CN116031036B - Fluorescent peptide-loaded magnetic nanomaterial and preparation method and application thereof - Google Patents
Fluorescent peptide-loaded magnetic nanomaterial and preparation method and application thereof Download PDFInfo
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- CN116031036B CN116031036B CN202211669017.7A CN202211669017A CN116031036B CN 116031036 B CN116031036 B CN 116031036B CN 202211669017 A CN202211669017 A CN 202211669017A CN 116031036 B CN116031036 B CN 116031036B
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- 230000005291 magnetic effect Effects 0.000 title claims abstract description 96
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 90
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims abstract description 15
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 14
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- RRUYWEMUWIRRNB-LURJTMIESA-N (2s)-6-amino-2-[carboxy(methyl)amino]hexanoic acid Chemical compound OC(=O)N(C)[C@H](C(O)=O)CCCCN RRUYWEMUWIRRNB-LURJTMIESA-N 0.000 description 2
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Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application provides a magnetic nano material loaded with fluorescent peptide, a preparation method and application thereof, and relates to the field of food safety research, in particular to a magnetic nano material loaded with fluorescent peptide, which is prepared by mixing dipeptide with zinc ion to obtain fluorescent peptide and grafting the fluorescent peptide onto a magnetic carrier; wherein the ratio of the amount of dipeptide to zinc ion is 4:1-1:4. By combining the fluorescent peptide with the magnetic nanomaterial, the precursor of the advanced glycosylation end product can be quickly combined and the content of the precursor can be indicated; the precursor material can be easily removed under the application of an externally applied magnetic field, thereby blocking the formation of advanced glycosylation end products. The magnetic nano material loaded with fluorescent peptide has the advantages of easily obtained raw materials, biological safety, solving the difficult problem that non-fluorescent target objects need to be analyzed by a large instrument in a complex and fussy way, greatly improving the efficiency of analysis and detection, and being applicable to the food industry.
Description
Technical Field
The application belongs to the field of food safety research, and particularly relates to a fluorescent peptide-loaded magnetic nanomaterial as well as a preparation method and application thereof.
Background
Advanced glycation end products (Advanced glycation end products, AGE) are a hazardous substance for promoting cell death, are closely related to various chronic diseases such as nephropathy, diabetes, alzheimer disease, atherosclerosis, tumor and the like and complications thereof, are important sources of exogenous advanced glycation end products in foods, and the reduction of the advanced glycation end products in foods can effectively reduce the threat of the advanced glycation end products to public health. Pyrroline, carboxymethyllysine, carboxyethyllysine are typical end products of advanced glycosylation, often as a reference indicator of the extent of glycosylation reaction. In food systems, inhibition of the formation of advanced glycation end products is mainly achieved by modifying the food processing mode and adding inhibitors.
Inhibitors generally block the formation of advanced glycation end products by capturing precursor substances of advanced glycation end products such as dicarbonyl compounds like 3-deoxyglucosone, glyoxal, methylglyoxal, etc. to form new addition byproducts, thereby reducing the chance of human exposure to advanced glycation end products. At present, natural compounds with antioxidant property, such as plant polyphenol and the like, are mainly used as inhibitors for capturing precursor substances. Chinese patent CN102883733B discloses an AGE formation inhibitor which contains an extract of cherokee rose (preferably flowers or leaves) and/or a treated product thereof as an active ingredient and can effectively inhibit AGE formation. However, extracts such as phenols are easily degraded under heating conditions, thereby being unfavorable for capturing AGEs precursor substances; in addition, the addition by-products and unreacted inhibitors remain in the food system and can cause undesirable off-flavors and even toxic effects.
The precursor species and the end-labeled advanced glycosylation products have non-fluorescent properties and require further quantitative analysis by large-scale instruments such as liquid chromatography-mass spectrometry. Chinese patent CN103070400B discloses the use of lotus procyanidins as an inhibitor of advanced glycosylation end product formation, which is a pharmaceutical composition using lotus procyanidins as main active ingredients, wherein the weight percentages of the extracts of various natural active ingredients are as follows: 70-80% of lotus procyanidine, 10-20% of synergistic agent such as VE, VC, gallocatechin gallate (EGCG) and cysteine, and 5-10% of other natural extracts such as folium Nelumbinis extract and folium Ginkgo extract; the method adopts high performance liquid chromatography to detect the clearance effect of lotus procyanidine and catechin as main structural unit on methylglyoxal, which is an important intermediate in the formation process of advanced glycosylation end products, and uses high performance liquid multistage mass spectrometry to identify 4 kinds of catechin and methylglyoxal adducts and 9 kinds of lotus procyanidine and methylglyoxal adducts. The results prove that the lotus procyanidine has good inhibition effect on the formation of advanced glycosylation end products in the simulated physiological environment and the simulated food system. The analysis efficiency is inevitably limited by expensive instrumentation, complicated sample pretreatment, lengthy time consumption, and manipulation by specialized personnel. The quantum dot, the carbon dot, the organic dye, the rare earth up-conversion nano material and the like can be used as the current common optical response element to prepare the sensing material, so that the purpose of rapid and sensitive quantitative analysis can be achieved, the detection analysis efficiency is greatly improved, and the application of the sensing material in a food system is still limited due to toxic effects.
There is a need for a more safe, faster and sensitive quantitative assay that can effectively detect precursor substances and inhibit the formation of labeled advanced glycation end products.
Disclosure of Invention
Aiming at the problems existing in the prior art, the application provides a fluorescent peptide-loaded magnetic nanomaterial, a preparation method and application thereof, wherein the fluorescent peptide comprises red fluorescent peptide and/or blue fluorescent peptide, and the fluorescent peptide and the magnetic nanomaterial are combined to quickly combine and indicate the content of a precursor substance of a late glycosylation end product, so that the precursor substance can be easily removed under the application of an external magnetic field, and the generation of the late glycosylation end product is blocked.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
the application provides a fluorescent peptide-loaded magnetic nanomaterial, which is prepared by mixing dipeptide with zinc ions to obtain fluorescent peptide and grafting the fluorescent peptide onto a magnetic carrier.
Specifically, grafting refers to the combination of branched chains or functional side groups with other molecules through chemical bonds, and the magnetic carrier passes throughThe hydrolysis realizes the coating of the silicon layer and further carries out the modification of the outer surface amino, and the carboxyl of the fluorescent peptide is stably connected with the exposed amino on the outer surface of the magnetic carrier material through the amide bond.
Preferably, the ratio of the amount of dipeptide to zinc ion species is 4:1 to 1:4.
Further preferably, the ratio of the amount of dipeptide to zinc ion species is from 2:1 to 1:2.
Most preferably, the ratio of the amount of dipeptide to zinc ion species is 1:2.
Preferably, the dipeptide is α -alanine-histidine, β -alanine-histidine, phenylalanine-histidine or histidine-histidine.
Further preferably, the dipeptide is β -alanine-histidine.
Preferably, the magnetic carrier is superparamagnetic gamma-Fe 2 O 3 Superparamagnetic alpha-Fe 2 O 3 Superparamagnetic Fe 3 O 4 Superparamagnetic CoFe 2 O 4 Superparamagnetic NiFe 2 O 4 At least one of them.
Further preferably, the magnetic carrier is superparamagnetic gamma-Fe 2 O 3 。
Preferably, the fluorescent peptide has a fluorescence emission wavelength of 600-700nm.
Further preferably, the fluorescent peptide has a fluorescence emission wavelength of 600-650nm.
Most preferably, the fluorescent peptide has a fluorescence emission wavelength of 610nm.
Preferably, the particle size of the fluorescent peptide is 2-14nm; the particle size of the magnetic carrier is 3-15nm, and the particle size of the fluorescent peptide-loaded magnetic nanomaterial is 10-60nm.
The fluorescent peptide comprises red fluorescent peptide or blue fluorescent peptide, and the magnetic nanomaterial loaded with the fluorescent peptide comprises a magnetic nanomaterial loaded with the red fluorescent peptide or a magnetic nanomaterial loaded with the blue fluorescent peptide.
Further preferably, the particle size of the red fluorescent peptide is 8.26nm, and the average particle size of the blue fluorescent peptide is 4.23nm; the particle size of the magnetic carrier is 8.42nm; the particle size of the magnetic nano material loaded with the red fluorescent peptide is 45.41nm, and the particle size of the magnetic nano material loaded with the blue fluorescent peptide is 40.73nm.
Preferably, the saturation magnetization of the magnetic carrier is 35-85emu g -1 The method comprises the steps of carrying out a first treatment on the surface of the The saturation magnetization intensity of the magnetic nano material loaded with the fluorescent peptide is 35-60emu g -1 。
Further preferably, the saturation magnetization of the magnetic carrier is 74.22emu g -1 The method comprises the steps of carrying out a first treatment on the surface of the The saturation magnetization intensity of the magnetic nano material loaded with the red fluorescent peptide is 55.78emu g -1 The method comprises the steps of carrying out a first treatment on the surface of the The saturation magnetization intensity of the magnetic nano material loaded with the blue fluorescent peptide is 57.51emu g -1 。
The application also provides a preparation method of the fluorescent peptide-loaded magnetic nanomaterial, which comprises the following steps:
s1, preparing fluorescent peptide: mixing dipeptide with zinc ions, heating, centrifuging, discarding supernatant, drying precipitate, and sieving to obtain fluorescent peptide;
s2, preparing a superparamagnetic carrier by a coprecipitation method;
s3, preparing a silicon layer stable superparamagnetism carrier: performing ultrasonic treatment on the superparamagnetic carrier obtained in the step S2, then mixing the superparamagnetic carrier with an organic solvent, sealing, heating in a water bath for overnight, washing, drying, mixing with toluene and 3-aminopropyl triethoxysilane, and heating in the water bath for overnight to obtain a silicon layer stable superparamagnetic carrier;
and S4, grafting the fluorescent peptide obtained in the step S1 onto the silicon layer stable superparamagnetic carrier obtained in the step S3 to obtain the fluorescent peptide-loaded magnetic nanomaterial.
Specifically, the coprecipitation method in step S2 is to disperse ferrous chloride and ferric chloride in an acidic aqueous solution, and then add ammonia water to stir and magnetically separate to obtain the magnetic material.
Preferably, the organic solvent in step S3 includes at least one of cetyltrimethylammonium bromide, ammonia water, and cyclohexane in which tetraethoxysilane is dissolved.
Further preferably, the organic solvent described in step S3 includes cetyltrimethylammonium bromide, aqueous ammonia, and cyclohexane dissolving tetraethoxysilane.
Preferably, the amount of the fluorescent peptide used in step S4 is 5-30mg.
It is further preferred that the amount of the fluorescent peptide used in step S4 is 20-30mg.
Most preferably, the amount of fluorescent peptide used in step S4 is 20mg.
The application also provides an application of the fluorescent peptide-loaded magnetic nanomaterial or the fluorescent peptide-loaded magnetic nanomaterial prepared by the preparation method in preparation of products for inhibiting generation of exogenous advanced glycosylation end products.
Compared with the prior art, the application has the following beneficial effects:
(1) The fluorescent peptide and the magnetic nanomaterial thereof have good fluorescent characteristics, can provide the precursor substance site for identifying and combining the advanced glycosylation end product, effectively capture the precursor substance of the advanced glycosylation end product, sensitively indicate the content of the precursor substance by fluorescent signals, avoid the possibility of autofluorescence interference signals in a complex matrix, and improve the accuracy of visual detection.
(2) The fluorescent peptide and the magnetic nano material thereof have the advantages of easily obtained raw materials, biological safety and application to the food industry.
(3) The fluorescent peptide and the magnetic nano material thereof solve the difficult problem that non-fluorescent targets need to be analyzed by a large instrument in a complex and fussy way, and greatly improve the efficiency of analysis and detection.
(4) The fluorescent peptide and the magnetic nano material of the application can adsorb precursor substances rapidly (within 10 min), and the adsorption capacity is up to 80.12mg g -1 The adsorption of the catalyst can be fast and high-capacity, so that the catalyst is convenient to remove fast and the residues of byproducts are avoided.
(5) The fluorescent peptide and the magnetic nano material thereof integrate detection and removal, and can effectively control the generation of harmful substances in the food processing process.
Drawings
FIG. 1 is a graph of fluorescence emission spectra of prepared peptides derived from different linear dipeptides; wherein A is the spectrum diagram of beta-alanine-histidine and zinc ion, B is the spectrum diagram of phenylalanine-histidine and zinc ion, C is the spectrum diagram of histidine-histidine and zinc ion, D is the spectrum diagram of alpha-alanine-histidine and zinc ion.
FIG. 2 is a graph of the optimal excitation and optimal emission spectra of the prepared beta-alanine-histidine and divalent zinc ions at different molar ratios to prepare peptide spots; wherein, the mole ratio of A is 4:1, the mole ratio of B is 2:1, the mole ratio of C is 1:1, the mole ratio of D is 1:1.5, the mole ratio of E is 1:2, and the mole ratio of F is 1:4; the expression represents the Excitation spectrum and the Emission spectrum.
FIG. 3 is a topography of the material prepared in the examples; wherein A is a morphology diagram of blue fluorescent peptide, B is a morphology diagram of red fluorescent peptide, and C is superparamagnetic gamma-Fe 2 O 3 And D is a morphology diagram of the blue fluorescent peptide grafted magnetic nanomaterial, and E is a morphology diagram of the red fluorescent peptide grafted magnetic nanomaterial.
FIG. 4 is a graph showing the particle size distribution of the materials prepared in the examples; wherein A is the particle size distribution diagram of blue fluorescent peptide, B is the particle size distribution diagram of red fluorescent peptide, and C is superparamagnetic gamma-Fe 2 O 3 The particle size distribution diagram of the nano material is that of the blue fluorescent peptide grafted magnetic nano material, and E is that of the red fluorescent peptide grafted magnetic nano material.
FIG. 5 is a graph of saturation susceptibility; wherein A is prepared superparamagnetic gamma-Fe 2 O 3 Nanomaterial and blue fluorescenceA saturation susceptibility map of the peptide grafted magnetic nanomaterial; b is prepared superparamagnetic gamma-Fe 2 O 3 Saturation susceptibility diagrams of nanomaterials and red fluorescent peptide grafted magnetic nanomaterials.
FIG. 6 is a graph of the linear relationship between fluorescent signal and precursor concentration for fluorescent peptide grafted magnetic nanomaterials prepared in the test example; wherein, the graph A is the fluorescence response spectrum of the blue fluorescent peptide grafted magnetic nano material to precursors with different concentrations, the graph B is the linear relation of the concentration of the precursors to the fluorescence intensity change of the blue fluorescent peptide grafted magnetic nano material according to the Stern-Volmer formula, the graph C is the fluorescence response spectrum of the red fluorescent peptide grafted magnetic nano material to the precursors with different concentrations, and the graph D is the linear relation of the concentration of the precursors to the fluorescence intensity change of the red fluorescent peptide grafted magnetic nano material according to the Stern-Volmer formula.
FIG. 7 is a graph of the optical response of fluorescent peptide grafted magnetic nanomaterials to precursors in a test example; wherein A is an optical response diagram of the blue fluorescent peptide grafted magnetic nano material to different precursors, and B is an optical response diagram of the red fluorescent peptide grafted magnetic nano material to different precursors.
FIG. 8 is a graph of adsorption capacity of fluorescent peptide grafted magnetic nanomaterial in a test example.
FIG. 9 is a graph showing the inhibition effect of magnetic nanomaterial, blue fluorescent peptide-grafted magnetic nanomaterial, and red fluorescent peptide-grafted magnetic nanomaterial on end-stage saccharification products in the test examples.
Detailed Description
The following description of the present application is provided by way of specific examples to facilitate understanding and grasping of the technical solution of the present application, but the present application is not limited thereto, and the described examples are only some, but not all, examples of the present application.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
Example 1
1. Preparation of fluorescent peptide:
dissolving the beta-alanine-histidine and the zinc chloride in a mixed solution with the volume ratio of methanol to water being 9:1 in a ratio of 1:2, heating in a water bath at 85 ℃, centrifuging at 10000rpm for 10min after water in an organic solvent is evaporated, discarding the supernatant, drying the precipitate, grinding and sieving to obtain the red fluorescent peptide (with the particle size of 8.26+/-1.7 nm).
2. Superparamagnetic gamma-Fe 2 O 3 Preparation of the nanomaterial:
dispersing 0.54M ferrous chloride and 0.54M ferric chloride in 36mL acidic aqueous solution with pH of 0.06, rapidly adding 11.4mL ammonia water under intense stirring, continuously stirring for reaction for 30min, washing twice with deionized water, separating by magnetic force, adding 4.9mL 2M nitric acid solution into the obtained magnetic material, stirring for 30min, separating by magnetic force to obtain black magnetic material, injecting 12mL 1.5M ferric nitrate solution, refluxing at 150deg.C for 30min, washing with deionized water and acetone, ultrasonic treating with 30mL nitric acid solution (pH=2) for 10min to redisperse nano particles, centrifuging at 10000rpm for 15min, dispersing the collected magnetic solid in 0.3M citric acid aqueous solution, refluxing at 150deg.C for 30min, washing with acetone, oven drying, and grinding to obtain super paramagnetic gamma-Fe 2 O 3 Nanometer material (particle size of 8.42+ -2.29 nm, saturation magnetization of 74.22emu g) -1 )。
3. Silicon layer stabilized superparamagnetic gamma-Fe 2 O 3 Preparing a nano material:
60mg of superparamagnetic gamma-Fe 2 O 3 Dispersing the nano material in 36mL deionized water, performing ultrasonic treatment for 10min, and adding 45mL
14.1mmol of cetyltrimethylammonium bromide in water, 65. Mu.L of aqueous ammonia solution, 12mL of cyclohexane in which 1.5mmol of tetraethoxysilane was dissolved, followed by sealing and reaction in a water bath at 65℃overnight with continuous stirring. Washing and drying, dispersing 60mg in 20mL toluene, dripping 260 μl 3-aminopropyl triethoxysilane under stirring, and reacting overnight in water bath at 60deg.C under continuous stirring to obtain silicon layer stable superparamagnetic gamma-Fe 2 O 3 A nanomaterial.
4. Fluorescent peptide grafted magnetic nanomaterial:
(1) Dispersing 20mg of red fluorescent peptide in 10mL of 0.1M PBS, then adding 20mg of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and incubating at room temperature for 30min to obtain solution 1;
(2) Simultaneously, 10mg of silicon layer is stabilized to superparamagnetic gamma-Fe 2 O 3 Adding 10mL of PBS into the nano material, and then treating the nano material for 10min under ultrasonic conditions to obtain a solution 2;
(3) Solution 1 and solution 2 above were thoroughly mixed and incubated for 12h. Finally, washing twice with PBS and drying at 50 ℃, grinding and sieving to obtain the red fluorescent peptide grafted magnetic nanomaterial (particle size 45.41nm, saturation magnetization 55.78emu g -1 )。
Example 2
1. Preparation of fluorescent peptide:
dissolving the substances of beta-alanine-histidine and zinc chloride in a mixed solution with the volume ratio of methanol to water being 9:1 in a ratio of 2:1, heating in a water bath at 85 ℃, centrifuging at 10000rpm for 10min after water in an organic solvent is evaporated, discarding the supernatant, drying the precipitate, grinding and sieving to obtain the blue fluorescent peptide (with the particle size of 4.23+/-0.92 nm).
2. Superparamagnetic gamma-Fe 2 O 3 Preparation of the nanomaterial:
dispersing 0.54M ferrous chloride and 0.54M ferric chloride in 36mL acidic water with pH of 0.06In the solution, 11.4mL of ammonia water is rapidly added under the condition of intense stirring, the stirring reaction is continued for 30min, the washing is carried out twice by deionized water, the magnetic separation is carried out by magnetic separation, 4.9mL of 2M nitric acid solution is added into the obtained magnetic material, the black magnetic material is obtained by magnetic separation after stirring for 30min, 12mL of 1.5M ferric nitrate solution is injected, the reflux treatment is carried out for 30min at 150 ℃, the washing is carried out by deionized water and acetone, the ultrasonic treatment is carried out for 10min by 30mL of nitric acid solution (pH=2) to redisperse nano particles, the centrifugation is carried out for 15min at 10000rpm, the collected magnetic solid is dispersed in 0.3M citric acid aqueous solution, the reflux is carried out for 30min at 150 ℃, the washing is carried out by acetone, the super paramagnetic gamma-Fe is obtained after the drying and grinding 2 O 3 Nanometer material (particle size of 8.42+ -2.29 nm, saturation magnetization of 74.22emu g) -1 )。
3. Silicon layer stabilized superparamagnetic gamma-Fe 2 O 3 Preparing a nano material:
60mg of superparamagnetic gamma-Fe 2 O 3 Dispersing the nano material in 36mL deionized water, performing ultrasonic treatment for 10min, and adding 45mL
14.1mmol of cetyltrimethylammonium bromide in water, 65. Mu.L of aqueous ammonia solution, 12mL of cyclohexane in which 1.5mmol of tetraethoxysilane was dissolved, followed by sealing and reaction in a water bath at 65℃overnight with continuous stirring. Washing and drying, dispersing 60mg in 20mL toluene, dripping 260 μl 3-aminopropyl triethoxysilane under stirring, and reacting overnight in water bath at 60deg.C under continuous stirring to obtain silicon layer stable superparamagnetic gamma-Fe 2 O 3 A nanomaterial.
4. Fluorescent peptide grafted magnetic nanomaterial:
(1) 5mg of fluorescent peptide was dispersed in 10mL of 0.1M PBS, followed by addition of 20mg of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and incubation at room temperature for 30min to give solution 1;
(2) Simultaneously, 10mg of silicon layer is stabilized to superparamagnetic gamma-Fe 2 O 3 Adding 10mL of PBS into the nano material, and then treating the nano material for 10min under ultrasonic conditions to obtain a solution 2;
(3) Mixing the above solution 1 and solution 2 thoroughly and incubatingIncubating for 12h. Finally, washing twice with PBS and drying at 50 ℃, grinding and sieving to obtain the blue fluorescent peptide grafted magnetic nanomaterial (particle size 40.73nm, saturation magnetization 57.51emu g -1 )。
Comparative example 1
In comparison with example 1, only steps 1 and 4 were omitted, and the other steps were the same as in example 1.
Test example 1 linear dependence of fluorescence on substance concentration
1mg of the fluorescent peptide-grafted magnetic nanomaterial in example 1 and example 2 were accurately weighed and fully mixed with 1mL of a standard working solution of 3-deoxyglucosone in a concentration range of 0.81-162.14mg L -1 Fluorescence measurement was performed after incubation for 30 min. According to the Stern-Volmer formula: f (F) 0 /F－1＝K[C]Wherein K is the fluorescence quenching constant, [ C]To quencher concentration, F 0 Fluorescence intensity of the system in the absence and presence of the quencher is denoted as F. The standard working solution concentration is taken as the abscissa, and the fluorescence intensity ratio F 0 and/F is the ordinate, and the linear relation between fluorescence and the concentration of the substance is established. The results are shown in FIG. 6, and the fluorescent peptide grafted magnetic nanomaterial in example 1 and example 2 can sensitively and accurately indicate the 3-deoxyglucosone content. The parameters of the fluorescent enzyme-labeled instrument are set: the excitation wavelength is set to 360nm, the emission wavelength ranges from 390 nm to 700nm, and the gain is 120.
Test example 2 fluorescence adsorption Capacity
1mg of the fluorescent peptide-grafted magnetic nanomaterial of example 1 and example 2 and 1mL of standard working solution of 3-deoxyglucosone, glyoxal, methylglyoxal and 2, 3-butanedione (concentration 162.14mg L) -1 ) After thorough mixing, fluorescence measurements were performed after 3-30min, respectively. By fluorescence intensity ratio F 0 And judging the adsorption balance degree according to the change of/F, wherein the parameters of the fluorescence enzyme label instrument are set as follows: excitation wavelength is 360nm, emission wavelength ranges from 390 nm to 700nm, and gain is 120.
As can be seen from the results of FIG. 7, the fluorescent peptide-grafted magnetic nanomaterial of example 1, example 2 was F after 10min 0 Almost no change in/F indicates that the fluorescent peptide grafted magnetic nanomaterialThe binding sites had been gradually occupied, adsorption had equilibrated, and residual 3-deoxyglucosone was detected by liquid chromatography, FIG. 8 shows that the adsorption capacity of red fluorescent peptide grafted magnetic nanomaterial can be as high as 60.68mg g -1 The adsorption capacity of the blue fluorescent peptide grafted magnetic nano material can reach 80.12mg g -1 . The above results show that the fluorescent peptide grafted magnetic nanomaterial can accurately indicate the content of 3-deoxyglucosone through the change of a fluorescent signal, and the shorter adsorption balance is time and satisfactory adsorption capacity, so that the adsorption of the 3-deoxyglucosone in a system can be realized rapidly, and the generation of corresponding end products is inhibited.
Test example 3 inhibition of late glycosylation end product formation in food systems
The dairy product contains abundant proteins, and a large amount of amino groups and high temperature can promote the generation of advanced glycosylation end products in the dairy product, so that a milk system is used as a chemical simulation system, and the inhibition effect of the fluorescent peptide grafted magnetic nano material on the generation of the advanced glycosylation end products in a food system is evaluated.
The chemical simulation system was established with the molar ratio of lysine to sugar in the real milk sample. Casein (30 g) and glucose (27 g) were dispersed in a 0.1M PBS solution (1 l, ph=6.8) and stirred continuously at room temperature (8 h) to completely dissolve to give a simulated system solution; then, 6mL of each of the simulation system solutions was put into a glass reaction tube, and 2mg of the fluorescent peptide-grafted magnetic nanomaterial in example 1 and example 2 and 2mg of the silicon layer-stabilized superparamagnetic gamma-Fe in comparative example 1 were added, respectively 2 O 3 The nanomaterial was incubated at 60 ℃ for 30min and then rapidly cooled in an ice-water bath to stop the reaction. And removing the magnetic nano material grafted by the fluorescent peptide attached to the precursor through magnetic separation under the action of an external magnetic field. And analyzing the production amount of typical advanced glycosylation end products of the pyrroline, the carboxymethyl lysine and the carboxyethyl lysine in a simulated system by using liquid chromatography-mass spectrometry, and evaluating the inhibition effect. The results are shown in FIG. 9, and the fluorescent peptide grafted magnetic nanomaterials and pairs in example 1 and example 2The silicon layer in proportion 1 stabilizes superparamagnetic gamma-Fe 2 O 3 Compared with the nano material, the nano material has higher inhibition rate and stronger inhibition effect on advanced glycosylation end products.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present application, and not for limiting the scope of the present application, and that the simple modification and equivalent substitution of the technical solution of the present application can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present application.
Claims (5)
1. A fluorescent peptide-loaded magnetic nanomaterial characterized in that: mixing dipeptide with zinc ions to obtain fluorescent peptide, and then grafting the fluorescent peptide onto a magnetic carrier to obtain a fluorescent peptide-loaded magnetic nanomaterial; wherein the ratio of the amount of dipeptide to zinc ion species is 1:2; the dipeptide is beta-alanine-histidine; the magnetic carrier is superparamagnetic gamma-Fe 2 O 3 Superparamagnetic alpha-Fe 2 O 3 Superparamagnetic CoFe 2 O 4 Superparamagnetic NiFe 2 O 4 At least one of (a) and (b);
the fluorescence emission wavelength of the fluorescent peptide is 600-700nm; the particle size of the fluorescent peptide is 2-14nm; the particle size of the magnetic carrier is 3-15nm, and the particle size of the fluorescent peptide-loaded magnetic nanomaterial is 10-60nm; the magnetic carrier is subjected to pretreatment, which comprises the steps of carrying out ultrasonic treatment on the magnetic carrier, mixing the magnetic carrier with an organic solvent, sealing, heating in a water bath for overnight, washing, drying, mixing with toluene and 3-aminopropyl triethoxysilane, and heating in the water bath for overnight to obtain the silicon layer stable superparamagnetic carrier; the organic solvent is cetyl trimethyl ammonium bromide, ammonia water and cyclohexane for dissolving tetraethoxysilane;
the grafting method comprises the following steps: incubating the fluorescent peptide with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the silicon layer stabilizes the superparamagnetic carrier for ultrasound; and mixing and incubating the incubated fluorescent peptide and the ultrasonic silicon layer stabilized superparamagnetic carrier to obtain the fluorescent peptide-loaded magnetic nanomaterial.
2. The fluorescent peptide-loaded magnetic nanomaterial of claim 1, wherein: the saturation magnetization of the magnetic carrier is 35-85emu g -1 The method comprises the steps of carrying out a first treatment on the surface of the The saturation magnetization intensity of the magnetic nano material loaded with the fluorescent peptide is 35-60emu g -1 。
3. A method for preparing a fluorescent peptide-loaded magnetic nanomaterial according to any one of claims 1 to 2, characterized in that: the method comprises the following steps:
s1, preparing fluorescent peptide: mixing dipeptide with zinc ion, heating, centrifuging, discarding supernatant, oven drying precipitate, and sieving to obtain fluorescent peptide;
s2, preparing a superparamagnetic carrier by a coprecipitation method;
s3, preparing a silicon layer stable superparamagnetism carrier: performing ultrasonic treatment on the superparamagnetic carrier obtained in the step S2, then mixing the superparamagnetic carrier with an organic solvent, sealing, heating in a water bath for overnight, washing, drying, mixing with toluene and 3-aminopropyl triethoxysilane, and heating in the water bath for overnight to obtain a silicon layer stable superparamagnetic carrier;
and S4, grafting the fluorescent peptide obtained in the step S1 onto the silicon layer stable superparamagnetic carrier obtained in the step S3 to obtain the fluorescent peptide-loaded magnetic nanomaterial.
4. A method of preparation according to claim 3, characterized in that: the amount of the fluorescent peptide in the step S4 is 5-30mg.
5. Use of the fluorescent peptide-loaded magnetic nanomaterial of any one of claims 1-2 or the fluorescent peptide-loaded magnetic nanomaterial prepared by the preparation method of any one of claims 3-4 in the preparation of a product for adsorbing 3-deoxyglucosone.
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