CN111337468A

CN111337468A - Multi-analysis snake venom mixture analysis fluorescent sensor

Info

Publication number: CN111337468A
Application number: CN202010334013.8A
Authority: CN
Inventors: 刘巍; 王帆; 李风煜
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-06-26
Anticipated expiration: 2040-04-24
Also published as: CN111337468B

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

Multi-analysis snake venom mixture analysis fluorescent sensor

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 human 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:

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 for analyzing a plurality of snake venom proteins and a mixture complex sample.

Secondly, the invention selects universal combined fluorescent dye according to the snake venom protein to be detected, studies the fluorescent quantitative relation, and in order to determine that the fluorescent intensity can generate specific change after the selected fluorescent dye is combined with the snake venom protein, the method comprises the following steps:

(1) representative protein stock preparation

Phospholipase A2, α -neurotoxin, cardiotoxin, hyaluronidase, thrombin and freeze-dried powder of thrombin are put in deionized water, and all solutions are put in a refrigerator at 4 ℃ in the dark for standby.

(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 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.

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).

And fourthly, through the difference of fluorescence 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.

And fifthly, through the combination degree and combination mode of different proteins to different fluorescent dyes, the expressed unique fluorescent fingerprint signals.

The multivariate analysis method comprises the following specific steps:

(1) preparation of representative protein solutions

The 6 representative proteins (phospholipase A2, α -neurotoxin, cardiotoxin, hyaluronidase, thrombin) were each formulated in deionized water at a concentration of 1mg/mL, all solutions were kept in the dark in a refrigerator at 4 ℃ for future use.

(2) Preparation of fluorescent dye solution and blank control solution

4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, dandan yellow) were placed in deionized water at concentrations of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively.

(3) Preparation of different concentrations of representative protein solutions

The deionized water is used for preparing hemagglutinating enzyme, cardiotoxin, α -neurotoxin, phospholipase A2, hyaluronidase and thrombin with different concentrations, and all the solutions are placed in a refrigerator at 4 ℃ for standby.

(4) Preparation of multiple concentration gradient representative protein solution

The thrombin, α -neurotoxin and phospholipase A2 powders were placed in deionized water at a concentration gradient and all solutions were kept in a 4 ℃ freezer for further use.

(5) Preparation of protein mixture solution

Phospholipase A2, hyaluronidase, cardiotoxin, α -neurotoxin, thrombin and thrombin are dissolved in deionized water and mixed in the same ratio to form 7 components, phospholipase A2 and α -neurotoxin, α -neurotoxin and thrombin, α -neurotoxin and thrombin, thrombin and thrombin, hyaluronidase, cardiotoxin and α -neurotoxin, hyaluronidase, cardiotoxin and thrombin, phospholipase A2, thrombin 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 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 is a representative protein solution, a representative protein solution with different concentrations, a representative protein solution with multiple concentration gradients, seven snake venom protein solutions of different types and seven multi-component mixed protein solutions which are respectively prepared in the first to fifth measures, 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: 480nm, CH: 505nm, CH: 535nm, CH: 570nm and CH: 605 nm).

(8) LDA and HCA analysis

The snake venom protein solution is subjected to discriminant analysis by 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 1 mg/mL.

The dyes eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B and danhuang used in the second and fourth measures (2) and (2) respectively have 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 α -neurotoxin, 0.5mg/mL of phospholipase A2, 1mg/mL of hyaluronidase and 5mg/mL of thrombin, respectively.

In the fourth step (4), the concentration gradients of the thrombin, α -neurotoxin and 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 powders of the present invention in step (5) described in measure five are all 0.001 g. After the protein solution is prepared, the concentration of the mixed complex protein of 7 components is 1 mg/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 sensing array designed by the inventor is also helpful to identify snake venom proteins with similar toxicity, and the fluorescence sensing array can also correctly identify protein mixtures 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.

Drawings

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) α -neurotoxin (1 × 10-3 mg/mL) was added, (b) haemagglutinase (1 × 10-3 mg/mL) was added, (c) α -neurotoxin (1 × 10-3 mg/mL) was added, (d) thrombin (1 × 10-3 mg/mL) was added.

Note: the plot was made using log10 because the fluorescence values in plot (a) were too variable.

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) HCA recognition results for 6 representative proteins by the fluorescence sensing array.

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 the LDA of the multi-concentration gradient protein and a graph of the Jackknifed classification matrix in example 4 of the present invention.

(a) Identification LDA results of the fluorescence sensing array on the multi-concentration gradient protein, (b) Jackknifed classification matrix chart of the fluorescence sensing array for distinguishing the multi-concentration protein, remarks are that T is thrombin, C is α -neurotoxin, P is phospholipase A2, D is 0.01mg/mL, E is 0.02 mg/mL, F is 0.05mg/mL, X is 0.1mg/mL, Y is 0.2mg/mL, and Z is 0.5mg/mL.

FIG. 5 is a LDA and HCA plots of 7 snake venom proteins in example 5 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.

FIG. 6 is LDA graph and Jackknifed sorting matrix graph of 7 different component protein mixtures in example 6 of the present invention.

(a) Identification LDA scattergrams of 6 proteins and two-component and three-component mixtures thereof, wherein A-F are phospholipase A2, hyaluronidase, cardiotoxin, α -neurotoxin, hemagglutinating enzyme and thrombin respectively, and the concentrations of the mixtures are all 1 × 10-3mg/mL, (b) Jackknifed classification matrix chart of fluorescence sensing array identification multi-component protein mixture.

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, α -neurotoxin, cardiotoxin, trypsin, thrombin, and thrombin) were placed in deionized water at a concentration of 1mg/mL, respectively, and 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, and daphnyellow) 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, and these 6 proteins were added to these 4 dyes, and their fluorescence intensities were measured at 2min intervals within 20min, and changes in fluorescence were observed.

Example 2: discriminant analysis of six representative proteins in snake venom

6 proteins (phospholipase A2, α -neurotoxin, cardiotoxin, trypsin, thrombin, hemagglutinating enzyme) were each dispensed at a concentration of 1mg/mL into deionized water, 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, daphnane yellow) 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, 40 μ L of the 4 dyes were added in columns to a 384 well plate, keeping one row as a single dye as a Blank (Blank), then 40 μ L of 6 representative protein solutions were added in columns, keeping one column as a protein-free dye, as a Control (Control), the prepared sensor array was placed in a multichannel discriminatory gel analysis system for discriminant analysis, and discrimination analysis was performed using linear analysis (LDA) for identification and stratification (HCA) for the relationship between six representative snake venom proteins in the cluster analysis.

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, α -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, eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B, danoflavin 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, 40 μ L of 4 dyes were dispensed in columns into a 384 well plate using a 16-channel pipette gun, keeping one row as a single dye, as a Blank sample (Blank), and then 40 μ L of 6 representative protein solutions were dispensed in columns, keeping one column as a dye without protein, as a Control sample (Control), the prepared fluorescence sensor array was put into a multichannel gel analysis system for discriminant analysis, and Linear Discriminant Analysis (LDA) was performed using linear discriminant analysis.

Example 4 discriminant analysis of three representative proteins in Snake venom at different concentration gradients

Thrombin, α -neurotoxin, phospholipase a2 powder were dispensed 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, 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, 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 Blank (Blank), and then 40 μ L of 6 representative protein solutions, keeping one column as a dye without protein, as Control (Control), the prepared sensor array was placed in a multichannel gel analysis system for discriminant analysis, and linear discriminant analysis (jak) and identification matrix were used for identification.

Example 5: discrimination analysis of 7 multicomponent mixed proteins

Phospholipase A2, hyaluronidase, cardiotoxin, α -neurotoxin, thrombin and hemocoagulase powders 0.001g were added to 1mL of deionized water at a concentration of 1mg/mL, respectively, and were mixed well and dissolved with a magnetic stirrer, after which they were mixed in the same ratio to combine 7 components of phospholipase A2 and α -neurotoxin, α -neurotoxin and hemocoagulase, α -neurotoxin and thrombin, thrombin and hemocoagulase, hyaluronidase, cardiotoxin and α -neurotoxin, hyaluronidase, cardiotoxin and hemocoagulase, phospholipase A2, hemocoagulase and thrombin, respectively, 4 dyes (eosin Y, fluorescein isothiocyanate I, sulforhodamine B, and daparine yellow) were dispensed into deionized water at concentrations of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL, 17.393 × 103mg/mL, respectively, 40 μ L of 4 dyes were added to 384 columns with a 16-channel pipette gun, a single column of dye was kept as a single line, a single line of dye was kept as a single line, and a gel was analyzed using a read-based on a analytical system (Jack).

Example 6: discriminant analysis of 7 Snake venom proteins

Adding 0.001g of Changbai mountain Agkistrodon halys venom protein, bungarus multicinctus venom protein, viper venom protein, cobra venom protein, Jiangzhe Agkistrodon venom protein and Ophiophagus hannah venom protein powder into 1mL of deionized water to obtain a concentration of 1mg/mL, adding 4 dyes (eosin Y, fluorescein isothiocyanate isomer I, sulforhodamine B and dadanhuang) into deionized water at concentrations of 6.054 × 103mg/mL, 194.690 × 103mg/mL, 14.516 × 103mg/mL and 17.393 × 103mg/mL, adding 40 μ L of 4 dyes into a 384-well plate in a row by using a 16-channel pipette, keeping a row as a single dye, using a Blank sample (Blank) and then adding 40 μ L of 6 representative protein solutions in a row, keeping a row as a dye without protein, using a Control sample (Control), putting the prepared sensor array into a multichannel gel analysis system for linear discrimination analysis, and performing seven-layer classification analysis by adopting LDA.

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

1. A multiplex assay fluorescent sensor for assaying snake venom mixtures, comprising: the fluorescence sensing array is characterized in that different fluorescent dye solutions are uniformly spotted on a substrate, representative proteins in snake venom are in-situ spotted on the substrate, and fluorescence sensing information of 6 channels (CH: 450nm, CH: 480nm, CH: 505nm, CH: 535nm, CH: 570nm and CH: 605 nm) is recorded under the conditions of excitation wavelengths of 302nm and 365nm, wherein the protein solution is composed of protein freeze-dried powder and a solvent, the concentration of the solution in the protein solution is 1mg/mL, and the solvent is deionized water.

2. The multiple-analysis snake venom mixture analysis fluorescence sensor according to claim 1, wherein the fluorescent dye solution is composed of dye powder and solvent, the substrate is 384 micro-well plate, and the concentration of eosin Y solution in the fluorescent dye solution is 6.054 × 10³The concentration of the fluorescein isothiocyanate isomer I solution is 194.690 × 10 in mg/mL³The concentration of the sulforhodamine B solution is 14.516 × 10³The concentration of the solution of the Dandan yellow is 17.393 × 10³mg/mL; the solvent is deionized water.

3. The multiplex assay snake venom mixture assay fluorescence sensor of claim 1, wherein: the protein is selected from snake venom, and six representative proteins are phospholipase A₂Hyaluronidase, cardiotoxin, α -neurotoxin, thrombin and thrombin.

4. The multiplex assay fluorescence sensor of claim 3, wherein said protein solutions have concentrations of hemagglutinin 0.01mg/mL, cardiotoxin 0.05mg/mL, α -neurotoxin 0.1mg/mL, phospholipase A₂0.5mg/mL, hyaluronidase 1mg/mL, and thrombin 5mg/mL.

5. The multiplex assay fluorescence sensor of claim 3, wherein said thrombin, α -neurotoxin, phospholipase A are introduced₂The powder was 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.

6. A multi-component analytical snake venom mixture analytical fluorescence sensor according to any of claims 1-5 wherein: the collected fluorescence sensing information is distinguished and analyzed by adopting LDA and HCA methods.

7. The multiplex assay snake venom mixture fluorescence sensor of any one of claims 1-5, wherein different concentrations of haemagglutinase, cardiotoxin, α -neurotoxin, phospholipase A are used₂Hyaluronidase, thrombin discrimination assay.

8. A snake venom mixture analysis technology based on a multivariate analysis method is characterized in that: the method comprises the following steps:

(1) coating proper fluorescent dye on 384 micro-porous plates according to a certain periodic point, and keeping a row of single dye as a Blank sample (Blank);

(2) respectively dispensing solutions containing different proteins into the different fluorescent dye solutions of the step (1), and keeping a row of dyes without proteins as a Control;

(3) fluorescence sensing information of 6 channels (CH 1: 450nm, CH 2: 480nm, CH 3: 505nm, CH 4: 535nm, CH 5: 570nm and CH 6: 605 nm) was recorded under two excitation lights of 302nm and 365nm by a multi-channel gel analysis system, and subjected to discriminant analysis by the methods of LDA and HCA.

9. The snake venom mixture analysis technique according to claim 9, wherein the protein solution is a multi-component protein mixture solution prepared by mixing a plurality of proteins, and the multi-component protein mixture solution is characterized in that: the multicomponent protein is divided into the following 7 components: phospholipase A₂And α -neurotoxin, α -neurotoxin and haemagglutinase, α -neurotoxin and thrombin, thrombin and haemagglutinase, hyaluronidase, cardiotoxin and α -neurotoxin, hyaluronidase, cardiotoxin and haemagglutinase, phospholipase A₂The concentrations of the components are 1 mg/mL.

10. The multi-analysis-method-based snake venom mixture analysis technique of claim 9, wherein: the snake venom proteins of Changbai mountain Agkistrodon halys and Baimei Agkistrodon halys, Agkistrodon acutus, Agkistrodon halys, Viperas, cobra venom, Jiangzhe Agkistrodon, and Ophiophagus hannah are disposed in deionized water at a concentration of 1 mg/mL.