CN110592091A - Aptamer functional material, preparation method thereof and application thereof in AFs detection - Google Patents
Aptamer functional material, preparation method thereof and application thereof in AFs detection Download PDFInfo
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Abstract
The invention belongs to the technical field of mycotoxin detection, and particularly relates to an aptamer functional material, a preparation method thereof and application thereof in AFs detection. The aptamer functional material comprises: carboxyl microsphere and 5' -NH bound on surface of carboxyl microsphere2‑Biotin‑T18‑22-ACTGCTAGAGATTTTCCACAT-3'; wherein carboxyl on the surface of the carboxyl microsphere forms an amido bond with amino, T18‑22Is 18-22T bases. The adapter bodyThe functional material has high specific recognition and binding effects on AFs, high adsorption capacity and good adsorption stability, and has good application prospects in AFs detection and analysis.
Description
Technical Field
The invention belongs to the technical field of mycotoxin detection, and particularly relates to an aptamer functional material, a preparation method thereof and application thereof in AFs detection.
Background
The Aptamer (Aptamer) can be specifically and affinity combined with a target substance, when the Aptamer approaches to the target substance, the Aptamer can be bent, coiled and folded, and complementary pairing of bases in a chain can form special three-dimensional structures (such as a hairpin, a stem loop, a false knot, a bulge loop, a G-tetrad and the like), and the three-dimensional structures are the structural basis of the affinity action of the Aptamer and the target substance and have a specific recognition function; the target substance or a part of the target substance can enter the three-dimensional structure, has a certain specific recognition site in the three-dimensional structure, and can be subjected to affinity interaction with the target substance through pseudo base stacking, hydrogen bonding, electrostatic interaction and other acting forces. In recent years, Aptamer has been reported to be applied to chromatographic purification of a target.
Aflatoxins (Aflatoxins) are attracting attention because of their wide distribution in grain, high detectable rate, large toxic output and strong toxicity. The detection method for aflatoxin is widely applied to ELISA, HPLC and HPLC-MS methods, but because food matrixes are complex and various and the content of mycotoxin is very low, a proper pretreatment means is required for enriching and purifying mycotoxin from a sample, such as a solid phase extraction technology. However, the traditional solid phase extraction material is limited by the adsorption selectivity and the adsorption capacity, and the immunoaffinity column which is most applied at present is expensive, difficult to produce and modify and easy to inactivate, so the development of a novel mycotoxin adsorption material is urgently needed.
AFs are analogues with a dihydrofuran oxanaphtalene orthoketone structure, wherein aflatoxin B1(AFB1), aflatoxin B2(AFB2), aflatoxin G1(AFG1) and aflatoxin G2(AFG2) are common in grain grains, and aflatoxin M1(AFM1) and aflatoxin M2(AFM2) are metabolites of B-family AFs in animals and are often detected in animal-derived foods; for example, AFM1 belongs to one of aflatoxins and other compounds with similar structures, can cause various toxic reactions, carcinogenesis, teratogenicity and mutation of human and animals, can accumulate residues in livestock and poultry bodies, is transmitted through a food chain to further harm human health, and brings serious food safety problems. Therefore, the research and development of selective adsorption materials for AFs are more urgent.
Disclosure of Invention
The invention aims to provide an aptamer functional material, a preparation method thereof and application thereof in AFs detection, and aims to solve the technical problem that specific selective adsorption materials of the conventional AFs are limited.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides an aptamer functional material comprising:
carboxyl microsphere and 5' -NH bound on surface of carboxyl microsphere2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3'; wherein carboxyl on the surface of the carboxyl microsphere forms an amido bond with amino, T18-22Is 18-22T bases.
The invention provides an aptamer functional material for specifically and selectively adsorbing AFs, wherein carboxyl magnetic beads are used as a carrier material, nucleic acid aptamers for specifically adsorbing AFs are used as functional monomers, and T is used as a functional monomer18-22The 5' -end connecting arm serving as the aptamer is formed by amide bond connection formed by carboxyl and amino; the aptamer functional material has good structural stability, and tests prove that the aptamer functional material only has a high-specificity recognition binding effect on AFs, is high in adsorption capacity and good in adsorption stability, and has a good application prospect in AFs detection and analysis.
The invention also provides a preparation method of the aptamer functional material, which comprises the following steps:
providing carboxyl microspheres, and dispersing the carboxyl microspheres in a buffer solution to obtain a carboxyl microsphere dispersion solution;
adding NHS and EDC into the carboxyl microsphere dispersion liquid to carry out carboxyl activation to obtain an intermediate;
reacting 5' -NH2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' is mixed with the intermediate to carry out amidation reaction to obtain the aptamer functional material.
The preparation method of the aptamer functional material provided by the invention has the advantages of simple process and low cost, and can directly prepare 5' -NH2-Biotin-T18-22ACTGCTAGAGATTTTCCACAT-3' and carboxyl activated carboxyl microspheres are subjected to amidation reaction, and the aptamer functional material obtained by the preparation method has a stable structure, has a high specific recognition and binding effect on AFs, and has a good application prospect in AFs detection.
Finally, the invention also provides an application of the aptamer functional material and/or the aptamer functional material prepared by the preparation method in AFs detection.
Drawings
FIG. 1 is an EIC diagram of the remaining solution before and after modification of the nucleic acid aptamer and the washing solution in example 1 of the present invention;
FIG. 2 is a SRM plot of the control solution, adsorbed test eluent and adsorbed test eluent for AFM1 in example 2 of the present invention;
FIG. 3 is a graph showing the results of an adsorption selectivity test of the aptamer functional material in example 3 of the present invention;
FIG. 4 is the isothermal adsorption curve of the aptamer functional material and unmodified carboxyl microspheres in example 4 of the present invention;
FIG. 5 is a graph showing the adsorption performance of the aptamer functional material in example 5 of the present invention as a function of time.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In one aspect, an embodiment of the present invention provides an aptamer functional material, where the aptamer functional material includes: carboxyl microsphere and 5' -NH bound on surface of carboxyl microsphere2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3'; wherein carboxyl on the surface of the carboxyl microsphere forms an amido bond with amino, T18-22Is 18-22T bases.
The embodiment of the invention provides an aptamer functional material for specifically and selectively adsorbing AFs, wherein carboxyl magnetic beads are used as a carrier material, a nucleic acid aptamer for specifically adsorbing AFs is used as a functional monomer, and T is used as18-22The 5' -end connecting arm serving as the aptamer is formed by amide bond connection formed by carboxyl and amino; the aptamer functional material has good structural stability, and tests prove that the aptamer functional material only has a high-specificity recognition binding effect on AFs, is high in adsorption capacity and good in adsorption stability, and has a good application prospect in AFs detection and analysis.
Specifically, in the aptamer functional material, the surface modification part of the carboxyl microsphere carrier: the 5' end is a connecting group of amino biotin NH2Biotin, 3' terminal DNA sequence for specifically recognizing AFs, and T18-22As aminobiotin NH2The spacer arm between Biotin and the DNA sequence of the specific recognition AFs can reduce the probability that the DNA sequence of the specific recognition is buried by the carrier, can fully stretch the DNA sequence, is favorable for ensuring the specific recognition effect of the immobilized DNA sequence, and further improves the recovery rate of the AFs. Without increasing T18-22Before the sequence, the recovery rate of the aptamer functional material to AFs is only 30% -40%, and the aptamer functional material provided by the embodiment of the invention increases T18-22After sequencing, the recovery rate reaches 60-80%.
In one embodiment, T18-22Is selected as A20(the connecting arm is 20T basic groups), namely the aptamer functional material is carboxyl microspheres and 5' -NH combined on the surfaces of the carboxyl microspheres2-Biotin-T20-ACTGCTAGAGATTTTCCACAT-3’。
The AFs adsorbed by the aptamer functional material of the embodiment of the invention are selected from at least one of AFB1, AFB2, AFG1, AFG2, AFM1 and AFM 2. The aptamer functional material has the same principle of identifying and combining with AFs, namely a special dihydrofuran oxanaphthalenone structure of a three-dimensional structure identification and combination with AFs is formed, and aflatoxin has similar structural composition, so that subsequent experiments take identification and combination with AFM1 as an example to carry out related detection.
In one embodiment of the invention, the nucleic acid aptamer (complete functional unit: 5' -NH) is characterized by a nanogold colorimetric method2-Biotin-T20-ACTGCTAGAGATTTTCCACAT-3') with AFM 1; the rule that the color change (the signal change of A650/A530) of Gold Nanoparticles (GNPs) changes along with factors such as aptamer and salt is researched through a single-factor experiment. The results show that: (1) the critical state of the GNPs under the action of various factors is considered through a single-factor test: when the addition amount of GNPs in a pH7 system is 200 mu L and 0.075nmol/L, the influence of the aptamer on the color change of the GNPs system shows a trend along with the addition amount of the aptamer, and the GNPs system can reach a relatively stable state only when the final concentration of the aptamer is more than 0.035 mmol/L; the trend of the influence of the induced coagulation effect of NaCl on the color change of the GNPs system has a critical point (35mmol/L NaCl) of an A650/A530 signal of the GNPs system, the induced coagulation effect of NaCl and the stabilizing effect of an aptamer are in a critical state of antagonistic balance in the range of the critical point and after the critical point, and the color of the GNPs system can be obviously changed by the change of any factor. (2) AFM1 is added into a GNPs system with induction effect and stabilization effect in an antagonistic equilibrium critical state through a nanogold colorimetric method, and A650/A530 signals are increased along with the increase of the addition amount, which shows that more and more nucleic acid aptamers and AFM1 generate affinity effect to generate a compound, so that the antagonistic balance tends to the induction coagulation effect of NaCl, and the addition of non-corresponding target substances has no obvious influence on the A650/A530 signals of the GNPs system; the result shows that the aptamer can specifically identify AFM1, and can be used as a functional monomer to design a novel aptamer functional adsorption material. Therefore, the synthesis of the aptamer and the carboxyl microsphere which are special for the embodiment of the inventionAptamer functional material is aptamer functionalized microspheres.
On the other hand, the embodiment of the invention also provides a preparation method of the aptamer functional material, which comprises the following steps:
s01: providing carboxyl microspheres, and dispersing the carboxyl microspheres in a buffer solution to obtain a carboxyl microsphere dispersion solution;
s02: adding NHS and EDC into the carboxyl microsphere dispersion liquid to carry out carboxyl activation to obtain an intermediate;
s03: reacting 5' -NH2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' is mixed with the intermediate to carry out amidation reaction to obtain the aptamer functional material.
The preparation method of the aptamer functional material provided by the embodiment of the invention has the advantages of simple process and low cost, and can directly prepare 5' -NH2-Biotin-T18-22ACTGCTAGAGATTTTCCACAT-3' and carboxyl activated carboxyl microspheres are subjected to amidation reaction, and the aptamer functional material obtained by the preparation method has a stable structure, has a high specific recognition effect on AFs, and has a good application prospect in AFs detection.
In the above step S01: the buffer used for dispersing the carboxyl microspheres was PBS buffer, and the pH of the buffer was 5.5. Specifically, the concentration was 20mmol/L PBS buffer.
In the above step S02: NHS (N-hydroxysuccinimide) and EDC ((1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)) are used as activating reagents for carboxyl groups, and intermediates obtained after activation of carboxyl microspheres are O-acylurea intermediates and N-hydroxysuccinimide ester intermediate mixtures.
And in step S03: reacting 5' -NH2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' with said intermediate mixing step, T18-22Is selected as T20。
Further, the 5' -NH2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' was added as a solution to the microsphere dispersion at a concentration of 0.02 mmol/L.
Preparation of aptamer solutionCan be as follows: a20 OD nucleic acid aptamer tube was centrifuged at 4000rpm at 4 ℃ for 2min, and 1.0mL of ultrapure water was added to prepare a stock solution of 0.1mmol/L nucleic acid aptamer. The standard stock solutions are stored in a refrigerator at the temperature of-30 ℃, and the low-concentration working solution is obtained by diluting the stock solutions with proper solvents step by step. The structural sequence of the aptamer specifically recognizing the AFs is as follows: 5' -N H2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3 ', which can be synthesized by Biotechnology engineering (Shanghai) GmbH and NH at the 5' end2Biotin modified spacer and HPLC purification.
Further, the amidation reaction time is 0.5-1h, and under the condition, the aptamer is completely modified on the surface of the carboxyl microsphere. After the amidation reaction, the reaction mixture may be further centrifuged, and after washing the reaction mixture 3 times with 20mmol/L PBS buffer (pH7.2), the aptamer-functional material may be stored at 4 ℃.
Preparing an aptamer functional material by EDC/NHs mediated amidation reaction; an analysis method of the aptamer functional material is established through UPLC-Q-Orbitrap MS/MS, the content of the nucleic aptamer in reaction liquid before and after modification reaction is analyzed, and as a result, no nucleic acid aptamer is detected in residual solution and washing liquid after modification reaction, which shows that 5' amino modified nucleic acid aptamer can be completely modified on carboxyl microsphere through EDC/NHs mediated amidation reaction.
Finally, the embodiment of the invention also provides an application of the aptamer functional material and/or the aptamer functional material prepared by the preparation method in the embodiment of the invention in AFs detection.
Specifically, the AFs are selected from at least one of AFB1, AFB2, AFG1, AFG2, AFM1 and AFM 2.
Specifically, the AFs detection comprises the following steps:
e01: providing an AFs sample solution;
e02: loading the aptamer functional material into a solid phase extraction column, and then enabling the AFs sample solution to flow through the solid phase extraction column for adsorption treatment;
e03: and (4) eluting the solid phase extraction column after adsorption treatment, and detecting the content of the AFs through chromatography-mass spectrometry.
In the step E01, the AFs sample solution may be the solution to be detected or the standard solution of AFs. In the present example, AFM1 solution was used.
In the step E02, the solid-phase extraction column for containing the aptamer functional material is an SPE hollow column, and the adsorption treatment is extraction adsorption.
In one embodiment, the adsorption treatment system contains 10mmol/L Mg2+Or 5mmol/L of K+(ii) a Experiments prove that when the adsorption treatment is carried out by using the extraction solvent for adsorption extraction, the extraction solvent does not contain Mg2+The adsorption rate of the ligand functional material to AFM1 in the aqueous solution is still about 97%. And with Mg in the system2+The content of (A) is increased, and the adsorption rate is increased, when Mg is in the system2+When the concentration of (A) is more than 10mmol/L, the amplification begins to slow down, and the adsorption rate can reach 99.5%. As the ligand functional material has carboxyl of the carboxyl microsphere as a retention enhancing site besides a specific recognition site, the retention effect on AFM1 in an aqueous solution is strong, and Mg2+The aptamer is induced and stabilized to form a special three-dimensional structure, the adsorption rate is not obviously increased when the aptamer serving as a connecting medium is used for increasing the interaction between the aptamer and AFM1, but the adsorption rate is up to 99.5 percent without Mg2+Cannot be achieved during induction.
In the step E02, the elution solvent used in the elution process is an acetonitrile aqueous solvent or a methanol aqueous solvent. The polar solvent of methanol and acetonitrile can destroy the acting force of the aptamer functional material on the AFM1, thereby achieving the purpose of desorbing the AFM 1. The desorption effect of the elution solvent on the AFM1 is enhanced along with the increase of the proportion of methanol or acetonitrile, when the volume ratio of the methanol or the acetonitrile reaches 50%, the elution rate on the AFM1 can reach 90%, a better desorption effect can be obtained, and when the volume ratio of the methanol or the acetonitrile is 75%, the elution rate is further improved. But the proportion of methanol or acetonitrile is not suitable for being higher than 75 percent, so as to avoid the influence of solvent effect on the pattern peak of the eluent in mass spectrum detection and the interference of accurate quantification. At lower proportions of methanol or acetonitrile, acetonitrile shows better desorption than methanol, which may be related to the fact that acetonitrile can destroy the specific spatial structure of the aptamer, resulting in a change in the conformation of the active recognition site. Therefore, depending on the actual sample, a low ratio of methanol-water may be used as the eluting solvent, and a 75% by volume aqueous solution of methanol or acetonitrile may be used as the eluting solvent.
In one embodiment, the chromatography-mass spectrometry is ultra performance liquid chromatography-tandem mass spectrometry; wherein the chromatographic conditions comprise: the chromatographic column was Thermo Hypersll GOLD C18, mobile phase a was 5mmol/L ammonium acetate in water (containing 0.1% formic acid), mobile phase B was acetonitrile: methanol (v/v, 1:1), flow rate 0.3mL/min, sample size 5. mu.L, column temperature 40 ℃. The mass spectrometry conditions include: the ion source is a heatable electrospray ion source, the scanning mode is a positive ion mode, the spraying voltage is 3.0kV, the gasification temperature is 400 ℃, the temperature of an ion transmission pipe is 300 ℃, the flow rate of sheath gas is 40L/min, and the flow rate of auxiliary gas is 10L/min.
The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.
Example 1 Synthesis of aptamer functional materials
a.O-acyl urea carboxyl activation reaction
Taking 3.0g of dry carboxyl microspheres, adding 50mL of water, fully stirring and dispersing, performing immersion washing, performing centrifugal separation on the carboxyl microspheres, repeatedly washing with water for 5 times to remove ethanol, washing and immersing for 1 time with 20mmol/L PBS buffer solution (pH5.5), and re-dispersing the carboxyl microspheres in 40mL PBS buffer solution for later use.
0.54g NHS and 4.5g EDC were weighed into a clean 150mL Erlenmeyer flask and dissolved well by adding 50mL of 20mmol/L PBS buffer (pH 5.5). Pouring the carboxyl microsphere suspension, mixing well, magnetically stirring at room temperature for 1h, centrifuging, and washing with water for 3 times to obtain reaction intermediate mixture (O-acylurea intermediate and N-hydroxysuccinimide ester intermediate).
b. Amidation reaction
1g of the above reaction intermediate on a dry weight basis was put into a clean 50mL beaker, and 0.1mL of 0.02mmol/L aptamer (5' -NH)2-Biotin-T20ACTGCTAGAGATTTTCCACAT-3') and adding 20mmol/L PBS buffer (pH7.2) to just soak (about 3mL), stirring at room temperature for 30min, centrifuging, and remaining the solution after reaction for analysis of aptamer. Washing the separated microspheres with 20mmol/L PBS buffer solution (pH7.2) for 3 times, and storing the aptamer functionalized microspheres, namely the aptamer functional material, at 4 ℃.
UPLC-Q-Orbitrap MS/MS analytical characterization
In order to characterize the modification of the aptamer on the carboxyl microsphere, the aptamer in the solution before the aptamer modification (i.e., the standard solution containing the aptamer), the remaining solution after the aptamer modification (i.e., the amidation reaction) and the washing solution was analyzed by UPLC-Q-Orbitrap MS/MS. The exact mass number of the sample through the primary mass spectrum is characterized by a data-dependent scanning mode (full MS-dd MS2), and further confirmed by its secondary fragment, and its signal intensity can be used as a quantitative index.
Chromatographic conditions are as follows: the chromatographic column is Thermo DNAPacTMRP (2.1 mmol/L. times.100 mmol/L, 4.0. mu.m); mobile phase a was 5mmol/Lol/L ammonium acetate in water and mobile phase B was acetonitrile, gradient elution (see mobile phase gradient program in table 1); flow rate: 0.2 mL/min; sample introduction amount: 5 mu L of the solution; column temperature: at 60 ℃.
Mass spectrum conditions: an ion source: a heatable electrospray ion source (HESI-II); scanning mode: a negative ion mode; spraying voltage: 3.0 kV; the gasification temperature: 350 ℃; ion transfer tube temperature: 320 ℃; sheath gas (N)2): 35L/min; auxiliary gas (N)2): 5L/min; lens voltage: 50V; using a data dependent scanning mode (Full MS-dd MS)2) Detection, the resolution of the first-stage scanning is 70000, and the AGC target is 1.0 multiplied by 106The maximum injection time is 100ms, and the scanning range is 200-2000; the secondary scanning resolution is 17500, and the AGC target is 1.0 multiplied by 105The maximum injection time is 50ms, the isolation window is 2M/z, the mass-to-charge ratio M/z of the scanning parent ions is 1638.78833 (the aptamer standard solution is injected into the mass spectrum through an injection pump system of a Q-Orbitrap mass spectrometer and is scanned on a primary mass spectrum thereof, and ions with the proper mass-to-charge ratio (M/z is 1638.78833 [. M-4H]4-) As parent ion for detection of aptamers) window range is 10 ppm.
TABLE 1
As shown in FIG. 1, in the ion flow chart (EIC) of the extracted ion channel with m/z of 1638.78833, no aptamer signal was detected in both the remaining solution and the washing solution of the amidation modification reaction, whereas the aptamer signal was detected in the standard solution of the aptamer before the amidation modification reaction, and further the primary mass spectrum and the secondary mass spectrum were compared, and the primary parent ion peak m/z, the secondary ion peak m/z and the mass spectrum information were matched. Therefore, the synthesis method can lead the aptamer to be completely modified on the surface of the carboxyl microsphere, and the adopted EDC/NHs amidation method is favorable for the amino at the 5' end of the aptamer to be bonded on the surface of the carboxyl microsphere through amidation reaction due to the high carboxyl coverage rate (more than or equal to 1mmol/L carboxyl/g) of the carboxyl microsphere.
Example 2 aptamer functional material enrichment isolation AFM1
0.25g of the aptamer functional material prepared in example 1 above was packed into a 3mL SPE hollow column, compacted, washed with water and soaked thoroughly. Balancing with an extraction solvent, controlling 2.5mL of AFM1 standard solution with a certain concentration to flow through a small column at a constant speed to finish AFM1 extraction, adsorption and enrichment, leaching with 2.5mL of the extraction solvent, eluting with 2.5mL of an elution solvent, and detecting AFM1 contents in an effluent, a leacheate and an eluent by UPLC-MS/MS.
Chromatographic conditions are as follows: the chromatographic column is Thermo Hypersll GOLD C18(2.1mmol/L × 100mmol/L,1.9 μm); mobile phase a was 5mmol/Lol/L ammonium acetate in water (containing 0.1% formic acid), mobile phase B was acetonitrile: methanol (v/v, 1:1), and the elution was carried out with a gradient (mobile phase gradient see table 2). Flow rate: 0.3 mL/min; sample introduction amount: 5 mu L of the solution; column temperature: at 40 ℃.
Mass spectrum conditions: ion(s)Source: a heatable electrospray ion source (HESI-II); scanning mode: a positive ion mode; spraying voltage: 3.0 kV; the gasification temperature: 400 ℃; ion transfer tube temperature: 300 ℃; sheath gas (N)2): 40L/min; auxiliary gas (N)2): 10L/min; the scan mode is SRM and the scan conditions are tabulated in table 3.
TABLE 2
TABLE 3
Where the symbol "+" is a quantitative ion.
Respectively taking 5 mu L of reference substance solution (namely AFM1 standard solution), the effluent of the adsorption of the aptamer functional material on the AFM1 sample, eluent and eluent to detect the AFM1 content on UPLC-MS/MS according to the chromatographic mass spectrum conditions, wherein the multi-ion reaction detection (SRM) spectrum is shown in figure 2; under the method, the linear equation of the AFM1 standard solution detected by UPLC-MS/MS is A-4697C(AFM1)+2.414 (linear range 0.1-2.0 ng/mL, R2＝0.9996)。
Example 3 specific adsorption of aptamer-functional materials to AFs
The column packing and the balance of the aptamer functional material are completed according to the method of the embodiment 2, 2.5mL of AFM1, AFB1, AFB2, AFG1, AFG2, ochratoxin A (OTA), Deoxynivalenol (DON) and zearalenone (Zea) standard solution (the structure of a sample compound is shown in the following) with certain concentration are respectively controlled, the solution flows through a small column at constant speed to complete adsorption enrichment, 2.5mL of extraction solvent is used for leaching, 2.5mL of elution solvent is used for eluting, and the contents of effluent, eluent and eluent are detected on UPLC-MS/MS.
The aptamer functional material can form a special dihydrofuran oxanaphthalenone structure with a three-dimensional structure recognition combination AFM1 in the process of recognition combination with AFM1, aflatoxins have similar structural compositions, and specific recognition capability verification is also carried out on toxins with other structural types such as OTA, DON and Zea in order to further research whether the aptamer functional material can also specifically recognize and combine a plurality of aflatoxins other than AFM 1. The result is shown in fig. 3, the aptamer functional material has strong specific adsorption to AFM1, AFB1, AFB2, AFG1 and AFG2, and the adsorption rate is higher than 90%, and on the contrary, the adsorption rate of the aptamer functional material to OTA, DON and Zea is only 50-68%, because AFM1, AFB1, AFB2, AFG1 and AFG2 have similar chemical structures, and the special stereo structure formed by the nucleic acid aptamers in the aptamer functional material can identify the dihydrofuran oxanaphthalene orthoketone structures thereof and is specifically combined with the nucleic acid aptamers through hydrogen bonding, pseudo-base accumulation, electrostatic interaction and the like, wherein the AFG2 with the largest difference in structure from AFM1 has relatively weaker action than the nucleic acid aptamers. Therefore, OTA, DON and Zea without a dihydrofuran oxanaphthalene orthoketone structure cannot be identified and combined by an aptamer functional material, only carboxyl of the microsphere can retain OTA, DON and Zea to a certain extent through hydrogen bond action, and almost completely elutes under the desorption action of an elution solvent. The adsorption selectivity test result shows that the nucleic acid aptamer in the aptamer functional material has specific recognition and retention effects on aflatoxin compounds with similar structures except for the specific recognition of AFM 1.
Example 4 adsorption equilibrium experiment of aptamer functional materials
And (3) filling 20mg of the aptamer functional material synthesized in the same batch into a 1mL SPE empty column, balancing with an extraction solvent, preparing AFM1 standard solutions with series concentrations, enabling the solutions to flow through each small column at a constant speed to finish AFM1 adsorption enrichment, collecting all effluent, and detecting the content of AFM1 on a UPLC-MS/MS.
The isothermal adsorption test of the aptamer functional material is completed according to the steps, the adsorption capacity (Q, mu g/g) of the aptamer functional material and the unmodified carboxyl microsphere is calculated according to the following formula 1, and the modification factor (K) is calculated according to the formula 2 so as to compare the extraction performance difference of the aptamer functional material and the unmodified carboxyl microsphere:
in the formula, CiFor adsorption of AFM1 concentration (. mu.g/L), C in the solution before the experimenttiIn order to obtain the concentration (mu g/L) of AFM1 in the solution after the adsorption experiment, V is the volume (L) of the added sample standard solution, w is the dosage (g) of the aptamer functional material and the unmodified carboxyl microsphere participating in the adsorption equilibrium experiment, and QApt-MAnd QCarboxyl-MThe adsorption capacities (mu g/g) of the aptamer functional material and the unmodified carboxyl microsphere are respectively.
As a result, as shown in FIG. 4, the adsorption capacities of the aptamer functional material and the unmodified carboxyl microspheres both showed an increase in the concentration of AFM 1. The two show the same change trend in the AFM1 concentration range of 2-1000 ng/mL, the adsorption capacity growth speed is higher, the adsorption rate is stable and is at a higher level, and therefore, the fact that in the range, the number of adsorption sites is far higher than that of AFM1 can be explained. In the AFM1 concentration range of 1000-15000 ng/mL, the increase of the adsorption capacity of the AFM1 and the adsorption capacity of the AFM1 start to gradually slow, in the process, the adsorption rate is limited by the relation between the amount of AFM1 and the number of effective adsorption sites, the adsorption rate starts to gradually decrease, when the AFM1 concentration is 15000ng/mL, the adsorption capacity of the AFM1 and the adsorption capacity of the AFM1 both approach to a saturation state, at the moment, the adsorption capacity is 233.1 mu g/g and 167.6 mu g/g respectively, and the saturation adsorption capacity is completely suitable for measuring the content of the AFM 1. In the whole process, the aptamer functional material always presents higher adsorption capacity, which is attributed to the specific recognition sites provided by the nucleic acid aptamer, and compared with the hydrogen bond adsorption sites covering a huge number of carboxyl microspheres, the few specific recognition sites have higher adsorption selectivity andstronger adsorption, as the concentration of AFM1 is increased, the matching relationship between the amount of AFM1 and the number of effective adsorption sites is more and more tense, and the advantages of the specific recognition sites provided by the aptamer are more and more increased at the moment, which is specifically represented by QApt-MAnd QCarboxyl-MThe difference in (A) is increasing, i.e.the modification factor (K) is increasing, the K value being about 1.391 as the adsorption capacity approaches saturation. Therefore, in the analysis process of AFM1 in a complex sample matrix, under the condition that the matching of the amount of AFM1 and the number of effective adsorption sites is difficult, the specific recognition sites provided by the nucleic acid aptamers in the aptamer functional material still have the advantages of selective adsorption and effective adsorption.
Example 5 stability of aptamer functional materials
Selecting the aptamer functional materials synthesized in the same batch and different batches to finish an AFM1 extraction experiment, and investigating the stability of the extraction performance of the aptamer functional materials in the synthesis batch and between batches; the synthesized aptamer functional material was stored in a 4 ℃ refrigerator, taken out every several days, and packed into a column to complete the AFM1 extraction adsorption experiment, and the time stability of the extraction performance of the aptamer functional material was studied, and the final results are shown in table 4 and fig. 5.
TABLE 4
In table 4, the adsorption rates RSD of the three batches of the aptamer functional material in the batch to complete the AFM1 adsorption test are not greater than 1.7%, and the adsorption rates RSD of the three batches of the aptamer functional material in the batch to complete the AFM1 adsorption test are 0.4% calculated by the average value of the adsorption rates in the batches, which indicates that the synthetic method of the aptamer functional material and the AFM1 adsorption test are simple, convenient and feasible in operation steps, high in repeatability, and high in batch-to-batch stability of the performance of the aptamer functional material adsorbing AFM 1. As can be seen from fig. 5, the synthesized aptamer functional material can exist stably within 30 days, and has no significant change in the adsorption performance of AFM1, but the affinity capacity of the synthesized aptamer functional material for AFM1 slightly decreases with the passage of time, and the extraction rate of the synthesized aptamer functional material for AFM1 in an aqueous solution is reduced to about 90% at about 60 days of synthesis, which still meets the requirement of analytical detection, indicating that the synthesized aptamer functional material has high stability.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. An aptamer functional material, wherein the aptamer functional material comprises:
carboxyl microsphere and 5' -NH bound on surface of carboxyl microsphere2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3'; wherein carboxyl on the surface of the carboxyl microsphere forms an amido bond with amino, T18-22Is 18-22T bases.
2. The aptamer functional material of claim 1, wherein the aptamer functional material is a carboxyl microsphere and 5' -NH is bound to the surface of the carboxyl microsphere2-Biotin-T20-ACTGCTAGAGATTTTCCACAT-3’。
3. A method for preparing an aptamer functional material is characterized by comprising the following steps:
providing carboxyl microspheres, and dispersing the carboxyl microspheres in a buffer solution to obtain a carboxyl microsphere dispersion solution;
adding NHS and EDC into the carboxyl microsphere dispersion liquid to carry out carboxyl activation to obtain an intermediate;
reacting 5' -NH2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' is mixed with the intermediate to carry out amidation reaction to obtain the aptamer functional material.
4. The process according to claim 3, wherein 5' -NH is added2-Biotin-T18-22-ACTGCTAGAGATTTTCCACAT-3' with said intermediate mixing step, T18-22Is selected as T20。
5. The method of claim 3, wherein the buffer is PBS buffer and the pH of the buffer is 5.5.
6. The process according to claim 3, wherein the amidation reaction time is 0.5 to 1 hour.
7. Use of the aptamer functional material of claim 1 or 2 and/or the aptamer functional material prepared by the preparation method of any one of claims 3 to 6 in the detection of AFs.
8. The use according to claim 7, wherein the AFs are selected from at least one of AFB1, AFB2, AFG1, AFG2, AFM1 and AFM 2.
9. The use according to claim 7, wherein the AFs detection comprises the steps of:
providing an AFs sample solution;
loading the aptamer functional material into a solid phase extraction column, and then enabling the AFs sample solution to flow through the solid phase extraction column for adsorption treatment;
and (4) eluting the solid phase extraction column after adsorption treatment, and detecting the content of the AFs through chromatography-mass spectrometry.
10. The use of claim 9, wherein the chromatography-mass spectrometry is ultra performance liquid chromatography-tandem mass spectrometry; wherein the content of the first and second substances,
the chromatographic conditions include: the chromatographic column was Thermo Hypersll GOLD C18, mobile phase a was 5mmol/L ammonium acetate in water (containing 0.1% formic acid), mobile phase B was acetonitrile: methanol (v/v, 1:1), flow rate of 0.3mL/min, sample size of 5 μ L, column temperature of 40 deg.C; and/or the presence of a gas in the gas,
the mass spectrometry conditions include: the ion source is a heatable electrospray ion source, the scanning mode is a positive ion mode, the spraying voltage is 3.0kV, the gasification temperature is 400 ℃, the temperature of an ion transmission pipe is 300 ℃, the flow rate of sheath gas is 40L/min, and the flow rate of auxiliary gas is 10L/min.
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