A fluorescent probe and its preparation method and application, and a method for detecting fumonisin content

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a fluorescent probe and its preparation method and application, and a method for detecting the content of fumonisin. The fluorescent probe provided by the present invention has an acyl chloride structure, can quickly complete a derivatization reaction with fumonisin, and the amide structure generated by the derivatization reaction can form a derivative of a larger conjugated system with the parent of the fluorescent probe. The derivative is more likely to generate a high fluorescent signal under ultraviolet light excitation, thereby improving the fluorescence efficiency of the fluorescent probe, and the detection limit reaches 0.12μg / L.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a fluorescent probe and a preparation method and application thereof, and a method for detecting the content of fumonisin. Background Art

[0002] Fumonisin FB1 is one of the fungal toxins produced by Fusarium rotundifolia and Fusarium rotundifolia, and is mainly found in animal foods such as corn. The severe and persistent toxic effects of fumonisins can cause esophageal cancer and neurological defects, and can also damage the brain, kidneys and lungs. The maximum content of fumonisins in corn products approved by the European Union is 200-4000 mg / kg. Horses are very sensitive to fumonisins, and the maximum recommended daily intake of fumonisins is 1 mg / kg. Therefore, for the detection of fumonisins, the sensitivity of the instrument analysis is required to be high enough, and the sample pretreatment should be simple to meet the requirements for rapid analysis and detection of grain crop samples.

[0003] At present, the analytical methods for fumonisin FB1 usually include thin layer chromatography, LC-MS / MS and HPLC. The above instrumental analysis has high sensitivity and reliable results. The detection limit of fumonisin analysis based on chromatography and mass spectrometry systems is 0.08-0.20 μg / L. Most quantitative methods for fumonisin require derivatization to graft chromophores on the fumonisin molecules so that they can be detected by emission fluorescence or absorption spectra. Traditional methods mostly use chromatography to analyze and detect fumonisin in food. Chromatography-based analysis technology has the characteristics of accuracy, sensitivity, and strong selectivity, and has been widely used in actual sample analysis. However, due to the large and expensive chromatographic analysis instruments, complex sample pretreatment, long response time of chromatographic technology, it is not conducive to the rapid detection of a large number of samples, and the sensitivity of fumonisin detection is not high enough, it is difficult to meet the market detection needs. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a fluorescent probe and its preparation method and application, and a method for detecting the content of fumonisin. The fluorescent probe provided by the present invention can quickly and accurately detect fumonisin in large quantities and has high sensitivity.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a fluorescent probe having a structure shown in any one of Formulas I to VI:

[0007]

[0008] The present invention also provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps:

[0009] A fluorescent compound, an acyl chloride compound, a catalyst and an organic solvent are mixed to carry out a substitution reaction to obtain a fluorescent probe;

[0010] The fluorescent compound includes rhodamine B, rhodamine 6G, rhodamine 123, coumarin, acrylamide or 2-naphthoic acid.

[0011] Preferably, the acyl chloride compound includes oxalyl chloride, benzoyl chloride, propionyl chloride, trifluoromethanesulfonyl chloride or acryloyl chloride.

[0012] Preferably, the catalyst comprises N,N-dimethylformamide or formamide.

[0013] Preferably, the organic solvent includes dichloromethane, chloroform, 1,2-dichloroethane, hexachloroethane or carbon tetrachloride.

[0014] Preferably, the temperature of the substitution reaction is 15-25° C. and the time is 2-3 h.

[0015] The present invention also provides the use of the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution in the detection of fumonisin.

[0016] The present invention also provides a method for detecting the content of fumonisin, comprising the following steps:

[0017] The sample to be tested, the fluorescent probe, the buffer solution and the polar solvent are mixed to carry out a derivatization reaction to obtain a derivatization reaction product; the fluorescent probe is the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution;

[0018] Using a filter membrane to perform solid phase extraction on the derivatization reaction product to obtain a filter membrane that adsorbs fumonisin derivatives; the filter membrane is a nylon membrane or a polyethersulfone membrane;

[0019] The filter membrane adsorbing the fumonisin derivative is subjected to fluorescence spectrum detection, and the fluorescence signal intensity is recorded. The content of fumonisin in the sample to be tested is obtained according to a predetermined fumonisin concentration-fluorescence intensity standard curve.

[0020] Preferably, the derivatization reaction time is 3 to 5 minutes; the derivatization reaction is carried out at room temperature in a dark environment.

[0021] Preferably, the wavelength of the fluorescence spectrum detection is 430-440 nm.

[0022] The present invention provides a fluorescent probe having a structure shown in any one of Formulas I to VI:

[0023]

[0024] The acyl chloride structure of the fluorescent probe provided by the present invention can quickly complete a derivatization reaction with the amino group of fumonisin. The amide structure generated by the derivatization reaction forms a fumonisin derivative with a large-structure conjugated system with the parent body of the fluorescent probe. The derivative is more likely to generate a high fluorescence signal under ultraviolet light excitation due to the large-structure conjugated system, thereby improving the fluorescence efficiency of the fluorescent probe and making the detection limit reach 0.12 μg / L.

[0025] The invention provides a method for detecting the content of fumonisin. The formed fumonisin derivatives can be enriched on a filter membrane through solid phase extraction. The filter membrane has a large number of amide groups and a large number of amino groups. These groups can form a large conjugated system with a multi-ring structure on the fumonisin derivative through coordination, and part of the groups can fix the derivatives on the filter membrane through the formation of hydrogen bonds, so that when a solution flows through a nylon membrane, the solution flows away while the fumonisin derivatives are adsorbed on the filter membrane, so that as many fumonisin derivatives as possible can be detected, thereby improving the accuracy of the detection result, and the content of fumonisin can be quantitatively detected by directly detecting the fluorescent signal of the fumonisin derivatives adsorbed on the filter membrane, so that rapid and large-scale quantitative detection of fumonisin can be easily achieved.

[0026] In addition, the preparation method of the fluorescent probe provided by the present invention is simple and rapid, the reaction conditions are mild, the reaction mechanism research is relatively mature, and the development cost is low. DETAILED DESCRIPTION

[0027] The present invention provides a fluorescent probe, characterized in that it has a structure shown in any one of Formulas I to VI:

[0028]

[0029]

[0030] Preferably

[0031]

[0032] The present invention also provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps:

[0033] A fluorescent compound, an acyl chloride compound, a catalyst and an organic solvent are mixed to carry out a substitution reaction to obtain a fluorescent probe;

[0034] The fluorescent compound includes rhodamine B, rhodamine 6G, rhodamine 123, coumarin, acrylamide or 2-naphthoic acid.

[0035] Unless otherwise specified, the present invention has no special requirements on the sources of the raw materials used in the preparation, and commercially available products known to those skilled in the art can be used.

[0036] The present invention mixes a fluorescent compound, an acyl chloride compound, a catalyst and an organic solvent.

[0037] In the present invention, the fluorescent compound includes rhodamine B, rhodamine 6G, rhodamine 123, coumarin, acrylamide or 2-naphthoic acid, preferably rhodamine B, rhodamine 6G or rhodamine 123; the acyl chloride compound preferably includes oxalyl chloride, benzoyl chloride, propionyl chloride, trifluoromethanesulfonyl chloride or acryloyl chloride, more preferably oxalyl chloride; the molar ratio of the fluorescent compound to the acyl chloride compound is preferably 1:(1.2-1.7), more preferably 1:(1.3-1.5); the catalyst preferably includes N,N-dimethylformamide or formamide, more preferably N,N-dimethylformamide; the organic solvent preferably includes dichloromethane, chloroform, 1,2-dichloroethane, hexachloroethane or carbon tetrachloride, more preferably dichloromethane.

[0038] In the present invention, the molar ratio of the fluorescent compound to the acyl chloride compound is preferably 1:(1.2-1.7), more preferably 1:(1.3-1.5); the molar ratio of the fluorescent compound to the catalyst is preferably 1:(0.03-0.07), more preferably 1:(0.04-0.06); the ratio of the mass of the fluorescent compound to the volume of the organic solvent is preferably 5g:(3-15)mL, more preferably 5g:(5-15)mL.

[0039] In the present invention, the mixing process of the fluorescent compound, the acyl chloride compound, the catalyst and the organic solvent is preferably to first mix Rhodamine B and dichloromethane, stir, then add the acyl chloride compound, continue stirring, and then dropwise add the catalyst.

[0040] After the mixing is completed, the present invention performs a substitution reaction on the mixed material.

[0041] In the present invention, the temperature of the substitution reaction is preferably 15-25°C, more preferably 18-20°C, and the time is preferably 2-3h, more preferably 2.4-2.6h; the substitution reaction is preferably carried out under stirring; the stirring rate is preferably 800-1500rpm, more preferably 800-1300rpm.

[0042] After the substitution reaction is completed, the present invention preferably filters, washes and dries the substitution reaction product in sequence to obtain a fluorescent probe.

[0043] In the present invention, the equipment used for filtration is preferably a nylon membrane; the pore size of the nylon membrane is preferably 0.22-0.45 μm, more preferably 0.22 μm; the washing liquid used for washing is preferably dichloromethane; the washing is preferably elution; the number of washings is preferably 3-5 times, more preferably 3 times; the amount of the washing liquid is not particularly limited and can be determined according to actual needs; the present invention preferably takes 3-5 minutes to continue the next washing after each washing is completed to allow the residual washing liquid to evaporate completely; the equipment used for drying is preferably a vacuum oven; the drying temperature is preferably 20°C, and the drying time is preferably 3-4 hours, more preferably 3 hours; the present invention preferably stores the fluorescent probe in a vacuum bottle.

[0044] In the present invention, taking rhodamine B as a fluorescent compound and oxalyl chloride as an acyl chloride compound as an example, the substitution reaction for preparing the fluorescent probe is as follows:

[0045]

[0046] The fluorescent compound and the acyl chloride compound undergo a substitution reaction to generate a fluorescent probe with an acyl chloride structure. The acyl chloride structure of the fluorescent probe can react quickly with the amino group on the fumonisin to form a stable amide structure.

[0047] The present invention also provides the use of the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution in the detection of fumonisin.

[0048] The present invention also provides a method for detecting the content of fumonisin, comprising the following steps:

[0049] The sample to be tested, the fluorescent probe, the buffer solution and the solvent are mixed to carry out a derivatization reaction to obtain a derivatization reaction product; the fluorescent probe is the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution;

[0050] Using a filter membrane to perform solid phase extraction on the derivatization reaction product to obtain a filter membrane that adsorbs fumonisin derivatives; the filter membrane is a nylon membrane or a polyethersulfone membrane, more preferably a nylon membrane;

[0051] The filter membrane adsorbing the fumonisin derivative is subjected to fluorescence spectrum detection, and the fluorescence signal intensity is recorded. The content of fumonisin in the sample to be tested is obtained according to a predetermined fumonisin concentration-fluorescence intensity standard curve.

[0052] The invention mixes a fumonisin sample, a fluorescent probe, a buffer solution and a polar solvent, performs a derivatization reaction and obtains a derivatization reaction product.

[0053] In the present invention, the molar ratio of the sample to be tested and the fluorescent probe is preferably 1:(1.2-3), more preferably 1:(1.5-2); the polar solvent is preferably acetonitrile, a mixed solvent of methanol and acetonitrile, or a mixed solvent of ethanol and acetonitrile, more preferably acetonitrile; the volume percentage of methanol in the mixed solvent of methanol and acetonitrile is preferably 10-20%, more preferably 15-18%; the volume percentage of ethanol in the mixed solvent of ethanol and acetonitrile is preferably 5-10%, more preferably 7-8%; the ratio of the mass of the fluorescent probe to the volume of the polar solvent is preferably 1g:10mL.

[0054] In the present invention, the mixing process of the sample to be tested, the fluorescent probe, the buffer solution and the polar solvent is preferably to dissolve the fluorescent probe in the polar solvent to prepare a fluorescent probe solution, add the buffer solution to the sample to be tested, and then add the fluorescent probe solution.

[0055] In the present invention, the fluorescent probe solution is preferably stored in a -4°C refrigerator; the buffer solution is preferably potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer solution or sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution; the pH value of the buffer solution is preferably 7-8; the volume ratio of the sample to be tested and the buffer solution is preferably 1:(0.5-1.5), more preferably 1:(0.5-1); the present invention has no special limitation on the concentration of the buffer solution, which can be adjusted according to the sample detection concentration level; in an embodiment of the present invention, the concentration of the buffer solution is preferably 0.5-100 μg / L, more preferably 0.5-10 μg / L.

[0056] In the present invention, the derivatization reaction time is preferably 3 to 5 minutes, more preferably 3.5 to 4 minutes; the derivatization reaction is preferably carried out at room temperature in a light-proof environment; the derivatization reaction is preferably carried out under stirring conditions; the stirring rate is preferably 500 to 1000 rpm, more preferably 750 to 800 rpm.

[0057] The polar solvent in the present invention provides a polar reaction environment to realize the derivatization reaction of the fluorescent probe and fumonisin.

[0058] In the present invention, taking the fluorescent probe and fumonisin prepared by substitution reaction of rhodamine B and oxalyl chloride as an example, the process of the derivatization reaction is as follows:

[0059]

[0060] The amide structure generated by the derivatization reaction of the fluorescent probe with fumonisin can form a larger conjugated system with the parent body of the fluorescent probe, thereby improving the fluorescence efficiency of the fluorescent probe. Not only is it easy to generate fluorescent signals, but the intensity of the fluorescent signals can also be enhanced, thereby improving the sensitivity of detection.

[0061] After obtaining the derivatization reaction product, the present invention uses a filter membrane to perform solid phase extraction on the derivatization reaction product to obtain a filter membrane that adsorbs fumonisin derivatives.

[0062] In the present invention, the filter membrane is preferably a nylon membrane; the pore size of the nylon membrane is preferably 0.22-0.45 μm, more preferably 0.22 μm; the solid phase extraction process is preferably filtering the derivatization reaction product through the filter membrane; the filtration rate is preferably 5-8 mL / min, more preferably 5.5-6 mL / min.

[0063] After obtaining the filter membrane adsorbing the fumonisin derivative, the present invention performs fluorescence spectrum detection on the filter membrane adsorbing the fumonisin derivative, records the fluorescence signal intensity, and obtains the fumonisin content in the sample to be tested according to the fumonisin concentration-fluorescence intensity standard curve.

[0064] In the present invention, the wavelength of the fluorescence spectrum detection is preferably 430-440 nm, more preferably 435 nm.

[0065] The filter membrane performs solid phase extraction on the derivatization reaction products, and the fumonisin derivatives are adsorbed on the filter membrane. The filter membrane has a large number of amide groups and a large number of amino groups, which are conducive to the specific adsorption of the fumonisin derivatives, so that the fumonisin derivatives do not flow away with the solution during solid phase extraction, but are adsorbed on the filter membrane, so that as many fumonisin derivatives as possible can be detected, thereby improving the accuracy of the detection.

[0066] In the present invention, the method for establishing the concentration-fluorescence intensity standard curve of fumonisin preferably comprises the following steps:

[0067] The fumonisin standard solutions of a series of concentrations are mixed with the fluorescent probe solution respectively to perform a derivatization reaction to obtain a derivatization reaction product; the concentration of the fluorescent probe solution is the same as the concentration of the fumonisin standard solutions of a series of concentrations;

[0068] Using a filter membrane to perform solid phase extraction on the derivatization reaction product to obtain a filter membrane that adsorbs fumonisin derivatives;

[0069] The filter membrane adsorbing the fumonisin derivative is subjected to fluorescence spectrum detection, and the fluorescence signal intensity is recorded. According to the series concentrations of the fumonisin standard solution and the fluorescence signal intensities corresponding to the series concentrations, a fumonisin concentration-fluorescence intensity standard curve is obtained.

[0070] In the present invention, the series concentration of the fumonisin standard solution is preferably 1×10 -3 g / L, 1×10 -4 g / L, 1×10-5 g / L, 1×10 -6 g / L, 1×10 -7 g / L; the concentration of the fluorescent probe solution is preferably 1×10 -3 g / L, 1×10 -4 g / L, 1×10 -5 g / L, 1×10 -6 g / L, 1×10 -7 g / L; the volume ratio of the fumonisin standard solution and the fluorescent probe solution is preferably (3-5):(1-2), more preferably (3-4.2):1; the mixing of the fumonisin standard solution and the fluorescent probe solution is preferably carried out under shaking conditions; the present invention has no special restrictions on the time and rate of the shaking, as long as the materials are mixed evenly; the temperature of the derivatization reaction is preferably room temperature, and the time is preferably 3-5 min, more preferably 3.5-4 min; the derivatization reaction is preferably carried out under static conditions; the contents of the solid phase extraction and fluorescence spectrum detection are consistent with the above and are not repeated here.

[0071] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0072] Example 1

[0073] Weigh 5 g of rhodamine B, add it to 11.0 mL of dichloromethane, stir at 800 rpm, then add 2 g of oxalyl chloride, continue stirring, and then drop 0.08 mL of N,N-dimethylformamide, keep stirring at 20°C for substitution reaction for 2.5 hours to obtain a substitution reaction product, filter the substitution reaction product with a nylon membrane with a pore size of 0.22 μm, and use 75 mL of dichloromethane to wash the filtered solid three times. After each washing is completed, wait 4 minutes before continuing the next washing, and then dry it in a vacuum oven at 20°C for 3 hours to obtain a fluorescent probe.

[0074] Application Example 1

[0075] 1.0 g of the fluorescent probe prepared in Example 1 was weighed and dissolved in 10 mL of acetonitrile to obtain a fluorescent probe solution, which was then gradiently diluted and placed in a -4 °C refrigerator as a stock solution for use. The concentration gradient series was as follows: 1 × 10 -3 g / L, 1×10 -4 g / L, 1×10 -5 g / L, 1×10 -6 g / L, 1×10 -7 g / L;

[0076] The concentration gradient was prepared as follows: 1×10 -3 g / L, 1×10 -4g / L, 1×10 -5 g / L, 1×10 -6 g / L, 1×10 -7 g / L fumonisin standard solution, take 5 mL of each concentration gradient of fumonisin standard solution, add 2 mL of the corresponding concentration gradient of fluorescent probe solution thereto, shake and mix evenly, and let stand for 4 minutes to obtain a derivatization reaction product;

[0077] The derivatization reaction product is filtered at a rate of 6 mL / min using a nylon membrane with a pore size of 0.22 μm to obtain a filter membrane adsorbing the fumonisin derivative, and then the filter membrane adsorbing the fumonisin derivative is subjected to fluorescence spectrum detection at 435 nm, and the fluorescence signal intensity is recorded. According to the fumonisin standard solution and its corresponding fluorescence signal intensity, a fumonisin concentration-fluorescence intensity standard curve is obtained: I=2029C+566.2, where I is the fluorescence intensity and C is the fumonisin concentration;

[0078] 2 mL of potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer solution with a pH of 7.5 was added to 10 mL of fumonisin sample, and then 1 mL of 0.5 g / mL fluorescent probe solution was added to carry out derivatization at 800 rp for 4 minutes at room temperature in a dark environment to obtain a derivatization reaction product; the derivatization reaction product was filtered with a nylon membrane with a pore size of 0.22 μm at a rate of 6 mL / min to obtain a filter membrane that adsorbs fumonisin derivatives, and then the filter membrane that adsorbs fumonisin derivatives was subjected to fluorescence spectrum detection at 435 nm, and the fluorescence signal intensity was recorded. According to the fumonisin concentration-fluorescence intensity standard curve: I=2029C+566.2, the fluorescence intensity was substituted to calculate the fumonisin content.

[0079] Application Example 2

[0080] The method of Application Example 1 was used to prepare 0.5, 0.8, 1.0, 2.0, 5.0, 8.0, 10.0 μg / L fumonisin standard solutions, and the derivatization reaction, solid phase extraction and solid phase fluorescence spectrum analysis were performed according to the method of Application Example 1, and a standard curve was established: I=2016C+577.9, where I is the fluorescence intensity and C is the fumonisin concentration. According to the established standard curve, the detection limit of this method for fumonisin is 0.12 μg / L.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting the content of fumonisin, characterized in that: The method comprises the following steps: mixing a sample to be tested, a fluorescent probe, a buffer solution and a polar solvent, and performing a derivatization reaction to obtain a derivatization reaction product; the fluorescent probe has a structure shown in Formula I: Formula I; The preparation method of the fluorescent probe comprises the following steps: mixing a fluorescent compound, an acyl chloride compound, a catalyst and an organic solvent, and performing a substitution reaction to obtain a fluorescent probe; The fluorescent compound is rhodamine B; The acyl chloride compound includes oxalyl chloride, benzoyl chloride, propionyl chloride, trifluoromethanesulfonyl chloride or acryloyl chloride; The catalyst includes N,N-dimethylformamide or formamide; The organic solvent includes dichloromethane, chloroform, 1,2-dichloroethane, hexachloroethane or carbon tetrachloride; the temperature of the substitution reaction is 15-25°C and the time is 2-3h; Using a filter membrane to perform solid phase extraction on the derivatization reaction product to obtain a filter membrane that adsorbs fumonisin derivatives; the filter membrane is a nylon membrane or a polyethersulfone membrane; Performing fluorescence spectrum detection on the filter membrane adsorbing the fumonisin derivative, recording the fluorescence signal intensity, and obtaining the fumonisin content in the sample to be tested according to a predetermined fumonisin concentration-fluorescence intensity standard curve; The derivatization reaction time is 3 to 5 minutes; the derivatization reaction is carried out at room temperature in a dark environment; The wavelength of the fluorescence spectrum detection is 430-440 nm; The filter membrane is a nylon membrane; the pore size of the nylon membrane is 0.22-0.45 μm; the solid phase extraction process is to filter the derivatization reaction product through the filter membrane; the filtration rate is 5-8 mL / min.

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