A fluorescent labeling method and quantitative analysis method for gelatin protein

Abstract

The present invention provides a fluorescent labeling method and quantitative analysis method for gelatin protein, wherein tetrazole-modified aggregation-induced luminescence fluorescent dye molecules are dissolved in an aqueous phase, an alcohol phase or a PBS buffer solution, and then mixed with a gelatin protein solution to obtain a fluorescent protein complex solution. The tetrazole-modified aggregation-induced luminescence fluorescent dye molecules do not emit light or emit light weakly when dissolved, but after being combined with gelatin protein, the fluorescence of the dye molecules is significantly enhanced, and can be used as a fluorescent labeling and quantitative detection method for gelatin protein.

Description

Technical Field

[0001] The invention relates to a fluorescent labeling method and a quantitative analysis method of gelatin protein, belonging to the technical field of gelatin protein quantitative analysis. Background Art

[0002] Gelatin is a water-soluble protein that can be extracted from collagen-rich tissues such as animal skin, tendons, ligaments and bones. As a natural bio-based material, gelatin has good biocompatibility, good biodegradability, cell adhesion and proliferation, non-toxicity, non-immunogenicity and low price. It has great potential applications in the fields of biomedicine and tissue engineering, such as medical dressings and repair of cartilage damage.

[0003] As a natural high molecular protein, gelatin has a wide source and a complex structure. As a hydrolysis product of collagen, the quantitative analysis of gelatin may be related to certain disease indicators, such as proximal interphalangeal joint enlargement caused by collagen precipitation, myocardial fibrosis caused by myocardial interstitial collagen precipitation, etc. Therefore, it is particularly important to achieve quantitative analysis of gelatin protein. At present, there are many means for qualitative analysis of gelatin protein, such as real-time fluorescence PCR to detect bovine and porcine components of gelatin, high performance liquid chromatography tandem mass spectrometry to detect peptide chain amino acid sequences, etc. Before quantitative analysis of gelatin, gelatin protein needs to be fluorescently labeled. However, traditional fluorescent labeling methods often face the problem of fluorescence attenuation caused by protein deposition, and gelatin as a protein requires mild labeling conditions. How to achieve effective labeling of gelatin protein without affecting the structure of gelatin itself (gelatin invariance) is the key to achieving quantitative analysis of gelatin.

[0004] Aggregation-induced emission (AIE) is a relatively new concept of responsive fluorescence. Unlike conventional luminophores, typical AIE luminophores have a propeller-shaped non-planar structure. In dilute solutions, AIE molecules undergo intermolecular rotation, dissipating energy through non-radiative pathways and rendering them non-luminescent. In the case of protein binding and encapsulation, the free rotation of dye molecules is restricted due to physical constraints from neighboring molecules, thereby inducing highly efficient fluorescence activity. This unique AIE property provides a responsive fluorescence light-up analysis strategy in chemical sensing, physical visualization, and bioimaging, and many AIE fluorescent dye systems have been demonstrated to be materials with high brightness, low background, and good photostability.

[0005] However, there is no report in the prior art on methods for labeling and quantitatively analyzing gelatin protein using AIE fluorescent dyes. Summary of the invention

[0006] The present invention provides a fluorescent labeling method and a quantitative analysis method for gelatin protein, which can effectively solve the above problems

[0007] The present invention is achieved in that:

[0008] A fluorescent labeling method for gelatin protein comprises dissolving tetrazolium-modified aggregation-induced luminescence fluorescent dye molecules in an aqueous phase, an alcohol phase or a PBS buffer solution, and then mixing the solution with a gelatin protein solution to obtain a fluorescent protein complex solution.

[0009] As a further improvement, the structural formula of the tetrazole-modified aggregation-induced emission fluorescent dye molecule is as follows:

[0010]

[0011] Wherein, at least one of R′, R″, R″′ and R″″ is a tetrazolyl group.

[0012] As a further improvement, the tetrazole-modified aggregation-induced emission fluorescent dye molecule is selected from one or more of TPE-4TA, TPE-3TA, TPE-2TA, and TPE-TA.

[0013] As a further improvement, the tetrazole-modified aggregation-induced emission fluorescent dye molecule is TPE-4TA.

[0014] As a further improvement, the concentration of the tetrazole-modified aggregation-induced luminescence fluorescent dye molecules dissolved in the aqueous phase, alcohol phase or PBS buffer solution is 1×10 -2 ~10 -4 mol / L.

[0015] As a further improvement, the alcohol phase is ethanol.

[0016] As a further improvement, the fluorescent labeling method of gelatin protein also includes irradiating the fluorescent protein complex solution with excitation light, and judging whether the gelatin protein is effectively labeled based on whether fluorescence is observed.

[0017] As a further improvement, the gelatin protein solution is prepared at a temperature of 35-45°C and a stirring time of 25-35 minutes.

[0018] A quantitative analysis method for gelatin protein comprises the following steps:

[0019] S1, preparing gelatin solutions with gradient concentrations, labeling the gelatin using the above method, and testing the fluorescence intensity;

[0020] S2, fitting the standard curve of the relationship between fluorescence intensity and gelatin protein concentration;

[0021] S3, obtaining a gelatin protein sample, testing the fluorescence intensity using the method of step S1, and then performing a quantitative analysis of the gelatin protein in the sample according to the standard curve.

[0022] As a further improvement, the excitation wavelength for testing the fluorescence intensity is 340-450 nm.

[0023] The beneficial effects of the present invention are:

[0024] The tetrazole-modified aggregation-induced luminescence fluorescent dye molecule used in the present invention has the main characteristic that the dye molecule does not emit light when dissolved in water or alcohol phase, but can be efficiently excited to emit fluorescence when combined with gelatin protein, so it can be applied to a simple and efficient method for fluorescent lighting labeling of gelatin protein and a quantitative analysis method.

[0025] The fluorescent labeling method of gelatin protein of the present invention is characterized by enhanced fluorescence emission of dyes due to protein encapsulation and aggregation-induced luminescence. Such dye labels have good stability and can be combined with gelatin protein for a long time.

[0026] The fluorescent labeling method of gelatin protein of the present invention only requires simple mixing of gelatin solution and fluorescent dye solution to achieve efficient labeling effect, mild reaction conditions, high safety, and no need for complex reaction conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is a schematic diagram of the lighting gelatin protein provided in Example 1 of the present invention.

[0029] Figure 2 It is a diagram showing the effects of lighting up gelatin protein provided in Example 1, Example 2, Example 3 and Comparative Example 1 of the present invention.

[0030] Figure 3 The fluorescence spectra and fluorescence lifetime diagrams are provided in Example 1, Example 2, Example 3 and Comparative Example 1 of the present invention. Among them, A is the fluorescence spectrum diagram, and B is the fluorescence lifetime diagram.

[0031] Figure 4 It is a fluorescence spectrum diagram of the illuminated gradient concentration gelatin protein provided by Examples 4 and 5 of the present invention.

[0032] Figure 5It is a linear relationship diagram for quantitative analysis of gelatin protein provided in Examples 4 and 5 of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] The embodiment of the present invention provides a fluorescent labeling method for gelatin protein, wherein tetrazolium-modified aggregation-induced luminescence fluorescent dye molecules are dissolved in an aqueous phase, an alcohol phase or a PBS buffer solution, and then mixed with a gelatin protein solution to obtain a fluorescent protein complex solution. The fluorescent labeling principle of this method is as follows Figure 1 As shown, the negatively charged tetrazolium anions of this type of tetrazolium-modified compounds bind to the amino cations in gelatin through electrostatic interactions, and the gelatin molecules wrap around the tetrazolium-modified tetraphenylethylene probe molecules, resulting in fluorescence lighting.

[0035] As a further improvement, the structural formula of the tetrazole-modified aggregation-induced emission fluorescent dye molecule is as follows:

[0036]

[0037] Among them, at least one of R′, R″, R″′ and R″″ is a tetrazolyl group, and the others are selected from one or more of hydrogen, heteroatom, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0038] The tetrazolyl group is selected from the following groups:

[0039]

[0040] As a further improvement, the tetrazole-modified aggregation-induced luminescence fluorescent dye molecule is selected from one or more of TPE-4TA, TPE-3TA, TPE-2TA, and TPE-TA, and the structural formula is as follows:

[0041]

[0042] As a further improvement, the tetrazole-modified aggregation-induced emission fluorescent dye molecule is TPE-4TA.

[0043] As a further improvement, the concentration of the tetrazole-modified aggregation-induced luminescence fluorescent dye molecules dissolved in the aqueous phase, alcohol phase or PBS buffer solution is 1×10 -2 ~10 -4 mol / L.

[0044] As a further improvement, the alcohol phase is ethanol.

[0045] As a further improvement, the fluorescent labeling method of gelatin protein also includes irradiating the fluorescent protein complex solution with excitation light, and judging whether the gelatin protein is effectively labeled based on whether fluorescence is observed.

[0046] As a further improvement, gelatin is a water-soluble polymer protein. When preparing its aqueous solution, the temperature should not be too high to avoid denaturation of the gelatin protein. The temperature of the gelatin protein solution is preferably 35-45°C. In addition, gelatin has foaming properties. It cannot be stirred for a long time when dissolved, and the stirring speed should not be too fast, otherwise a large number of bubbles will be generated. The stirring time of the gelatin protein solution is preferably 25-35 minutes.

[0047] The embodiment of the present invention also provides a quantitative analysis method of gelatin protein, comprising the following steps:

[0048] S1, preparing gelatin solutions with gradient concentrations, labeling the gelatin using the above method, and testing the fluorescence intensity;

[0049] S2, fitting the standard curve of the relationship between fluorescence intensity and gelatin protein concentration;

[0050] S3, obtaining a gelatin protein sample, testing the fluorescence intensity using the method of step S1, and then performing a quantitative analysis of the gelatin protein in the sample according to the standard curve.

[0051] As a further improvement, the excitation wavelength for testing the fluorescence intensity is 340-450 nm.

[0052] The gelatin used in the examples is type A gelatin, bloom 300 g, sourced from Sigma-Aldrich Group.

[0053] Example 1

[0054] This embodiment realizes the fluorescent labeling of gelatin protein, and the specific process is as follows:

[0055] (1) Weigh 0.005 g of gelatin protein into deionized water, heat it in a water bath to 40°C, stir it for about 30 minutes until it is completely dissolved, make up to 25 mL, and let it stand in a hot water bath for about 30 minutes to obtain a 200 mg / L gelatin protein aqueous solution.

[0056] (2) Weigh 0.006 g of TPE-4TA and dissolve it in 10.00 mL of ethanol to prepare 1×10 -3 mol / L TPE-4TA ethanol solution.

[0057] (3) Take 2.97 mL of the gelatin solution in step (1), add 0.03 mL of the TPE-4TA solution in step (2), and mix well.

[0058] Example 2

[0059] This embodiment realizes fluorescent labeling of gelatin protein. The difference from embodiment 1 is that the added TPE-4TA is replaced with TPE-2TA. 0.005 g of TPE-2TA is weighed and dissolved in 10 mL of ethanol to prepare 1×10 -3 mol / L TPE-2TA ethanol solution, and the rest of the process is reference to Example 1.

[0060] Example 3

[0061] This embodiment realizes fluorescent labeling of gelatin protein. The difference from embodiment 1 is that the added TPE-4TA is replaced with TPE-TA. 0.004 g of TPE-TA is weighed and dissolved in 10 mL of ethanol to prepare 1×10 -3 mol / L TPE-TA ethanol solution, and the rest of the process is reference to Example 1.

[0062] When the mixed solution is irradiated with 350nm excitation light, blue-green fluorescence emission can be observed. Figure 2 As shown. The comparison of fluorescence intensity was conducted by comparing the emission peak of the fluorescence spectrum. The instrument used was the Edinburgh Steady-State Transient Fluorescence Spectrometer FLS1000, with a fixed excitation wavelength of 350nm, and comparing the peak size corresponding to the characteristic emission peak of TPE-4TA (499nm). The fluorescence lifetime was also measured by the Edinburgh Steady-State Transient Fluorescence Spectrometer FLS1000, with a fixed emission wavelength of 499nm, and the fluorescence lifetime graph was measured. The fluorescence spectrum and fluorescence lifetime graph are shown in Figure 3 shown.

[0063] Depend on Figure 2 and Figure 3It can be seen that the AIE molecule TPE-4TA has a good recognition and lighting effect on gelatin protein, with a blue-green fluorescence and a maximum emission wavelength at 499nm. As the concentration of gelatin protein increases, the fluorescence also shows an enhanced trend, and a significant contrast from no fluorescence to luminescence can be observed with the naked eye.

[0064] Depend on Figure 2 and Figure 3 It can be seen that TPE-2TA also has a good recognition and lighting effect on gelatin protein, with blue fluorescence and a maximum emission wavelength at 484nm. The fluorescence will also increase with the increase of gelatin protein concentration. However, the lighting effect of TPE-2TA on gelatin protein is not as good as that of TPE-4TA. This is because the four tetrazole groups in TPE-4TA make it interact more strongly with the protein, and compared with TPE-2TA with only two tetrazole modifications, it has more significant fluorescence emission.

[0065] Depend on Figure 2 and Figure 3 It can be seen that TPE-TA also has a certain recognition and lighting effect on gelatin protein, but it only contains one tetrazole group, and the fluorescence after binding with gelatin protein is weaker than that of Example 1 and Example 2.

[0066] Comparative Example 1

[0067] The difference between this comparative example and Example 1 is that no dye is added. The rest of the process is reference to Example 1.

[0068] Comparative Example 2

[0069] The difference between this comparative example and Example 1 is that NaSCN is added. In step (1) of Example 1, 0.30 g of NaSCN powder is added to the gelatin aqueous solution and mixed evenly. The rest of the process is similar to Example 1.

[0070] NaSCN denatures proteins, and TPE-4TA cannot significantly illuminate denatured gelatin, which means that when the spatial structure of the protein is destroyed, TPE-4TA no longer has a lighting effect on it. That is, TPE-4TA can only illuminate gelatin with a specific spatial structure.

[0071] Comparative Example 3

[0072] The difference between this comparative example and Example 1 is that gelatin protein is replaced by fibronectin, elastin and Candida antarctica lipase.

[0073] Compared with gelatin, TPE-4TA has no obvious fluorescence change after mixing with fibronectin, elastin and lipase. This is because the cavity structure of gelatin has a good encapsulation effect on TPE-4TA, while the spatial stereoscopic structure of the other three proteins is different from that of gelatin, and their cavity structures cannot encapsulate TPE-4TA dye molecules. In addition, there is an electrostatic interaction between gelatin and TPE-4TA, which further restricts the movement of dye molecules, resulting in a good recognition lighting effect.

[0074] Example 4

[0075] This embodiment realizes the quantitative analysis of gelatin protein fluorescence labeling. The specific process is:

[0076] (1) Preparation of gelatin solutions with gradient concentrations: weigh 0.005 g of gelatin in deionized water, heat to 40°C in a water bath, stir for about 30 minutes until completely dissolved, and dilute to 25 mL to obtain a 200 mg / L gelatin stock solution. Take appropriate amounts and dilute to the required concentrations, which are 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, and 40 mg / L, respectively.

[0077] (2) Take 0.03 mL (1×10 -3 mol / L) TPE-4TA ethanol solution was added to 2.97 mL of gradient concentration gelatin solution and mixed evenly. The excitation / emission slit parameters of the fluorescence spectrometer were fixed to 5 nm, and the optimal excitation wavelength was set to excitation (λ TPE-4TA =346nm), and the fluorescence intensity was tested. According to the peak value of the fluorescence intensity and the concentration, a fitting curve was obtained, such as Figure 5 As shown, the molecule has good quantitative effect within a certain concentration range (such as Figure 4 The linear range of TPE-4TA for gelatin protein quantification is 0.1-2 mg / L (as shown in Figure 5 The minimum test detection limit is as low as 0.1 mg / L.

[0078] Example 5

[0079] This embodiment realizes the quantitative analysis of fluorescent labeling of gelatin protein. The difference from the embodiment 4 is that the added TPE-4TA is replaced with TPE-2TA, λTPE-2TA=325nm. The rest of the process is referred to the embodiment 4.

[0080] like Figure 4 and Figure 5 As shown in the figure, the linear range of TPE-2TA for gelatin protein quantification is 0.1-5 mg / L, and the minimum test detection limit is as low as 0.1 mg / L.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for fluorescent labeling of gelatin, characterized in that: The tetrazolium-modified aggregation-induced emission fluorescent dye molecules are dissolved in an aqueous phase, an alcohol phase or a PBS buffer solution, and then mixed with a gelatin protein solution to obtain a fluorescent protein complex solution; The structural formula of the tetrazole-modified aggregation-induced emission fluorescent dye molecule is as follows: wherein at least one of R′, R″, R″′ and R″″ is a tetrazolyl group; The tetrazole-modified aggregation-induced emission fluorescent dye molecules are selected from one or more of TPE-4TA, TPE-3TA, TPE-2TA and TPE-TA.

2. The method for fluorescent labeling of gelatin according to claim 1, characterized in that: The tetrazole-modified aggregation-induced emission fluorescent dye molecule is TPE-4TA.

3. The method for fluorescent labeling of gelatin according to claim 1, characterized in that: The concentration of the tetrazole-modified aggregation-induced luminescence fluorescent dye molecules dissolved in water, alcohol or PBS buffer solution is 1×10 -2 ~10 - 4 mol / L.

4. The method for fluorescent labeling of gelatin according to claim 1, characterized in that: The alcohol phase is ethanol.

5. The method for fluorescent labeling of gelatin according to claim 1, characterized in that: The method also includes irradiating the fluorescent protein complex solution with excitation light and judging whether the gelatin protein is effectively labeled based on whether fluorescence is observed.

6. The method for fluorescent labeling of gelatin according to claim 1, characterized in that: The gelatin protein solution is prepared at a temperature of 35-45° C. and a stirring time of 25-35 min.

7. A quantitative analysis method for gelatin protein, characterized in that: The following steps are involved: S1, preparing a gelatin solution with gradient concentrations, labeling the gelatin using the method described in any one of claims 1 to 6, and testing the fluorescence intensity; S2, fitting the standard curve of the relationship between fluorescence intensity and gelatin protein concentration; S3, obtaining a gelatin protein sample, testing the fluorescence intensity using the method of step S1, and then performing a quantitative analysis of the gelatin protein in the sample according to the standard curve.

8. The quantitative analysis method of gelatin protein according to claim 7, characterized in that: The excitation wavelength for testing the fluorescence intensity is 340-450 nm.

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