CN110699082A - Preparation method and application of 2-aminobenzimidazole false template molecularly imprinted fluorescent sensing material - Google Patents
Preparation method and application of 2-aminobenzimidazole false template molecularly imprinted fluorescent sensing material Download PDFInfo
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Abstract
The invention discloses a preparation method of a 2-aminobenzimidazole false template molecularly imprinted fluorescent sensing material, which adopts 2-aminobenzimidazole with a structure similar to that of aminoimidazole nitrogen heterocyclic amine molecules (AIAs) as false template molecules, CdSe/ZnS quantum dots as fluorescent probes, 3-aminopropyltriethoxysilane as functional monomers, tetraethoxysilane as a cross-linking agent, ammonia water as a catalyst and absolute ethanol as a solvent, synthesizes a molecularly imprinted polymer on the surfaces of the quantum dots by using a sol-gel method, and detects the aminoimidazole nitrogen heterocyclic amine molecules (AIAs) by observing the fluorescence quenching condition of the CdSe/ZnS quantum dots. The detection method provided by the invention is simple and convenient to operate, low in cost, high in accuracy and repeatability, and capable of detecting the aminoimidazole nitrogen heterocyclic amine molecules (AIAs) in food with high selectivity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of optical sensing materials and molecular imprinting, and particularly relates to a preparation method and application of a 2-aminobenzimidazole dummy template molecular imprinting fluorescent sensing material.
Background
Heterocyclic Amines (HAAs) are Aromatic compounds having a polycyclic structure comprising carbon, nitrogen and hydrogen atoms, most of which contain more than two Aromatic rings, at least one N atom being present in the ring and the ring being externally linked to a-NH-group2They can be classified into aminoimidazole nitrogen-based heterocyclic amine molecules (AIAs) and aminocarboline-based heterocyclic amine molecules (ACS) according to the mechanism of their formation. AIAs is formed at the temperature of 100 ℃ and 300 ℃, is formed by precursor such as free amino acid, creatine, hexose and the like through complex Maillard reaction, mainly comprises IQ, IQx, MeIQ, MeIQx,4,8-DiMeIQx, PhIP and the like, and can be generated under common cooking conditions and has stronger pathogenic capability. When the heat treatment temperature is higher than 300 ℃, proteins and single amino acids are pyrolyzed to form ACS, which has pathogenicity lower than AIAs and mainly comprises AAC, MeAAC, Harman, Norharman, Trp-P-1, Glu-P-l and the like.
The heterocyclic amine has trace amount in food, mutagenic activity and potential carcinogenicity; it is not detectable in unheated food products, generally formed by heating, and its main cause depends on cooking temperature, heating conditions and heat treatment time. The detection methods commonly used at present mainly include the following types: detecting by High Performance Liquid Chromatography (HPLC) in combination with ultraviolet detector (UV), fluorescence detector (FLD), Diode Array Detector (DAD), etc.; liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS); gas Chromatography (GC) and gas chromatography-mass spectrometry (GC-MS); capillary Electrophoresis (CE); liquid chromatography-electrochemical method (LC-ECD); enzyme linked immunosorbent assay (ELISA).
The Molecular Imprinted Polymer (MIP) is designed by immunology, and is synthesized by forming a complex between a functional monomer and a template molecule through covalent or non-covalent interaction in the presence of the template molecule (Imprinted molecule) according to the principle of antigen-antibody specific binding, and then further stabilizing and crosslinking the complex by a crosslinking agent and a solvent (porogen). Removal of the template molecule results in the formation of recognition holes in the highly crosslinked polymer that match the shape, size, and spatial arrangement of the template molecule. The molecular engram polymer has high affinity and selectivity, is similar to a natural antibody, and can be used for replacing the antibody; the stability is strong and is obviously superior to natural biological molecules; the preparation of MIP is simple and convenient to operate, and has a much higher production speed compared with the production speed of antibodies; which is easily adapted to the specific analysis and the actual application of the sensor.
Heterocyclic amines have mutagenic activity and potential carcinogenicity, which can result in the production of food products during thermal processing and can be harmful to humans. At present, the detection of heterocyclic amine mostly depends on large-scale tandem mass spectrometry equipment, and the method has complex pretreatment on a sample and longer analysis time. In addition, as the heterocyclic amine has various standard varieties and high manufacturing cost, the requirements of the usage amount of template molecules in the synthesis process of the molecularly imprinted polymer can not be met; meanwhile, in order to simultaneously analyze and detect the aminoimidazole nitrogen heterocyclic amine molecules (AIAs) with strong pathogenicity, a dummy template molecule with a structure similar to that of the substance is selected for material synthesis, the method is fast in research and development, good in sensitivity, capable of detecting the aminoimidazole nitrogen heterocyclic amine molecules (AIAs) with high selectivity and high sensitivity, and provides a new idea for heterocyclic amine detection.
Disclosure of Invention
The invention aims to provide a CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material for detecting aminoimidazole nitrogen heterocyclic amine molecules (AIAs), and a preparation method and application thereof.
The invention is realized by the following technical scheme:
the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs) adopts 2-amino benzimidazole with a structure similar to that of the amino imidazole nitrogen heterocyclic amine molecules (AIAs) as a dummy template molecule, CdSe/ZnS quantum dots as a fluorescent probe, 3-aminopropyltriethoxysilane as a functional monomer, tetraethoxysilane as a cross-linking agent, ammonia water as a catalyst and absolute ethyl alcohol as a solvent, and a sol-gel method is used for synthesizing a molecularly imprinted polymer on the surface of the quantum dots. After the 2-aminobenzimidazole as a false template molecule of a polymer is eluted, a recognition hole and a binding site which are matched with the structure of the molecular imprinting polymer are reserved on the molecular imprinting polymer, and when amino imidazole nitrogen heterocyclic amine molecules (AIAs) which are similar to the false template structure are used for adsorption, the heterocyclic amine molecules can be close to the hole of the polymer and combined with the recognition site to a certain degree due to the similarity of the structures of the two molecules, so that the fluorescence intensity of quantum dots is influenced, and the heterocyclic amine can be used for detecting the heterocyclic amine.
The identification capability and the fluorescence response of the fluorescent sensing material are influenced by the following conditions: the proportion of the molecular engram polymer false template molecule, the functional monomer and the cross-linking agent, and the addition amount of CdSe/ZnS quantum dots. Therefore, the invention optimizes and screens the conditions to ensure that the detection effect of the prepared fluorescence sensing material reaches the best.
The preparation of the CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material for detecting the aminoimidazole nitrogen heterocyclic amine molecules (AIAs) comprises the following steps:
(1) preparing a CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material:
0.133g of dummy template molecule 2-aminobenzimidazole, 941 muL of functional monomer 3-Aminopropyltriethoxysilane (APTES), CdSe/ZnS quantum dots (oil-soluble, 8 muM in concentration), Tetraethoxysilane (TEOS) as a cross-linking agent and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethyl alcohol, and after 15min of nitrogen degassing, the mixture is stirred at 600rpm and subjected to light-shielding polymerization for 24 h. Wherein 915-1831 mu L of TEOS is added, and 60-150 mu L of CdSe/ZnS quantum dots are injected to obtain the CdSe/ZnS quantum dots-molecularly imprinted fluorescent sensing material; ammonia water is used as a catalyst. APTES is a chemical substance with molecular formula of H2NCH2CH2CH2Si(OC2H5)3Boiling point 217 ℃, relative density (ρ 25 ℃): 0.946, refractive index 1.420, light yellow liquid, toxic to inhalation. APTES is easy to hydrolyze, and ethanol is released to generate a corresponding silanol condensation compound. C-NH in the molecule2The in-bond amino group can be reacted with an acid, a carboxylic acid ester, an aldehyde, a ketone, a halogenated hydrocarbon, an amide, a nitrile, and the like.
(2) Removal of the dummy template molecule 2-aminobenzimidazole:
and (3) removing the pseudo template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
Preferably, in the step (1), the CdSe/ZnS quantum dots are oil-soluble, the concentration is 8 μ M, the optimal addition amount is 100 μ L, and the optimal addition amount of TEOS is 1372 μ L.
The invention also provides an application of the fluorescent sensing material, which is used for quantitative detection of aminoimidazole nitrogen heterocyclic amine molecules (AIAs) in a sample to be detected. The method comprises the following specific steps:
placing 1.0mg of the fluorescence sensing material in 3.0mL of absolute ethanol solution containing an aminoimidazole nitrogen heterocyclic amine standard substance (0.5-20.0 mu g/L), placing the solution in a fluorescence cuvette after adsorption reaction for 15-120 min, measuring the fluorescence intensity of the material under the excitation wavelength of 280nm, and quantifying the detection substance according to the calculation of a fluorescence quenching equation.
Preferably, the reaction time of the fluorescence sensing material for adsorbing standard aminoimidazole nitrogen heterocyclic amine molecules (AIAs) is 60min optimally.
The invention has the beneficial effects that:
according to the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting the amino imidazole nitrogen heterocyclic amine molecules (AIAs), 2-aminobenzimidazole similar to the amino imidazole nitrogen heterocyclic amine molecules (AIAs) in structure is used as a dummy template molecule for synthesizing polymers, and the heterocyclic amine molecules have mutagenic activity and carcinogenicity, so that safety problems are easily caused; secondly, the heterocyclic amine molecular standard is expensive, and a large amount of template molecules are needed when materials are synthesized; in addition, template molecules of the molecularly imprinted polymer can form high cross-linking in the preparation process, so that the template molecules are difficult to completely remove during elution, the leakage of the template molecules is caused, and the authenticity of the detection result of an actual sample is influenced, so that the pseudo template molecules are selected to replace aminoimidazole nitrogen heterocyclic amine molecules (AIAs) to be used as the template molecules for synthesizing the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material. After the pseudo template molecules are eluted, the amino imidazole nitrogen heterocyclic amine molecules (AIAs) are used for adsorption, and can be combined with specific recognition sites on molecularly imprinted polymers in materials, so that the fluorescence of CdSe/ZnS quantum dots is quenched, and therefore, the amino imidazole nitrogen heterocyclic amine molecules (AIAs) can be detected, the aim of quickly and quantitatively detecting the amino imidazole nitrogen heterocyclic amine molecules (AIAs) in food is fulfilled, and the detection limit is 0.25 mug/L.
Drawings
FIG. 1 is a flow chart of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material preparation.
FIG. 2 is an infrared spectrum of the pseudotemplate molecules 2-aminobenzimidazole (a), CdSe/ZnS @ MIP (b) and CdSe/ZnS @ NIP (c).
FIG. 3 is a scanning electron micrograph of CdSe/ZnS @ MIP (A) and CdSe/ZnS @ NIP (B).
FIG. 4 is a graph of the adsorption kinetics of CdSe/ZnS @ MIP and CdSe/ZnS @ NIP.
FIG. 5 is a graph of the fluorescence response of CdSe/ZnS @ MIP (A) and CdSe/ZnS @ NIP (B) for different concentrations of aminoimidazole nitrogen-based heterocyclic amine molecules (4, 8-DiMeIQx).
FIG. 6 is a schematic diagram showing the chemical structures of 2-aminobenzimidazole with IQ, MeIQ, 4,8-DiMeIQ, 7,8-DiMeIQ, TriMeIQ, PhIP, Harman, and Norharman.
FIG. 7 shows the selectivity of CdSe/ZnS @ MIP and CdSe/ZnS @ NIP for IQ, IQx, MeIQ, MeIQx,4,8-DiMeIQx,7,8-DiMeIQx, TriMeIQx, PhIP, Harman and Norharman.
Detailed Description
The present invention is further illustrated by the following examples.
The reagents used in the following examples were all commercially available, with CdSe/ZnS quantum dots being oil soluble and having a concentration of 8 μ M, a mass fraction of 2-aminobenzimidazole of 97%, a mass concentration of 3-Aminopropyltriethoxysilane (APTES) of 98%, and a mass concentration of Tetraethoxysilane (TEOS) of 98%.
Example 1
The invention provides a preparation method and application of a CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs), and the specific preparation route is as follows:
(1) preparation of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material of amino imidazole nitrogen heterocyclic amine molecules (AIAs): 0.133g of dummy template molecule 2-aminobenzimidazole, 941 muL of functional monomer 3-aminopropyl triethoxysilane (APTES), 100 muL of CdSe/ZnS quantum dots (oil-soluble, concentration is 8 muM), 1372 muL of crosslinking agent Tetraethoxysilane (TEOS) and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethyl alcohol, and after 15min of nitrogen degassing, the mixture is stirred at 600rpm and is kept away from the sun for polymerization for 24 h. Dummy template molecule 2-aminobenzimidazole: APTES: TEOS (molar ratio) is 1:4:6, and the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material (CdSe/ZnS @ MIP) is obtained;
(2) removal of the dummy template molecule 2-aminobenzimidazole: and (3) removing the template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
Example 2
The invention provides a preparation method and application of a CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs), and the specific preparation route is as follows:
(1) preparation of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material of amino imidazole nitrogen heterocyclic amine molecules (AIAs): 0.133g of dummy template molecule 2-aminobenzimidazole, 941 muL of functional monomer 3-Aminopropyltriethoxysilane (APTES), 100 muL of CdSe/ZnS quantum dots (oil-soluble, concentration of 8 muM), 915 muL of crosslinking agent Tetraethoxysilane (TEOS) and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethyl alcohol, and after 15min of nitrogen degassing, the mixture is stirred at 600rpm and is subjected to polymerization reaction for 24h in the dark. Dummy template molecule 2-aminobenzimidazole: APTES: TEOS (molar ratio) is 1:4:4, and the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material (CdSe/ZnS @ MIP) is obtained;
(2) removal of the dummy template molecule 2-aminobenzimidazole: and (3) removing the template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
Example 3
The invention provides a preparation method and application of a CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs), and the specific preparation route is as follows:
(1) preparation of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material of amino imidazole nitrogen heterocyclic amine molecules (AIAs): 0.133g of 2-aminobenzimidazole as a dummy template molecule, 941 muL of 3-Aminopropyltriethoxysilane (APTES) as a functional monomer, 100 muL of CdSe/ZnS quantum dots (oil-soluble and 8 muM in concentration), 1831 muL of Tetraethoxysilane (TEOS) as a crosslinking agent and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethanol, degassed with nitrogen for 15min, and then stirred at 600rpm in the dark for polymerization for 24 h. Dummy template molecule 2-aminobenzimidazole: APTES: TEOS (molar ratio) is 1:4:8, and the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material (CdSe/ZnS @ MIP) is obtained;
(2) removal of the dummy template molecule 2-aminobenzimidazole: and (3) removing the template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
According to the method, 2-aminobenzimidazole without adding a dummy template molecule is used for preparing the corresponding non-imprinted fluorescent sensing material (CdSe/ZnS @ NIP) of the invention respectively.
The molar ratio optimization of the pseudo template molecules, functional monomers and cross-linking agents in the polymerization system is shown in table 1. The other addition amounts and reaction conditions in the immobilization system were changed to 1:4:4, 1:4:6, and 1:4:8, respectively, and the fluorescence responses of the synthesized imprinted and non-imprinted materials were measured (F)0/F) to obtain the blotting factor (IF). From the table, when the pseudo template molecule 2-aminobenzimidazole: APTES: the imprinting factor IF is maximal at a ratio of 1:4:6 for TEOS (molar ratio), 3.24. When the molar ratio of the three is 1:4:4, 0.133g of 2-aminobenzimidazole, 941 muL of 3-Aminopropyltriethoxysilane (APTES) and 915 muL of Tetraethoxysilane (TEOS) are added into the dummy template molecule; when the molar ratio of the three is 1:4:6, 0.133g of 2-aminobenzimidazole, 941 muL of 3-Aminopropyltriethoxysilane (APTES) and 1372 muL of Tetraethoxysilane (TEOS) are added into the dummy template molecule; when the molar ratio of the three is 1:4:8, 0.133g of 3-Aminopropyltriethoxysilane (APTES)941 muL and tetraethylOxoxysilane (TEOS) was added at 1831. mu.L.
TABLE 1 false template molecule, functional monomer and crosslinker molar ratio optimization
Example 4
A preparation method and application of a CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs) are disclosed, wherein the specific preparation route is as follows:
(1) preparation of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material of amino imidazole nitrogen heterocyclic amine molecules (AIAs): 0.133g of dummy template molecule 2-aminobenzimidazole, 941 muL of functional monomer 3-aminopropyl triethoxysilane (APTES), 60 muL of CdSe/ZnS quantum dots (oil-soluble, concentration is 8 muM), 1372 muL of crosslinking agent Tetraethoxysilane (TEOS) and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethyl alcohol, and after 15min of nitrogen degassing, the mixture is stirred at 600rpm and is kept away from the sun for polymerization for 24 h. Dummy template molecule 2-aminobenzimidazole: APTES: TEOS (molar ratio) is 1:4:6, and the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material (CdSe/ZnS @ MIP) is obtained;
(2) removal of the dummy template molecule 2-aminobenzimidazole: and (3) removing the template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
Example 5
A preparation method and application of a CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material for detecting amino imidazole nitrogen heterocyclic amine molecules (AIAs) are disclosed, wherein the specific preparation route is as follows:
(1) preparation of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material of amino imidazole nitrogen heterocyclic amine molecules (AIAs): 0.133g of dummy template molecule 2-aminobenzimidazole, 941 muL of functional monomer 3-aminopropyl triethoxysilane (APTES), 150 muL of CdSe/ZnS quantum dots (oil-soluble, concentration is 8 muM), 1372 muL of crosslinking agent Tetraethoxysilane (TEOS) and 100 muL of ammonia water are dissolved in 10.0mL of absolute ethyl alcohol, and after 15min of nitrogen degassing, the mixture is stirred at 600rpm and is kept away from the sun for polymerization for 24 h. Dummy template molecule 2-aminobenzimidazole: APTES: TEOS (molar ratio) is 1:4:6, and the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material (CdSe/ZnS @ MIP) is obtained;
(2) removal of the dummy template molecule 2-aminobenzimidazole: and (3) removing the template molecules by using 50% (v/v) ethanol-ultrapure water solution through ultrasonic-assisted elution until no 2-aminobenzimidazole can be detected, and drying in vacuum at 60 ℃.
The addition amount of CdSe/ZnS quantum dots in the polymerization system was optimized as shown in Table 2. The fluorescence response of the synthesized imprinted and non-imprinted materials is measured by changing the addition of CdSe/ZnS quantum dots while keeping the other addition amounts and reaction conditions in the system unchanged (F)0/F) to obtain the blotting factor (IF). As shown in the table, when the amount of CdSe/ZnS quantum dots added was 100. mu.L, the imprinting factor IF was the largest and was 6.15.
TABLE 2 optimization of CdSe/ZnS Quantum dot addition
FIG. 1 shows a preparation process of a CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material for detecting aminoimidazole nitrogen heterocyclic amine molecules (AIAs).
In order to analyze the structure of the fluorescent sensing material prepared by the invention, Fourier infrared spectrum characterization and scanning electron microscope characterization are respectively carried out on the material.
FIG. 2 is a Fourier infrared spectrum of the pseudotemplate molecules 2-aminobenzimidazole, CdSe/ZnS @ MIP and CdSe/ZnS @ NIP. In the graph (2a), 1661cm-1And 1562cm-1The absorption peak is the skeleton stretching vibration of benzene ring, 731cm-1The absorption peak of (A) proves that the benzene ring has ortho-disubstituted, 1452cm-1Is the stretching vibration peak of the imidazole ring; 1057cm in the graph (2c)-1The absorption peak of (a) is the asymmetric expansion and contraction of Si-O-Si, 791cm-1And 457cm-1The two characteristic peaks are vibration peaks of Si-O, and the characteristic absorption peaks indicate the formation of a Si-O-Si structure; 1641cm-1The absorption peak of (a) is the oscillation peak of the carbonyl group in the amide bond, 1545cm-1The peak of C-N stretching vibration and the peak of N-H bending vibration of amido bond are shown. Meanwhile, the characteristic peaks of b and c in FIG. 2 are substantially consistent, indicating that the structural compositions of CdSe/ZnS @ MIP and CdSe/ZnS @ NIP are similar.
The appearance of CdSe/ZnS @ MIP and CdSe/ZnS @ NIP was characterized and observed by scanning electron microscope, respectively, and the results are shown in FIG. 3. In the figure, A is CdSe/ZnS @ MIP, and B is CdSe/ZnS @ NIP, and the surface of the prepared material is relatively smooth and is microspherical.
In order to evaluate the adsorption performance of the fluorescent sensing material prepared by the invention, an adsorption kinetics experiment, a balance adsorption combination experiment and an adsorption selectivity experiment are respectively carried out on the material.
FIG. 4 shows the adsorption kinetics experiment result of CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material. CdSe/ZnS @ MIP p-aminoimidazole nitrogen heterocyclic amine standard (4,8-DiMeIQx,10.0 mu g L)-1) The fluorescence response of the fluorescent material is obviously higher than that of CdSe/ZnS @ NIP; at 60min, the imprinted polymer reached adsorption equilibrium, while the non-imprinted polymer took about 85min to equilibrate. The imprinted polymer has specific holes and recognition sites, so that the imprinted polymer has higher adsorption capacity and stronger fluorescence response to a target object, the mass transfer rate is accelerated, and the adsorption balance can be achieved in a shorter time.
The equilibrium adsorption binding experiment of CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material is carried out by adding aminoimidazole nitrogen heterocyclic amine standard (4,8-DiMeIQx,0.5-20.0 mu g L) with different concentrations into 1.0mg material-1) And measuring the fluorescence intensity of the material after adsorption and combination. FIGS. 5A and B show equilibrium adsorption binding curves and standard curves for materials CdSe/ZnS @ MIP and CdSe/ZnS @ NIP, respectively. According to the graph, as the concentration of the target substance is increased, the fluorescence of the CdSe/ZnS quantum dots is gradually quenched, and the fluorescence intensity of imprinted and non-imprinted polymers is gradually reduced. In addition, CdSe/ZnS @ MIP is used as a fluorescent sensing material for repeatedly adsorbing and measuring quintic aminoimidazole nitrogen heterocyclic amine standard substance (4,8-DiMeIQx,10 mu g L)-1) Relative Standard Deviation (RSD), i.e. precision of 2.41%. For the non-imprinted polymer CdSe/ZnS @ NIP, the fluorescence quenching degree is far less than that of the imprinted polymerThe reason is that a hole amorphous structure formed by adding no false template molecule 2-aminobenzimidazole during synthesis of the non-imprinted polymer cannot be correspondingly adsorbed and combined with a target molecule, so that the adsorption amount of CdSe/ZnS @ NIP is reduced, the fluorescence quenching degree of CdSe/ZnS quantum dots is correspondingly reduced, and the linear result is poor. From the fluorescence quenching equation, F0/F＝KSVC +1, according to the above equation, in the present invention, F0The fluorescence intensity of the molecularly imprinted polymer when the aminoimidazole nitrogen heterocyclic amine molecule (4,8-DiMeIQx) is not adsorbed is shown, and F shows the fluorescence intensity of the polymer after the heterocyclic amine molecule is adsorbed and combined; kSVThe fluorescence quenching constant is expressed by using an Imprinting Factor (IF) of the molecularly imprinted polymer, namely, the molecularly imprinted polymer is used for evaluating the adsorption performance of the polymer; c represents the concentration of the target aminoimidazole nitrogen heterocyclic amine standard (4,8-DiMeIQx,0.5-20.0 mu g L-1). When the concentration of the aminoimidazole nitrogen heterocyclic amine molecules is 0.5-20.0 mu g L-1The fluorescence quenching equation of the imprinted polymer CdSe/ZnS @ MIP is F0and/F is 0.0443C +1.0613, and the correlation coefficient is 0.9968.
After the dummy template molecules are eluted, in order to evaluate whether holes in the fluorescence sensing material can selectively identify and adsorb aminoimidazole nitrogen heterocyclic amine molecules (AIAs), several AIAs (IQ, IQx, MeIQ, 4,8-DiMeIQx,7,8-DiMeIQx, TriMeIQx, PhIP) and two aminocarboline heterocyclic amine molecules (ACS) (Harman, Norharman) are selected, and the fluorescence change conditions of CdSe/ZnS @ MIP and CdSe/ZnS @ NIP after the heterocyclic amine standard substance is adsorbed are respectively measured to examine the selectivity of the polymer. The structures of the pseudo-template molecule 2-aminobenzimidazole with several heterocyclic amine molecules adsorbed are shown in figure 6. FIG. 7 shows the result of adsorption selectivity experiment of the material, which shows that the selectivity of the imprinted polymer to the several aminoimidazole nitrogen heterocyclic amine molecules (AIAs) is basically similar because the structures of IQ, MeIQx,4,8-DiMeIQx, etc. are similar to the dummy template molecules; in contrast, Harman and Norharman have structures that are less similar to the pseudotemplate molecules and therefore less selective for the polymer. In conclusion, it is shown that CdSe/ZnS @ MIP has good selectivity for aminoimidazole nitrogen-based heterocyclic amine molecules (AIAs) and poor selectivity for aminocarboline-based heterocyclic amine molecules (ACS).
The CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material is used for detecting actual food samples, and the dried beef floss and the baked fish fillet are selected as the actual samples to carry out a labeling recovery experiment. The specific operation steps are as follows: respectively placing 1.0g of food sample in a 50mL centrifuge tube, adding 4,8-DiMeIQx as aminoimidazole nitrogen heterocyclic amine standard substance to make the standard concentration of 5, 10, 20 mu g L-1Vortex at 3000r/min for 5min and then let stand overnight. Then adding 5.0mL of ethyl acetate and 0.5mL of ammonia water, whirling at 3000r/min for 5min, centrifuging at 10000r/min for 5min, and taking the supernatant for later use; the mixture was extracted with 3.0mL of ethyl acetate again, and the two supernatants were combined and the solvent was blown dry with nitrogen at 50 ℃. After 3.0mL of absolute ethyl alcohol is used for redissolving, 1.0mg of CdSe/ZnS @ MIP is added for oscillation and adsorption for 60min, and the fluorescence quenching effect is measured. As shown in Table 3, the CdSe/ZnS @ MIP samples prepared according to the present invention were used to detect the actual samples, and the concentrations of the added heterocyclic amine standard (4,8-DiMeIQx) were 5, 10, and 20. mu.g kg-1The recovery rate of the sample obtained by detection is 82.8-110.8%, and the Relative Standard Deviation (RSD) is 1.4-5.5%.
TABLE 3 detection of heterocyclic amine spiking recovery rate in practical samples by CdSe/ZnS quantum dot-molecularly imprinted material
Although the invention has been described with reference to specific embodiments and examples shown in the accompanying drawings, it is not intended to limit the scope of the invention, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the scope of the invention.
Claims (6)
1. A preparation method of a 2-aminobenzimidazole false template molecularly imprinted fluorescent sensing material is characterized by comprising the following steps:
(1) preparing a CdSe/ZnS quantum dot-molecular imprinting fluorescent sensing material:
dissolving CdSe/ZnS quantum dots, a dummy template molecule 2-aminobenzimidazole similar to an amino imidazole nitrogen heterocyclic amine molecular structure, a functional monomer 3-aminopropyl triethoxysilane, a cross-linking agent tetraethoxy silane and ammonia water in absolute ethyl alcohol, degassing by nitrogen, and carrying out polymerization reaction at room temperature in a dark place under stirring to obtain the CdSe/ZnS quantum dot-molecularly imprinted fluorescent sensing material;
(2) removal of the dummy template molecule 2-aminobenzimidazole:
removing the 2-aminobenzimidazole of the dummy template molecule by using ethanol-ultrapure water solution with ultrasonic assistance until the dummy template molecule can not be detected, and drying in vacuum at 60 ℃.
2. The preparation method according to claim 1, wherein the CdSe/ZnS quantum dots in step (1) are oil-soluble, have a concentration of 8 μ M and an addition amount of 60 to 150 μ L, the dummy template molecule 2-aminobenzimidazole is added in an amount of 0.133g, the 3-aminopropyltriethoxysilane is added in an amount of 941 μ L, the tetraethoxysilane is added in an amount of 915 to 1831 μ L, the ammonia is used in an amount of 100 μ L, the absolute ethanol is added in an amount of 10mL, the nitrogen is degassed for 15min, the stirring rate is 600rpm, and the polymerization reaction time is 24 h.
3. The production method according to claim 1, wherein the concentration of the ethanol-ultrapure water of the elution solution in the step (2) is 50% (v/v).
4. The preparation method according to claim 1, wherein step (1) tetraethoxysilane is added at 1372 μ L and CdSe/ZnS quantum dots are added at 100 μ L.
5. The application of the 2-aminobenzimidazole false template molecularly imprinted fluorescent sensing material prepared by the preparation method of any one of claims 1 to 4 in the field of detection of aminoimidazole nitrogen heterocyclic amine molecules.
6. The application of the 2-aminobenzimidazole pseudo-template molecularly imprinted fluorescent sensing material in the field of detection of aminoimidazole nitrogen heterocyclic amine molecules, which is disclosed by claim 5, is characterized in that: the specific method comprises the steps of placing 1.0mg of the fluorescent sensing material in 3.0mL of anhydrous ethanol solution containing the aminoimidazole nitrogen heterocyclic amine molecular standard, placing the solution in a fluorescent cuvette after adsorption reaction for 15-120 min, measuring the fluorescence intensity of the material under the excitation wavelength of 280nm, and performing qualitative and/or semi-quantitative determination on the detected substance according to the fluorescence quenching condition and the calculation of a fluorescence quenching equation.
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