CN-121995060-A - Rapid detection method for protein content of rubber latex based on fluorescent probe labeling

CN121995060ACN 121995060 ACN121995060 ACN 121995060ACN-121995060-A

Abstract

The invention belongs to the field of protein content detection, and discloses a method for rapidly detecting the protein content of rubber gum emulsion based on fluorescent probe labeling, which comprises the steps of selecting a latex sample for pretreatment and dilution; the method comprises the steps of dyeing by using protein fluorescent dye, collecting fluorescent signals of dyed latex samples to be detected, selecting latex proteins with different qualities as standard samples for protein quantification, collecting the fluorescent signals by using the same parameters as that of sample detection, performing linear fitting by taking the abscissa as the mass of the latex proteins and the ordinate as fluorescent values to obtain a latex protein linear equation, substituting the measured latex fluorescent signals into the latex protein linear equation, and calculating the content of the latex proteins. The invention accurately and rapidly detects the protein content in the rubber tree latex by a fluorescent probe marking technology, and realizes rapid detection of the protein content in the latex so as to meet the modern industrial production requirements of high-quality latex.

Inventors

Fan Ruishen
WU QING
YANG WENFENG
QIU JIAN
WEI FANG
XIE QINGBIAO
GAO HONGHUA

Assignees

中国热带农业科学院橡胶研究所

Dates

Publication Date: 20260508
Application Date: 20260204

Claims (10)

1. A method for rapidly detecting the protein content of rubber latex based on fluorescent probe labeling is characterized by comprising the following steps: S1, selecting and preprocessing a sample, namely collecting fresh latex in the field, transporting and preserving the latex on ice to prevent the latex from coagulating, filtering the latex by using a screen to remove impurities, accurately weighing the latex sample in a centrifuge tube, adding distilled water for dilution, wherein the mass of the latex is M N , the unit is mg, and the dilution multiple is N; S2, dyeing a sample, namely adding a protein fluorescent dye into the diluted latex obtained in the step S1, and standing for more than 30 minutes after slightly and uniformly mixing to enable the dye to be fully dyed, wherein the final volume is V and the unit is ml, and the protein fluorescent dye comprises a cyanine dye Cy5, a CD3 mouse monoclonal antibody and recombinant streptavidin; S3, detecting a sample, namely subpackaging 0.2mL of the dyed latex obtained in the step S2 into a micro-pore plate, performing fluorescence detection by using a multifunctional enzyme-labeling instrument, and recording a fluorescence value Y L ; S4, establishing a standard curve, namely selecting latex proteins with different qualities as standard samples for protein quantification, acquiring fluorescent signals by using the same parameters as those of sample detection, and obtaining a linear equation of the latex protein quality as Y=ax+b by taking an abscissa X as the latex protein quality and an ordinate Y as a fluorescent value; S5, calculating the protein mass in the latex, namely calculating the protein mass of the latex according to the following formula, namely M L =（Y L -b)/a, wherein M L is the protein mass of the latex, the unit is mug, and Y L is the fluorescence value of the latex to be detected; and S6, calculating the protein content of the latex according to the following formula, wherein F= (M L ×N×V）/（M N multiplied by 0.2) multiplied by 100%, F is the protein content of the latex, M L is the protein mass of the latex, N is the dilution factor of the latex, V is the total volume of the dyed latex in the step S2, the unit is mL, and M N is the mass of the latex, and the unit is mg.
2. The method for rapidly detecting protein content of gum latex according to claim 1, wherein in the step S1, 1mL of the latex is accurately weighed in a centrifuge tube, the dilution ratio is 50-200, the dilution ratio is preferably 100, and the dilution ratio is preferably 0.1×pbs buffer.
3. The method for rapidly detecting protein content of gum latex according to claim 1, wherein in the step S2, the protein fluorescent dye is preferably Cy5, the working concentration of the Cy5 dye is 0.5-3.0 μm, the dyeing time is 60-150 min, more preferably, the optimal working concentration of Cy5 is 2.0 μm, and the optimal dyeing time is 100 min.
4. The method for rapidly detecting protein content of gum emulsion according to claim 1, wherein in the step S3, a black bottom transparent microplate is selected, excitation light is 650nm, emission light is 670nm, and the working temperature of the multifunctional enzyme-labeled instrument is 25 ℃.
5. The method for rapidly detecting the protein content of rubber latex according to claim 1, wherein in the step S4, the method for preparing the standard latex protein liquid comprises the following steps: s41, mixing the latex with a protein extraction solvent according to a proportion, and vibrating and uniformly mixing; S42, adding a phenylmethylsulfonyl fluoride protease inhibitor into the mixture in the step S41; S43, standing the mixture obtained in the step S42 at room temperature to be not less than 1 h; S44, centrifuging and layering the mixed solution obtained in the step S43 at the temperature of 4 ℃; s45, sucking the intermediate clear liquid of the liquid obtained in the step S44, placing the intermediate clear liquid into a new centrifuge tube, and centrifuging and layering the intermediate clear liquid at the temperature of 4 ℃; S46, sucking the intermediate clear liquid of the liquid obtained in the step S45, and placing the intermediate clear liquid into a new centrifuge tube to obtain a latex protein extract, wherein the protein extract contains a particle detergent, and the protein content is detected by using a BCA method.
6. The method according to claim 5, wherein in the step S41, the protein extraction solvent is 7M urea, 2M thiourea, 40mM Tris, 4% triton X-100, 0.07% β -mercaptoethanol, and the volume ratio of the latex to the protein extract is 1:3; The latex is added dropwise and vibrated in time.
7. The method according to claim 5, wherein in the step S42, the concentration of PMSF is 100 mM and the volume ratio of the mixture in the step S41 is 1:200.
8. The method for rapid detection of protein content of gum emulsion according to claim 5, wherein in step S43, the standing time is 1-2 h.
9. The method for rapid detection of protein content of gum emulsion according to claim 5, wherein in step S44, the centrifuge rotation speed is 14000-18000rpm, and the centrifugation time is 45-60min; preferably, the centrifuge speed is 18000rpm and the centrifugation time is 60min.
10. The method for rapid detection of protein content of gum emulsion according to claim 5, wherein in step S45, the centrifuge rotation speed is 14000-18000rpm, and the centrifugation time is 15-30min; preferably, the centrifuge speed is 14000rpm and the centrifugation time is 15min.

Description

Rapid detection method for protein content of rubber latex based on fluorescent probe labeling Technical Field The invention relates to the technical field of protein content detection, in particular to a method for rapidly detecting the protein content of rubber latex based on fluorescent probe labeling. Background Natural rubber is an important industrial raw material, and is prepared by processing latex discharged after cutting the mammary duct cells of a rubber tree, and the main component of the natural rubber is polyisoprene and contains a small amount of non-rubber components. The protein is used as a non-rubber component with higher content in the latex, plays a role similar to an auxiliary agent in natural rubber, and the content has obvious influence on improving the processability, mechanical property, aging resistance and the like of the rubber. The current method for measuring the protein content of the latex adopts a Kjeldahl nitrogen determination method specified in GB/T8088-2008 'determination of nitrogen content of natural raw rubber and natural latex'. The method uses a catalyst to digest a sample by concentrated sulfuric acid, so that organic nitrogen is decomposed into ammonia, and then the ammonia is transferred into a solution to be combined with sulfuric acid to generate ammonium bisulfate, and then the ammonia is distilled by heating with sodium hydroxide. The evolved ammonia is absorbed with sulfuric acid or boric acid solution, the excess acid is titrated with the corresponding calibration solution, the nitrogen content in the latex sample is calculated from the amount consumed, and converted to protein content. Some detection methods such as the Fu Lin Fen method, the biuret method, the Coomassie brilliant blue method and the BCA method are commonly used in protein detection, the methods need pretreatment of samples, the time consumption is long, the analysis process is complicated, and part of the methods can generate toxic or harmful gas in the reaction process and are easy to cause harm to human bodies and the environment. In addition, the methods have high personnel experiment skills and experiment conditions, are only suitable for laboratory implementation, and are not suitable for large-batch and rapid latex protein detection in industrial production processes. The cell fluorescence method has the advantages of accurate data, rapid detection, economy, environmental protection and the like due to high sensitivity and high specificity, and is widely applied to quantitative analysis of proteins in cells at present. However, the following technical challenges are faced in the detection of the rubber latex protein by fluorescent dye technology: 1. The fresh latex is extremely easy to solidify under normal temperature or improper treatment (acid condition), so that the sample is not uniform, the sample adding equipment is blocked, and stable fluorescence detection cannot be performed. 2. Parameters of fluorescent dye (such as dye type, concentration and staining time) in cell protein detection cannot be directly sleeved in latex systems with completely different components and morphologies. A completely new set of optimal detection conditions for latex protein-fluorescent dye reaction systems needs to be established to ensure the sensitivity, linear range and reproducibility of the detection. 3. Quantitative standard preparation and standard curve establishment difficulties require extraction of pure standards from complex latices that represent the overall characteristics of the latex protein. The preparation method of the standard substance is required to be ensured to be reliable and good in reproducibility, and the fluorescence behavior of the standard substance is consistent with that of protein in a latex sample to be detected, so that a standard curve for accurate quantification can be established. Disclosure of Invention In order to solve the technical problems, the invention aims to provide a method for rapidly detecting the protein content of rubber latex based on fluorescent probe marking, which can accurately and rapidly detect the protein content of rubber latex by using a fluorescent probe marking technology, thereby realizing rapid detection of the protein content of the latex and meeting the modern industrial production requirements of high-quality latex. In order to achieve the aim, the technical scheme adopted by the invention is that the method for rapidly detecting the protein content of the rubber latex based on fluorescent probe marking comprises the following steps: S1, selecting and preprocessing a sample, namely collecting fresh latex in the field, and transporting and storing on ice to prevent the latex from coagulating. After removing impurities by using an 80-mesh screen to filter latex, accurately weighing a latex sample in a centrifuge tube, adding distilled water for dilution, wherein the mass of the latex is M N, the unit is mg, and the dilution mu