CN111337468B - Multi-analysis snake venom mixture analysis fluorescent sensor - Google Patents
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Abstract
The invention discloses a multivariate analysis technology for multiple snake venom proteins. In particular to a method for analyzing a plurality of snake venom proteins and a complex sample of a mixture by combining proteins and fluorescent dyes by utilizing different fluorescent signals generated by different proteins and different dyes due to different combination degrees and combination modes. The invention prepares a multivariate analysis fluorescence sensing array based on differential snake venom protein fluorescence staining. The discrimination analysis of various snake venom proteins is successfully realized by the unique fluorescent fingerprints of the snake venom proteins, and the discrimination analysis of 100% of six representative proteins in snake venom can be successfully realized, and different types of snake venom proteins and mixed snake venom proteins with different components can be obviously distinguished. The characteristic of snake venom species is judged by multivariate analysis fluorescence sensor array detection of snake venom mixture, and the advantages of simple, quick and accurate detection of snake venom and the like of the sensor array enable the sensor array to be used for timely and accurate treatment of patients bitten by snake venom and screening research of snake venom anticancer and antitumor drugs.
Description
Technical Field
The invention belongs to a fluorescence detection analysis method in the biochemical field, and particularly relates to a multivariate fluorescence analysis technology for analyzing complex samples of various snake venom proteins and mixtures.
Background
Snake venom is a fluid secreted from the toxic glands of a poisonous snake. Its toxic components are mainly toxic proteins with enzymatic activity, and the enzymes and toxins contain about twenty kinds. In addition, it contains some small molecule peptides, amino acids, carbohydrates, lipids, nucleosides, biogenic amines and metal ions. The components of snake venom are very complex, and the toxicity, pharmacological action and toxicological action of different snake venom are respectively characterized. The action of snake venom on organisms is more complex, and different snake venom species contain different proteins and polypeptides, and can be classified into nerve venom, blood venom and mixed venom according to the toxicological effects of toxic components of the snake venom. According to their properties, they can be classified into neurotoxins, cardiotoxins, hemolytic toxins, thrombotoxins and anticoagulant toxins. This provides the possibility of identifying and distinguishing between different types of snake venom intoxication.
In nature, the human olfactory and gustatory systems can discriminate between a variety of odorant molecules in the surrounding environment, which benefits from the non-specific response of odorant receptor cells. Scientists of Anslyn et al have developed a new sensing pattern-sensing array by simulating the olfactory and gustatory recognition processes. The sensing array is based on a 'many-to-many' mode, is composed of a plurality of sensing compound molecules which are orderly arranged, and finally realizes high-throughput 'fingerprint identification' of a plurality of detection substrates through analysis of the differential response results of the plurality of series of sensing molecules.
In recent years, the cross-response sensing array provides a convenient and efficient means and method for aspects closely related to people's life, such as food detection, environmental monitoring, drug screening, clinical diagnosis and the like. In this context, we have achieved easy identification of 7 snake venom proteins by their specific response signals exhibited by different protein fluorochromes binding to different proteins, which selectively bind to the snake venom proteins, thereby altering their fluorescence intensity. This provides a simple, rapid and accurate new method for distinguishing snake venom proteins.
Disclosure of Invention
The invention aims to provide a method which is simple to operate, flexible to use and capable of effectively distinguishing snake venom proteins. The aim of the invention is achieved by the following measures:
1. the invention provides a fluorescence sensing array, which comprises a substrate, a fluorescent dye and a snake venom protein solution on the substrate, wherein the fluorescence sensing array is prepared and used for analyzing a plurality of snake venom proteins and a mixture complex sample.
2. The invention selects universal combined fluorescent dye according to the snake venom protein to be detected, studies the fluorescent quantitative relation, and determines that the fluorescent intensity of the selected fluorescent dye can generate specific change after the selected fluorescent dye is combined with the snake venom protein, wherein the method comprises the following steps:
(1) Representative protein stock preparation
6 representative proteins: phospholipase A2, alpha-neurotoxin, cardiotoxin, hyaluronidase, thrombin and freeze-dried powder of thrombin are configured in deionized water. All solutions were kept in the dark in a refrigerator at 4 ℃ until use.
(2) Arrangement of dyes
4 dyes: eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B and danhuang are configured in deionized water. All solutions were kept in the dark and placed in a refrigerator at 4 ℃ until use.
(3) Measurement of dye absorption and emission wavelengths
And respectively putting the prepared dye solution and a blank (only containing deionized water) solution into an ultraviolet-visible spectrophotometer to measure and record the absorption wavelength of the dye solution and the blank solution. And then respectively putting the prepared dyes into a fluorescence spectrophotometer, and measuring the emission wavelength and the original fluorescence intensity of the dyes according to the absorption wavelength of the dyes.
(4) Relationship between time taken for binding of protein and dye and fluorescence intensity
The fluorescence intensity of 4 dyes after 6 representative proteins were added every 2 minutes was set for 20 minutes, and the change in fluorescence intensity was observed.
3. The invention provides a preparation method of a fluorescence sensing array for distinguishing snake venom proteins, which comprises the following steps:
40 μ L of 4 dyes were added to 384 well plates in columns using a 16-channel pipette, keeping one row as a single dye, as a Blank (Blank). Then 40 μ L of snake venom protein solution was added in columns, and a column of protein-free dye was maintained as Control (Control).
4. Through the difference of fluorescent signals shown after different proteins are combined with different fluorescent dyes, the snake venom mixture analysis technology based on the multivariate analysis method can analyze and distinguish different representative proteins with the same concentration, representative proteins with different concentrations and representative proteins with different concentration gradients in snake venom.
5. Through the unique fluorescent fingerprint signals expressed by different binding degrees and different binding modes of different proteins to different fluorescent dyes, the multi-component mixed protein in snake venom and seven different snake venom proteins can be analyzed and distinguished by the snake venom mixture analysis technology based on the multivariate analysis method.
4. The multivariate analysis method comprises the following specific steps:
(1) Preparation of representative protein solutions
The 6 representative proteins (phospholipase A2, alpha-neurotoxin, cardiotoxin, hyaluronidase, thrombin) were each formulated in deionized water at a concentration of 1mg/mL. All solutions were kept in the dark and placed in a refrigerator at 4 ℃ until use.
(2) Preparation of fluorescent dye solution and blank control solution
4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, dadan yellow) were dispensed in deionized water at a concentration of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively. The blank control solution was deionized water. All solutions were kept in the dark in a refrigerator at 4 ℃ until use.
(3) Preparation of different concentrations of representative protein solutions
The deionized water is used for preparing the components with different concentrations, namely, the hemagglutinating enzyme, the cardiotoxin, the alpha-neurotoxin, the phospholipase A2, the hyaluronidase and the thrombin. All solutions were kept in a refrigerator at 4 ℃ until use.
(4) Preparation of multiple concentration gradient representative protein solution
The thrombin, the alpha-neurotoxin and the phospholipase A2 powder are respectively configured in deionized water according to certain concentration gradient. All solutions were kept in a refrigerator at 4 ℃ until use.
(5) Preparation of protein mixture solution
Phospholipase A2, hyaluronidase, cardiotoxin, alpha-neurotoxin, thrombin and hemagglutination enzyme powder are respectively dissolved in deionized water and then mixed according to the same proportion to form the following 7 components: phospholipase A2 and alpha-neurotoxin, alpha-neurotoxin and haemagglutinase, alpha-neurotoxin and thrombin, thrombin and haemagglutinase, hyaluronidase, cardiotoxin and alpha-neurotoxin, hyaluronidase, cardiotoxin and haemagglutinase, phospholipase A2, haemagglutinase and thrombin.
(6) Preparation of seven snake venom protein solutions
Respectively preparing venom proteins of Agkistrodon halys in Changbai mountain, agkistrodon Halys, viperas, cobra venom proteins, jiangzhe Agkistrodon Halys, and Ophiophagus hannah venom protein powder in deionized water. All solutions were kept in the dark and placed in a refrigerator at 4 ℃ until use.
(7) The multi-gel analysis system is used for analyzing and detecting the fluorescence sensing array.
Preparing a fluorescence sensing array, wherein the protein solution provided by the invention is the representative protein solution, the representative protein solution with different concentrations, the representative protein solution with multiple concentration gradients, the seven snake venom protein solutions with different types and the seven multi-component mixed protein solution in measures from one to five, analyzing and detecting the fluorescence sensing array by using a multi-gel analysis system under the conditions of excitation wavelengths of 302nm and 365nm, and recording fluorescence sensing information of 6 channels (CH: 450nm, CH.
(8) LDA and HCA analysis
The snake venom protein solution is subjected to discriminant analysis by a method of Linear Discriminant Analysis (LDA) and Hierarchical Cluster Analysis (HCA).
The concentration of the representative protein solution used in the second step (1) and the fourth step (1) of the present invention was 1mg/mL.
The dyes eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B and danhuang used in measures two (2) and four (2) of the present invention are respectively used at the concentrations of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL and 17.393 × 103 mg/mL.
The concentrations of the representative protein solutions used in step (3) of step four of the present invention are 0.01mg/mL of hemocoagulase, 0.05mg/mL of cardiotoxin, 0.1mg/mL of alpha-neurotoxin, 0.5mg/mL of phospholipase A2, 1mg/mL of hyaluronidase and 5mg/mL of thrombin, respectively.
In the step (4) of the fourth step, the concentration gradients of the thrombin, the alpha-neurotoxin and the phospholipase A2 solution are 0.01mg/mL, 0.02 mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL and 0.5mg/mL.
The seven snake venom protein powders used in step (6) of measure five of the present invention are each 0.001g, and are each prepared as a 1mg/mL snake venom protein solution.
The combined protein powder of step (5) of measure five of the present invention was 0.001g. After the protein solution is prepared, the concentration of the mixed complex protein of 7 components is 1mg/mL.
Has the advantages that: the invention designs a fluorescence sensing array by utilizing the complexation of fluorescent dye and snake venom proteins, and is used for cross-response recognition of six representative proteins in snake venom and 7 snake venom proteins in the nature. The fluorescence sensor array designed by the inventor is also helpful for identifying snake venom proteins with similar toxicity, and the fluorescence sensor array can also correctly identify the snake venom proteins with different concentrations and different components. The integrated sensing array of response and signal processing units facilitates efficient multi-analyte detection and complex system analysis, and will promote clinical pursuit for rapid clinical diagnosis and the development of drugs for the treatment of cancer, tumors.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 fluorescence spectra of fluorescence intensity over time for 4 dye solutions in accordance with the invention in example 1, in response to different representative proteins over 20 minutes.
FIG. 1 (a) alpha-neurotoxin (1X 10-3 mg/mL) was added. (b) adding the hemagglutinating enzyme (1X 10-3 mg/mL). (c) alpha-neurotoxin (1X 10-3 mg/mL) was added. (d) adding thrombin (1X 10-3 mg/mL).
Note: the plot was made using log10 because of the excessive change in fluorescence values in plot (a).
FIG. 2 is a LDA graph and HCA graph of six representative proteins in snake venom according to example 2 of the present invention.
(a) And (3) identification LDA results of the fluorescence sensing array on 6 representative proteins. (b) Recognition of HCA results by the fluorescence sensor array for 6 representative proteins.
FIG. 3 is a graph of LDA of representative proteins at 6 different concentrations in example 3 of the present invention.
FIG. 4 is a graph of LDA and Jackknifed classification matrix of the multi-concentration gradient protein in example 4 of the present invention.
(a) And (3) identifying the LDA result of the multi-concentration gradient protein by using the fluorescence sensing array. (b) The fluorescence sensing array discriminates jackknife classification matrix maps of multiple concentrations of protein. Remarking: t: thrombin, C: α -neurotoxin, P: phospholipase A2, D:0.01mg/mL, E:0.02 mg/mL, F:0.05mg/mL, X:0.1mg/mL, Y:0.2mg/mL, Z:0.5mg/mL.
FIG. 5 is a LDA graph and a Jackknifed classification matrix graph of a mixture of 7 different component proteins in example 5 of the present invention.
(a) Identification LDA scattergrams of 6 proteins and their two-component and three-component mixtures. A-F are phospholipase A2, hyaluronidase, cardiotoxin, alpha-neurotoxin, hemagglutinating enzyme, and thrombin, respectively. The concentrations of the mixtures were all 1X 10-3mg/mL. (b) The fluorescence sensing array discriminates the Jackknifed classification matrix map of the multi-component protein mixture.
FIG. 6 is a LDA and HCA plots of 7 snake venom proteins according to example 6 of the present invention.
(a) And (3) identifying LDA results of 7 snake venom proteins by using a fluorescence sensing array. (b) HCA recognition results of 7 snake venom proteins by a fluorescence sensing array.
Detailed Description
The technical scheme of the invention is further explained by combining the accompanying drawings as follows:
the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, which ranges of values should be considered as specifically disclosed herein
Example 1: fluorescence spectra of fluorescence intensity over time for 4 dye solutions in response to different representative proteins within 20 minutes
6 proteins (phospholipase A2, alpha-neurotoxin, cardiotoxin, trypsin, thrombin, and thrombin) were placed in deionized water at a concentration of 1mg/mL, and 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, and dandan yellow) were placed in deionized water at a concentration of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, and 17.393 × 103mg/mL, respectively. The 6 proteins were added to the 4 dyes, and the fluorescence intensity was measured every 2min for 20min to observe the change in fluorescence.
Example 2: discriminant analysis of six representative proteins in snake venom
6 proteins (phospholipase A2, alpha-neurotoxin, cardiotoxin, trypsin, thrombin, hemagglutinating enzyme) were placed in deionized water at 1mg/mL, 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, dadan yellow) were placed in deionized water at 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively, and 40 μ L of the 4 dyes were added in columns to a 384 well plate using a 16-channel pipette gun, keeping one row as a single dye as a Blank (Blank). Then 40. Mu.L of 6 representative protein solutions were added in columns, maintaining a column of dye without protein as a Control (Control). And putting the prepared sensing array into a multi-channel gel analysis system for discrimination analysis, and adopting Linear Discriminant Analysis (LDA) for identification and Hierarchical Clustering Analysis (HCA) for analyzing the genetic relationship among six representative proteins in snake venom.
Example 3: discrimination analysis of six representative proteins in snake venom at different concentrations
Hemocoagulase at a concentration of 0.01mg/mL, cardiotoxin at a concentration of 0.05mg/mL, alpha-neurotoxin at a concentration of 0.1mg/mL, phospholipase A2 at a concentration of 0.5mg/mL, hyaluronidase at a concentration of 1mg/mL, thrombin at a concentration of 5mg/mL, and 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, and dadanhuang) were dispensed into deionized water at a concentration of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively, and 40 μ L of the 4 dyes were added to a 384 well plate in columns using a 16-channel pipette gun, keeping one row as a single dye as a Blank (Blank). Then 40. Mu.L of 6 representative protein solutions were added in columns, maintaining a column of dye without protein as a Control (Control). And putting the prepared fluorescence sensing array into a multi-channel gel analysis system for discrimination analysis, and identifying by adopting Linear Discriminant Analysis (LDA).
Example 4 discriminant analysis of three representative proteins in Snake venom at different concentration gradients
Thrombin, alpha-neurotoxin, phospholipase A2 powder were placed in deionized water at a concentration gradient of 0.01mg/mL, 0.02 mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, respectively, and 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, dadan yellow) were placed in deionized water at a concentration of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively, and 40 μ L of 4 dyes were added to 384 well plates in columns using a 16-channel pipette gun, keeping the Blank as a single line of dye as a sample (Blank). Then 40. Mu.L of 6 representative protein solutions were added in columns, keeping one column with no protein dye as Control (Control). And (3) putting the prepared sensing array into a multichannel gel analysis system for discrimination analysis, and identifying by adopting Linear Discriminant Analysis (LDA) and a Jackknifed classification matrix.
Example 5: discrimination analysis of 7 multicomponent mixed proteins
0.001g of phospholipase A2, hyaluronidase, cardiotoxin, alpha-neurotoxin, thrombin and hemagglutination enzyme powder are respectively added into 1mL of deionized water, and the concentration is 1mg/mL. Stirring with magnetic stirrer to mix and dissolve. Then, they were mixed in the same ratio to combine the following 7 components: phospholipase A2 and a-neurotoxin, a-neurotoxin and a thrombin, thrombin and a thrombin, hyaluronidase, cardiotoxin and a-neurotoxin, hyaluronidase, cardiotoxin and a thrombin, phospholipase A2, a thrombin and a thrombin; separately, 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, dandan yellow) were dispensed in deionized water at a concentration of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively, and 40 μ L of the 4 dyes were added in columns to 384 well plates using a 16-channel pipette gun, keeping one row as a single dye, as a Blank (Blank). Then 40. Mu.L of the seven-component mixed protein solution was added in columns, keeping one column with the protein-free dye as Control (Control). And (3) putting the prepared sensing array into a multichannel gel analysis system for discrimination analysis, and identifying by adopting Linear Discriminant Analysis (LDA) and a Jackknifed classification matrix.
Example 6: discriminant analysis of 7 Snake venom proteins
Respectively taking 0.001g of Changbai mountain Agkistrodon halys venom protein, coral snake venom protein, agkistrodon halys venom protein, viper venom protein, cobra venom protein, jiangzhe Agkistrodon venom protein and elaeagnus mollis venom protein powder, adding into 1mL deionized water to obtain a concentration of 1mg/mL, respectively placing 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B and dadanhuang) in the deionized water at the concentrations of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL and 17.393 × 103mg/mL, respectively, adding 40 μ L of 4 dyes into a 384-well plate according to columns by using a 16-channel pipette gun, and keeping one row as a single dye to be used as a Blank sample (Blank). Then 40. Mu.L of 6 representative protein solutions were added in columns, keeping one column with no protein dye as Control (Control). And putting the prepared sensing array into a multichannel gel analysis system for discrimination analysis, identifying by adopting Linear Discriminant Analysis (LDA) and analyzing the genetic relationship among the seven snake venom proteins by adopting Hierarchical Clustering Analysis (HCA). .
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (8)
1. A multiplex assay fluorescent sensor for assaying snake venom mixtures, comprising: the fluorescence sensor comprises a substrate and a solution of a fluorescent dye and a snake venom protein on the substrate; the fluorescent sensor is characterized in that four different fluorescent dye solutions are uniformly spotted on a 384 micro-porous plate serving as a substrate according to columns, a row of single dyes is kept as a blank sample, then solutions containing different snake venom representative proteins are spotted on the 384 micro-porous plate which is spotted with the fluorescent dye solutions according to columns, a row of dyes without proteins is kept as a reference sample, and therefore the snake venom mixture analysis fluorescent sensor is obtained;
the four different fluorescent dye solutions are respectively 6.054X 10 3 Eosin Y solution at a concentration of 194.690X 10 in mg/mL 3 mg/mL fluorescein isothiocyanate isomer I solution with concentration of 14.516X 10 3 mg/mL sulforhodamine B solution and the concentration of 17.393 multiplied by 10 3 mg/mL dandan yellow solution;
the representative snake venom proteins are selected from six representative snake venom proteins, namely phospholipase A 2 Hyaluronidase, cardiotoxin, alpha-neurotoxin, thrombin and thrombin;
the multicomponent mixed protein in the snake venom can be analyzed and distinguished through unique fluorescent fingerprint signals expressed by different binding degrees and different binding modes of different proteins to different fluorescent dyes.
2. The multiplex assay snake venom mixture assay fluorescence sensor of claim 1, wherein: the fluorescent dye solution is composed of dye powder and a solvent, and the solvent is deionized water.
3. The multiplex assay snake venom mixture assay fluorescence sensor of claim 1, wherein: the solution of the representative snake venom protein consists of protein freeze-dried powder and a solvent, wherein the concentration of the solution in the solution of the representative snake venom protein is 1mg/mL, and the solvent is deionized water.
4. The multiplex assay snake venom mixture assay fluorescence sensor of claim 1, wherein: the solutions of the representative snake venom proteins are 0.01mg/mL of hemagglutinin, 0.05mg/mL of cardiotoxin, 0.1mg/mL of alpha-neurotoxin, and phospholipase A 2 0.5mg/mL, hyaluronidase 1mg/mL, and thrombin 5mg/mL.
5. The multiplex assay snake venom mixture assay fluorescence sensor of claim 1, wherein: mixing thrombin, alpha-neurotoxin, phospholipase A 2 The powders are respectively configured in deionized water with concentration gradients of 0.01mg/mL, 0.02 mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL and 0.5mg/mL to obtain solutions of the snake venom representative proteins with multiple concentration gradients, so that the fluorescence sensor can distinguish and analyze proteins with multiple concentration gradients.
6. A multi-analyte snake venom mixture analytical fluorescence sensor according to any one of claims 1-5, wherein: for treating different concentrations of hemagglutinin, cardiotoxin, alpha-neurotoxin, and phospholipase A 2 Hyaluronidase, thrombin discrimination assay.
7. A snake venom mixture analysis method based on multivariate analysis is characterized in that: the method comprises the following steps:
(1) Respectively spot-coating four different fluorescent dye solutions on a 384 micro-porous plate according to columns, and keeping a row of single dye as a blank sample; the four different fluorescent dye solutions are 6.054 x 10 3 mg/mL eosin Y solution at a concentration of 194.690X 10 3 mg/mL fluorescein isothiocyanate isomer I solution with concentration of 14.516X 10 3 The concentration of the solution of the sulforhodamine B is 17.393 multiplied by 10 in mg/mL 3 mg/mL dandan yellow solution;
(2) Respectively dispensing different snake venom protein solutions into 384 micro-porous plates dispensed with the four different fluorescent dye solutions, and keeping a row of dyes without protein as a control sample; said snake venom protein is selected from phospholipase A 2 The snake venom proteins are selected from Changbai mountain Agkistrodon blomhoffii snake venom protein, bungarus multicinctus snake venom protein, agkistrodon acutus snake venom protein, viperidae snake venom protein, agkistrodon acutus venom protein and Ophiophagus hannaeus snake venom protein;
(3) CH1 was recorded by a multichannel gel analysis system under two excitation lights, 302nm and 365 nm: 450nm, CH2:480nm, CH3:505nm, CH4:535nm, CH5:570nm, CH6:605nm and 6 channels, and distinguishing and analyzing representative proteins in snake venom by Linear Discriminant Analysis (LDA) and Hierarchical Clustering Analysis (HCA) according to the collected fluorescence sensing information.
8. The method of claim 7, wherein the snake venom protein solution is a multi-component protein mixture solution comprising a plurality of proteins, and the multi-component protein mixture solution is divided into the following 7 components: phospholipase A 2 And alpha-neurotoxin, alpha-neurotoxin and haemagglutinase, alpha-neurotoxin and thrombin, thrombin and haemagglutinase, hyaluronidase, cardiotoxin and alpha-neurotoxin, hyaluronidase, cardiotoxin and haemagglutinase, phospholipase A 2 Hemagglutinating enzyme and coagulationA blood enzyme.
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