CN117343726A - Fluorescent probe for rapidly and nondestructively detecting food freshness and preparation method thereof - Google Patents
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 74
- 235000013305 food Nutrition 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 241000251468 Actinopterygii Species 0.000 claims abstract description 15
- 235000013372 meat Nutrition 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- 238000012360 testing method Methods 0.000 claims description 34
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 12
- 239000011541 reaction mixture Substances 0.000 claims description 11
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 10
- 229940125904 compound 1 Drugs 0.000 claims description 9
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
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- 239000000203 mixture Substances 0.000 claims description 4
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 4
- 238000009659 non-destructive testing Methods 0.000 claims description 4
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 abstract description 31
- 230000000035 biogenic effect Effects 0.000 abstract description 31
- 239000000523 sample Substances 0.000 abstract description 30
- 230000004044 response Effects 0.000 abstract description 21
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- 238000011065 in-situ storage Methods 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
- 230000003287 optical effect Effects 0.000 abstract description 2
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 description 78
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 239000000243 solution Substances 0.000 description 10
- 238000010521 absorption reaction Methods 0.000 description 9
- 235000019688 fish Nutrition 0.000 description 9
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 241000238557 Decapoda Species 0.000 description 5
- 235000015278 beef Nutrition 0.000 description 5
- 239000005700 Putrescine Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
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- 230000003595 spectral effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- APJYDQYYACXCRM-UHFFFAOYSA-N tryptamine Chemical compound C1=CC=C2C(CCN)=CNC2=C1 APJYDQYYACXCRM-UHFFFAOYSA-N 0.000 description 2
- DZGWFCGJZKJUFP-UHFFFAOYSA-N tyramine Chemical compound NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
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- 206010030113 Oedema Diseases 0.000 description 1
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- 206010006451 bronchitis Diseases 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 125000001309 chloro group Chemical group Cl* 0.000 description 1
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- 239000003925 fat Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 210000003026 hypopharynx Anatomy 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229960003732 tyramine Drugs 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
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- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a fluorescent probe for rapidly and nondestructively detecting the freshness of food by using a compound Cl-BODIPY, and accordingly, a corresponding preparation method and a detection method are also established. The probe generates the change of color and fluorescence dual optical signals through the specific interaction of the recognition group and the volatile biogenic amine, and provides a simple and convenient detection means for the rapid detection of the volatile biogenic amine. The fluorescent probe provided by the invention is simple to prepare, low in cost, high in sensitivity and selectivity and quick in response, can be used for quickly detecting volatile biogenic amine generated by the spoilage of fish and meat foods in situ, and judging the freshness (biogenic amine content) of the fish and the meat through colorimetric and fluorescent dual visual signal change, so that an effective means is provided for the quick nondestructive detection of the freshness of the foods, and the fluorescent probe has a great application prospect and great popularization significance.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a fluorescent probe for rapidly and nondestructively detecting food freshness and a preparation method thereof.
Background
Food quality and safety are closely related to public health. According to World Health Organization (WHO) statistics, ingestion of spoilage foods can lead to over 200 diseases such as diarrhea and cancer. Meat food is an important nutrition source and is rich in various proteins and fats required by human body; however, they are also susceptible to deterioration during storage and transportation due to enzymatic reactions and microbial contamination. Various biogenic amines are typically produced during spoilage of protein foods (meats, fish and seafood), such as: cadaverine, putrescine, histamine, tyramine, tryptamine, spermine, and spermidine. Wherein, cadaverine and putrescine have high volatility, can be inhaled, eaten or absorbed by human body through skin, and have strong irritation to eyes, mucous membrane, skin and respiratory tract. Inhalation of volatile biogenic amines may cause laryngopharynx, bronchitis, cramps, oedema, pulmonary oedema or chemical pneumonia and even death. Therefore, the content of cadaverine and putrescine with volatility is the most remarkable indicator of the freshness of foods, and some countries have tried to give a limited standard according to the characteristics of different foods. Therefore, in order to ensure the quality and safety of meat products, simple and effective means must be developed to detect the freshness of fish and meat products.
Currently, food safety issues are increasingly gaining attention to the community. The guarantee of food safety depends on reliable technical detection means. Methods for detecting volatile biogenic amines to date include gas chromatography-mass spectrometry, electrochemical methods, spectrophotometry and liquid chromatography. However, these methods still suffer from a number of deficiencies. Such as complex sample preparation processes, expensive instruments, low sensitivity, inability to achieve in situ non-destructive testing, etc. In recent years, fluorescent probe technology has been evaluated as a powerful tool for detecting biogenic amines due to its advantages of high sensitivity, high selectivity, real-time detection, and ease of operation. Because biogenic amine can not emit fluorescence, the determination of biogenic amine by using a fluorescence photometry is mainly based on the reaction of a reaction site of a fluorescent probe and amino groups of biogenic amine under a certain condition to generate a product with different self-fluorescence properties from the fluorescent probe, and the content of biogenic amine can be detected according to the change of double signals of fluorescence and color. In view of the importance of food safety, it is very important and challenging to develop new fluorescent probes for rapid, high sensitivity, nondestructive visualization of volatile biogenic amine content in fish and meat products.
Therefore, the need for developing a simple and cost effective means of technical detection for detecting volatile biogenic amines in fish and meat is urgent.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fluorescent probe for rapidly and nondestructively detecting food freshness with stable structure, excellent performance and high sensitivity and a preparation method thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
application of compound Cl-BODIPY in rapid nondestructive testing of food freshness, wherein the structural formula of the compound Cl-BODIPY is
The food is fish or meat.
The compound Cl-BODIPY is used as a fluorescent probe for rapidly and nondestructively detecting the freshness of food.
The preparation method of the fluorescent probe comprises the following synthetic routes:
the preparation method of the fluorescent probe comprises the following steps:
(1) Preparation of Compound 1: pyrrole, N-diisopropylethylamine and an organic solvent 1, 2-dichloroethane are sequentially added into a three-necked round bottom flask, and then the reaction mixture is stirred for 20min in a nitrogen atmosphere; triphosgene is dissolved in 1, 2-dichloroethane and the above mixture is added dropwise at 0 ℃; after 2 hours of reaction, pyrrole is added, and the mixture is heated and refluxed for 1 hour at 70 ℃; removing redundant solvent under reduced pressure, and purifying the obtained crude product by a silica gel chromatographic column to obtain a white solid as a target compound 1;
(2) Preparation of fluorescent probes: sequentially adding the compound 1, 2-dichloroethane and phosphorus oxychloride into a three-neck round bottom flask, and then heating, refluxing and stirring the reaction mixture in a nitrogen atmosphere at 80 ℃ for 3-5h; after the TCI detection reaction was complete, the reaction mixture was cooled in an ice bath and triethylamine was slowly added; stirring for 5min, and slowly adding boron trifluoride diethyl etherate; then the reaction mixture is heated to room temperature and the reaction is continued for 2-5h; and (3) removing the redundant solvent under reduced pressure, and purifying the obtained crude product by a silica gel chromatographic column to obtain a red solid serving as a target fluorescent probe.
In the step (1), the molar ratio of triphosgene to pyrrole to N, N-diisopropylethylamine is 1-5.
The molar ratio of the compound 1 to phosphorus oxychloride, triethylamine and boron trifluoride diethyl etherate in the step (2) is 0.5-15.
And the test paper is loaded with the fluorescent probe for rapidly and nondestructively detecting the freshness of food.
The method for detecting the freshness of the food in a rapid and nondestructive way comprises the steps of placing the test paper in a closed dish, and placing fish or meat samples in the dish.
The temperature in the closed dish was-16 ℃, 0 ℃, 25 ℃.
Aiming at the difficult problem that the on-site rapid nondestructive visual detection of the freshness of foods such as fish, meat and the like cannot be carried out at present, the inventor uses a compound Cl-BODIPY as a fluorescent probe for rapid nondestructive detection of the freshness of foods, and accordingly, a corresponding preparation method and a detection method are also established. The probe generates the change of color and fluorescence dual optical signals through the specific interaction of the recognition group and the volatile biogenic amine, and provides a simple and convenient detection means for the rapid detection of the volatile biogenic amine. The fluorescent probe provided by the invention is simple to prepare, low in cost, high in sensitivity and selectivity and quick in response, can be used for in-situ quick detection of volatile biogenic amine generated by fish and meat food spoilage, judges the freshness (biogenic amine content) of fish and meat through colorimetric and fluorescent dual-visual signal change, provides an effective means for quick nondestructive detection of food freshness, has a great application prospect, has great popularization significance, is a preferred method for quick detection of the volatile biogenic amine content in fish and meat food in a period of time in the current and future, and provides a powerful technical guarantee for self-monitoring and market supervision of food manufacturers. The prepared test paper can realize high-sensitivity nondestructive visual monitoring of the freshness of fish and meat, and can become a mainstream technology for on-site rapid detection of volatile biogenic amine in food.
The basic principle of the invention is as follows: the chlorine atom in the compound Cl-BODIPY can be used as a recognition group to perform substitution reaction with the amino group of the volatile biogenic amine, and the signal is transmitted to the BODIPY fluorescent group through the change of the molecular structure to promote the change of the fluorescence emission wavelength and the solution color, so that the compound can be used for colorimetric and fluorescence dual signal response to the volatile biogenic amine. Specifically: the fluorescent probe had a distinct absorption peak at 499 nm. When cadaverine was added, a new absorption peak appeared at 395 nm; as cadaverine concentration increases, the original absorption peak of the probe gradually decreases and the absorption peak at 395 nm gradually increases, and a clear color change from green to yellowish can be observed. In addition, under excitation of the excitation light, the probe has obvious green fluorescence at about 512 nanometers. After cadaverine is added, the recognition group is combined with the amino, so that the original electron transfer process of the probe molecule is destroyed, the green fluorescence of the probe at about 512 nanometers is weakened, and the blue fluorescence at about 452 nanometers is enhanced, thereby realizing the ratio type fluorescence detection of the cadaverine. In the ratio type detection and identification event, the influence of external factors on the fluorescence signal intensities of the two detection and identification events is consistent, so that the external factors hardly influence the ratio of the two fluorescence intensities, and the mode can greatly reduce the influence of the external factors and improve the detection precision and accuracy.
Drawings
FIG. 1 is a one-dimensional hydrogen nuclear magnetic spectrum of a fluorescent probe of the present invention, in which: the abscissa is chemical shift and the ordinate is signal intensity.
FIG. 2 is a graph of UV titration spectra of a fluorescent probe of the invention against cadaverine, wherein: the abscissa is wavelength and the ordinate is absorption intensity.
FIG. 3 is a graph of fluorescence titration spectra of a fluorescent probe of the present invention against cadaverine, wherein: the abscissa is wavelength and the ordinate is fluorescence intensity.
FIG. 4 is a graph of the response time results of a fluorescent probe of the present invention to cadaverine, wherein: the abscissa is the time difference and the ordinate is the fluorescence intensity.
FIG. 5 is a graph showing the selectivity results of fluorescent probe-loaded test paper of the present invention for volatile biogenic amines.
FIG. 6 is a graph showing the response of the fluorescent probe-loaded test strips of the present invention to cadaverine vapors at various concentrations (0, 3,5,7,11,14,22,27,68,137,274,685 ppm).
FIG. 7 is a graph showing the results of the freshness measurement of beef samples at different temperatures and at different times for the test strip loaded with fluorescent probes according to the present invention, wherein: a is a picture taken under 365nm of a fluorescent lamp, b is a volatile biogenic amine content histogram obtained by the test paper and TVBN method in (a) at 25 ℃, c is a volatile biogenic amine content histogram obtained by the test paper and TVBN method in (a) at 0 ℃, and d is a volatile biogenic amine content histogram obtained by the test paper and TVBN method in (a) at-16 ℃.
FIG. 8 is a graph showing the results of measuring freshness of shrimps at different temperatures for different times by using the fluorescent probe-loaded test paper of the present invention.
FIG. 9 is a bar graph of the fluorescence response of the fluorescent probe of the invention and other BODIPY derivatives to cadaverine, wherein: cl-BODIPY is the fluorescent probe of the invention.
FIG. 10 is a flow chart showing the preparation of the fluorescent probe Cl-BODIPY of the present invention.
Detailed Description
EXAMPLE 1 preparation of fluorescent Probe Cl-BODIPY
The chemical formula of the fluorescent probe Cl-BODIPY for rapidly and nondestructively detecting the freshness of food is C 9 H 6 BClF 2 N 2 The structural formula is as follows:
as shown in FIG. 10, the preparation steps of Cl-BODIPY are as follows:
compound 1 (1 mmol) and phosphorus oxychloride (8 mmol) were dissolved in 1, 2-dichloroethane (16 mL) and the reaction mixture was stirred under reflux with heating at 80deg.C under nitrogen for 4h. After completion of the TCI detection reaction, the reaction mixture was cooled in an ice bath and triethylamine (10 mmol) was slowly added. Boron trifluoride diethyl etherate (10 mmol) was slowly added after stirring for 5 min. The reaction mixture was then warmed to room temperature and the reaction continued for 4h. And (3) removing the redundant solvent under reduced pressure, and purifying the obtained crude product by a silica gel chromatographic column to obtain a red solid serving as a target fluorescent probe. The one-dimensional hydrogen nuclear magnetic spectrum diagram is shown in figure 1.
1 H NMR(500MHz,CDCl 3 ):d＝7.89(brs,2H),7.41(d, 3 J＝4.3Hz,2H),6.58ppm(d, 3 J＝4.3Hz,2H).
Preparing a detection reagent:
(1) Preparation of probe stock solution: 5.0 mg of the probe was weighed and dissolved in dry methylene chloride to prepare a probe solution having a probe concentration of 5 mM.
(2) Cadaverine stock solution preparation: 5.0 ml of 1, 5-diaminopentane was aspirated and dissolved in HPLC grade acetonitrile to prepare a 1mM solution.
(3) Preparation of a probe-loaded test strip: the filter strip (3X 3 cm) was immersed in a 1mM probe solution for 30min, then taken out with tweezers, and left in a dark place for natural air drying to prepare a probe-loaded test strip.
Example 2 UV titration of fluorescent probe against cadaverine
The ultraviolet spectral response of the fluorescent probe to cadaverine was verified using the fluorescent probe in example 1. FIG. 2 shows fluorescent probes (concentration 0.5X10) -5 Moles/liter) in acetonitrile, the ultraviolet wavelength of the solution was changed when cadaverine was added in an amount of 0.78 times the molar amount of the fluorescent probe. The probe originally has an obvious absorption peak at 499 nanometers, and when cadaverine is added, a new absorption peak appears at 395 nanometers; as cadaverine concentration increases, the original absorption peak of the probe gradually decreases and the absorption peak at 395 nm gradually increases, and a clear color change from green to yellowish can be observed. The fluorescent probe can realize colorimetric detection of cadaverine.
Example 3 fluorescence titration of fluorescent probe against cadaverine
The fluorescent probe of example 1 was used to verify its fluorescence spectral response to cadaverine. FIG. 3 shows fluorescent probes (concentration 0.5X10) -5 Moles/liter) in acetonitrile, the change in fluorescence wavelength of the solution when cadaverine was added in an amount 0.78 times the molar amount of the fluorescent probe. The emission wavelength of the fluorescent probe was changed from 512 nm to 452 nm, and a clear change in fluorescence from green to blue was observed under an ultraviolet lamp. The fluorescent probe pair can realize the ratio-type fluorescent detection of cadaverine.
Example 4 response time of fluorescent probe to cadaverine
The response time of the fluorescent probe to cadaverine was evaluated using the fluorescent probe in example 1. FIG. 4 shows fluorescent probes (concentration 0.5X10) -5 Moles/liter) in acetonitrile, the fluorescence intensity of the solution varied with time when cadaverine was added in an amount of 0.78 times the molar amount of the fluorescent probe. The excitation wavelength of the fluorescent probe is 392 nanometers, and the emission wavelength is 452 nanometers. After the cadaverine with the molar quantity of 0.78 is added into the fluorescent probe solution, the fluorescent intensity is stable within 120 seconds, which proves that the fluorescent probe has good response speed and practical applicability to the cadaverine.
Example 5 selectivity of fluorescent probes for volatile biogenic amines
The fluorescent probe of example 1 was used to evaluate its selectivity for volatile biogenic amines. Fluorescent probe (concentration 1.0X10) -3 Moles/liter) of probe-loaded test paper was prepared in methylene chloride solution, and FIG. 5 shows that when various common biogenic amine related interfering substances (e.g., EA, DEA, TEA, NH) were added 3 、N 2 H 4 、H 2 O 2 、HCOOH、SO 2 、H 2 S, HCHO, TMP, tyr, his, put and Cad) changes in fluorescence and colorimetry of the test paper upon steaming. When the test paper loaded with the probe responded to cadaverine and putrescine, a clear change in fluorescence of the test paper from green to blue and a colorimetric change of the test paper from dark brown to pale yellow were observed. When various common biogenic amine interfering species are added, no obvious fluorescence and colorimetric change are observed in the test paper loaded with the probe, which indicates that the fluorescent probe has the functions of cadaverine and putrescineGood selective recognition and practical applicability.
Example 6 colorimetric and fluorescent response of test strips loaded with fluorescent probes to different concentrations of cadaverine vapors
The fluorescent probe-loaded test strips of example 1 were used to evaluate the colorimetric and fluorescent response of cadaverine vapors at different concentrations. As shown in fig. 6, as cadaverine vapor concentration increased, a clear test strip was observed that gradually changed in fluorescence from green to blue and a test strip gradually changed in color from dark brown to light yellow. The results show that: the test paper loaded with the probe can realize the rapid visual detection of cadaverine steam, and shows good practical analysis applicability.
Example 7 test paper with probes measurement of freshness of beef samples for different time periods at different temperatures
The fluorescent probe of example 1 was used to evaluate the response of the probe-loaded test strips to beef samples at different temperatures for different time periods. FIG. 7 shows the fluorescent response of probe-loaded test paper to beef after 0,1,3,5,7,9,12 and 24 hours of placement in a closed dish at three temperatures, namely-16 ℃, 0 ℃ and 25 ℃. The result shows that the test paper loaded with the probe has good practical applicability, and can realize nondestructive visual detection of the freshness of the beef sample.
Example 8 test paper with Probe loaded measurement of shrimp freshness at different temperatures for different time periods
The response of the probe-loaded test strips to shrimp samples at different temperatures and for different time periods was evaluated using the fluorescent probes of example 1. FIG. 8 shows the fluorescent response of probe-loaded test paper to shrimp after 0,1,3,5,7,9,12 and 24h of placement in a closed dish at three temperatures, -16 ℃, 0 ℃ and 25 ℃. The result shows that the test paper loaded with the probe has good practical applicability and can realize nondestructive visual detection of the freshness of the prawn sample.
EXAMPLE 9 fluorescent response of various BODIPY derivatives to cadaverine
To verify the specificity of the fluorescent probe Cl-BODIPY of the present invention, the inventors also performed fluorescence spectral response tests on cadaverine for other BODIPY derivatives as follows.
FIG. 9 shows other BODIPY derivatives (concentration 0.5X10) -5 Moles/liter) in acetonitrile, the change in fluorescence intensity of the solution when cadaverine was added in an amount of 0.78 times the molar amount of the fluorescent probe. The emission wavelength of the fluorescent probe Cl-BODIPY is changed from 512 nanometers to 452 nanometers, and obvious fluorescence change from green to blue is observed under an ultraviolet lamp, and the rest BODIPY derivatives have no obvious wavelength change. It shows that cadaverine only responds to the fluorescent probe Cl-BODIPY of the invention, and the rest of BODIPY derivatives have no colorimetric response or fluorescent response to volatile biogenic amines.
Claims (10)
1. The application of the compound Cl-BODIPY in the rapid nondestructive testing of the freshness of foods is characterized in that: the structural formula of the compound Cl-BODIPY is
2. The use according to claim 1, characterized in that: the food is fish or meat.
3. The compound Cl-BODIPY is used as a fluorescent probe for rapidly and nondestructively detecting the freshness of food.
4. A method for preparing a fluorescent probe according to claim 3, which is characterized by the following synthetic route:
5. the method for preparing a fluorescent probe according to claim 4, comprising the steps of:
(1) Preparation of Compound 1: pyrrole, N-diisopropylethylamine and an organic solvent 1, 2-dichloroethane are sequentially added into a three-necked round bottom flask, and then the reaction mixture is stirred for 20min in a nitrogen atmosphere; triphosgene is dissolved in 1, 2-dichloroethane and the above mixture is added dropwise at 0 ℃; after 2 hours of reaction, pyrrole is added, and the mixture is heated and refluxed for 1 hour at 70 ℃; removing redundant solvent under reduced pressure, and purifying the obtained crude product by a silica gel chromatographic column to obtain a white solid as a target compound 1;
(2) Preparation of fluorescent probes: sequentially adding the compound 1, 2-dichloroethane and phosphorus oxychloride into a three-neck round bottom flask, and then heating, refluxing and stirring the reaction mixture in a nitrogen atmosphere at 80 ℃ for 3-5h; after the TCI detection reaction was complete, the reaction mixture was cooled in an ice bath and triethylamine was slowly added; stirring for 5min, and slowly adding boron trifluoride diethyl etherate; then the reaction mixture is heated to room temperature and the reaction is continued for 2-5h; and (3) removing the redundant solvent under reduced pressure, and purifying the obtained crude product by a silica gel chromatographic column to obtain a red solid serving as a target fluorescent probe.
6. The method of preparing a fluorescent probe according to claim 5, wherein the molar ratio of triphosgene to pyrrole to N, N-diisopropylethylamine in the step (1) is 1-5.
7. The method of preparing a fluorescent probe according to claim 5, wherein the molar ratio of the compound 1 to phosphorus oxychloride, triethylamine and boron trifluoride etherate in the step (2) is 0.5-15.
8. A test strip for rapid nondestructive testing of food freshness loaded with the fluorescent probe of claim 3.
9. A method for quickly and nondestructively detecting the freshness of food, which is characterized in that the test paper according to claim 4 is placed in a closed dish, and fish or meat samples are placed in the dish.
10. The method of detecting according to claim 6, wherein: the temperature in the closed dish was-16 ℃, 0 ℃, 25 ℃.
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