA solution, 960 μg / ml BSA solution, 1280 μg / ml BSA solution, and 1600 μg / ml BSA solution with 400 μg / ml BSA solution at a volume ratio of 1:1 to obtain spiked sample b.

[0063] 4-3) Mixed staining of the above solutions: mix the above samples, spiked samples, and G-250 solution at a volume ratio of 10 μl:190 μl in a 96-well plate. Each concentration is tested in five groups in parallel. After mixing, incubate at 37°C for 20 min. After mixing, the BSA concentration of unspiked sample a is 10 μg / ml, and the BSA concentration of spiked sample b is 18 μg / ml, 26 μg / ml, 34 μg / ml, 42 μg / ml, and 50 μg / ml from small to large.

[0064] 5) Blank sample preparation:

[0065] The same as Example 1;

[0066] 6) Scanning the absorbance value of the standard solution mixture, unspiked sample a, spiked sample b, and blank sample:

[0067] Place the 96-well plate in the microplate reader and scan the absorbance value in the range of 300 nm to 800 nm.

[0068] Subtract the blank from the unspiked sample a and spiked sample b at 595 nm absorbance value and 595 nm / 465 nm absorbance value ratio, respectively, and substitute them into the corresponding standard curve linear equation (same as Example 1) to obtain the BSA concentration determination value of unspiked sample a and spiked sample b.

[0069] 6) Calculation of the standard addition recovery rate

[0070] According to the standard addition recovery rate formula, as follows:

[0071] Standard addition recovery rate = (determination value of spiked sample b - determination value of unspiked sample a) / addition amount, wherein the addition amount is the theoretical value of spiked sample b - the theoretical value of unspiked sample a.

[0072] By comparing the standard addition recovery rates obtained from the 595 nm and 595 nm / 465 nm standard curves (see Table 1, Table 2), it can be seen that the present application uses a new method of (595 nm / 465 nm) ratio type probe, which has a better recovery rate, greatly improving the accuracy and precision of the analysis results.

[0073] Table 1: Standard curve of 595 nm standard addition recovery test results (n = 5)

[0074]

[0075] Table 2: Standard curve of 595 nm / 465 nm standard addition recovery test results (n = 5)

[0076]

[0077] Example 3

[0078] An application of a method for improving the accuracy of protein detection using Coomassie brilliant blue ratio type probe for detecting protein content in milk powder, specifically comprising the following steps:

[0079] 1) Preparation of BSA standard solution with different concentrations:

[0080] Same as Example 1;

[0081] 2) Preparation of G-250 solution:

[0082] Same as Example 1;

[0083] 3) Preparation of sample solution:

[0084] 3-1) Select a milk powder sample, marked as sample No. 1. According to the milk powder nutrient composition table of the sample, it is known that the sample contains 21.5 grams of protein per 100 grams.

[0085] 3-2) 0.0186 grams of the sample is weighed and dissolved in 1 ml of PBS to prepare a sample solution with a protein concentration of 4 mg / ml.

[0086] 3-3) 50 μl of 4 mg / ml (the sample solution concentrations below are all in terms of protein concentration) sample solution is taken, diluted with PBS to 200 μl of 1000 μg / ml sample solution. In turn, half-diluted to 500 μg / ml sample solution, 250 μg / ml sample solution, 125 μg / ml sample solution, 62.5 μg / ml sample solution, and 31.25 μg / ml sample solution.

[0087] 4) Standard solution mixed staining:

[0088] Concentration, operation same as Example 1;

[0089] 5) Sample solution mixed staining:

[0090] In a 96-well plate, 190 μl of filtered G-250 solution is injected. 10 μl of the above six concentrations of sample solution is injected into the wells with G-250 solution, with five parallel experiments for each concentration. After mixing, incubate at 37°C for 20 min. After mixing, the protein concentration of the solution is 1.5625 μg / ml, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / ml, and 50 μg / ml, in order from small to large.

[0091] 6) Blank sample preparation

[0092] Same as Example 1;

[0093] 7) Absorbance value scanning of standard solution mixture, sample, and blank sample

[0094] The 595 nm absorbance value of the sample after deducting the blank is substituted into the linear curve (same as Example 1) to obtain the BSA concentration determination value of the sample.

[0095] 8) Analysis of sample determination results

[0096] After data processing, the error and bias values between the determination value and the true value, and the data mean value are calculated. By comparing the errors obtained by the two methods, the differences between the bias values (see Tables 3 and 4), it can be seen that the ratio probe method described in the present method has better accuracy and precision in the actual sample determination within the linear range.

[0097] Table 3: 595nm / 465nm standard curve actual sample determination results (n=5)

[0098] Number Standard value (μg / ml) Measured average value (μg / ml) RSD (%) 1 3.125 4.327 0.65 2 6.25 6.99 1.73 3 50 50.337 0.02

[0099] Table 4: 595nm standard curve actual sample determination results (n=5)

[0100] Number Standard value (μg / ml) Measured average value (μg / ml) RSD (%) 1 3.125 4.637 16.75 2 6.25 8.713 16.21 3 50 49.306 0.43

[0101] The ratio probe provided by the present application is based on the fact that the absorbance values at two wavelengths are significantly related to the concentration of the measured substance, and the linear increase at 595nm and the linear decrease at 465nm. Under the same system and the same determination conditions, the absorbance ratio can eliminate the common errors in experiments, including light source fluctuation, cuvette (or 96-well plate) difference (light path change), solution turbidity (scattered light) or background absorption, etc.

[0102] The above description of the embodiments is to facilitate those skilled in the art to understand and use the present application. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without creative labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art according to the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A method for improving protein detection accuracy using a Coomassie brilliant blue ratiometric probe, characterized in that: The method is as follows: Bovine serum albumin standard solutions of different concentrations were mixed with Coomassie Brilliant Blue solution for staining. At the same time, a good solvent PBS and Coomassie Brilliant Blue solution were mixed at the same volume ratio as a blank sample. After incubation and staining, the absorbance of the mixed solution from 300 to 800 nm was scanned using a microplate reader. After confirming that the staining was successful, the peak values ​​of the two peaks of solutions of different concentrations at 465 nm and 595 nm were detected. After subtracting the blank, the linear relationship between the peak value ratio of the two peaks and the bovine serum albumin concentration in the mixed staining system was used to construct a method for improving the accuracy of protein detection using a Coomassie Brilliant Blue ratio probe.

2. The method according to claim 1, characterized in that The bovine serum albumin solution is prepared by dissolving bovine serum albumin (BSA) in a good solvent, PBS, and diluting the solution to prepare bovine serum albumin solutions with different concentrations, which are 10, 20, 40, 80, 160, 320, 640, 960, and 1280 μg / ml.

3. The method according to claim 1 or 2, characterized in that The good solvent PBS was prepared as follows: 8.0 g of sodium chloride, 0.2 g of potassium chloride, 0.29 g of sodium dihydrogen phosphate dihydrate, and 2.96 g of disodium hydrogen phosphate dodecahydrate were weighed and dissolved in distilled water to a volume of 1000 ml.

4. The method according to claim 1, wherein The preparation method of the Coomassie Brilliant Blue solution is as follows: 5 mg of Coomassie Brilliant Blue is added to 2.5 ml of 95% ethanol, and after complete dissolution, 5 ml of 85% phosphoric acid is slowly stirred and added dropwise, and the volume is made up to 50 ml with deionized water. The resulting solution is 0.1 mg / ml, and the solution is stored at low temperature in the dark and needs to be filtered before mixed staining.

5. The method according to claim 1, wherein The volume ratio of Coomassie brilliant blue solution to bovine serum albumin solutions of different concentrations was 19:

1.

6. The method according to claim 1, characterized in that The heat preservation dyeing refers to the heat preservation time at 37° C. for 5-40 minutes.

7. The method according to claim 1 or 6, characterized in that The heat preservation dyeing refers to the heat preservation time at 37°C for 20 minutes.

8. An application of the method for improving protein detection accuracy using a Coomassie Brilliant Blue ratiometric probe according to any one of claims 1 to 7, characterized in that: For protein detection.

9. The use according to claim 8, characterized in that The protein detection method is as follows: the sample to be tested is prepared into a solution, and the sample solution to be tested is mixed and stained at a ratio of 10 μl of the sample to be tested and 190 μl of a 0.1 mg / ml G-250 solution. The good solvent PBS and the G-250 solution are mixed in the same volume ratio as a blank sample. The absorbance values ​​of the mixed solution at 465 nm and 595 nm are detected. After subtracting the blank, the absorbance values ​​are substituted into the obtained linear relationship to obtain the protein concentration of the sample in the sample solution to be tested.

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