CN115181297B - AlTCPP@MOF membrane based on porous alumina nano channel and preparation method and application thereof - Google Patents
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Abstract
The invention provides an AlTCPP@MOF film based on a porous alumina nano channel, and a preparation method and application thereof. According to the invention, aluminum ions are provided through the AAO film, the aluminum ions are bonded with the organic ligand to form the MOF structure, the MOF structure is uniformly filled in the nano channel of the AAO film, and more grafting sites are provided. According to the method for detecting microcystin, disclosed by the invention, the effect of target specificity of MOF is improved through surface modification/functionalization of the aptamer, so that the sensitivity of microcystin detection is improved. The nucleic acid aptamer reacts with the carboxyl of AlTCPP@MOF by virtue of the amino modified at the tail end to form an amide bond, and is covalently fixed on the MOF; the target binds to the aptamer-specific structure, resulting in a change in MOF transmembrane current. The method is simple to operate, has small sample quantity, can detect the specificity of microcystin in the environment, has strong specificity and has sensitivity as high as 0.004ng/mL.
Description
Technical Field
The invention relates to the technical field of environmental analysis and detection, in particular to an AlTCPP@MOF membrane based on a porous alumina nano channel, and a preparation method and application thereof.
Background
Microcystins are used as secondary metabolites of blue algae and cyclic heptapeptide blue algae toxins, are one of main marine toxins in a large Liu Xing water ecosystem, and more than 100 microcystins have been identified at present, wherein MC-LR is the most important type because of high toxicity and wide distribution, and seriously threatens human and animal health. Current detection of MC-LR includes conventional high performance liquid chromatography, enzyme-linked immunosorbent, thin layer chromatography, gas chromatography-mass spectrometry, etc., however, these methods have disadvantages such as low sensitivity and specificity, expensive operation equipment, need for experienced operators, and difficulty in sample preparation. For this reason, it is particularly important to develop a method for detecting microcystins rapidly, sensitively, accurately and at low cost.
In recent years, researchers have widely studied solid state nanochannels for intelligent control of ion and molecular transport, which have great potential for application in advanced nanofluids, molecular sieves, energy conversion and biological analysis. Among the diverse solid-state nanochannels, nanoporous anodic aluminum oxide films (AAOs) are very popular due to their high stability, durability, ordered nanochannels, narrow pore size distribution, etc. In particular, the high surface to volume ratio of AAO, which significantly increases the fixed number of guests, has been used as a versatile substrate for constructing many bioanalytical platforms. In addition, large-scale production of AAO becomes efficient, economical, controllable, reproducible, and easily upgradeable by mature production technologies.
Although the AAO nanochannel has a plurality of advantages, the research of the AAO nanochannel in the field of electrochemical biosensing is still in a starting stage, and particularly, the application of the AAO nanochannel in-situ forming of a metal organic framework in the field of electrochemical biosensing is rarely reported.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an AlTCPP@MOF membrane based on a porous alumina nano channel, and a preparation method and application thereof, so as to realize rapid, sensitive and accurate detection of microcystin. The technical proposal is as follows:
the invention provides a preparation method of an AlTCPP@MOF film based on a porous alumina nano channel, which is characterized in that a nucleic acid aptamer of an exposed carboxyl group and a modified amino group on an MOF structure grown in situ in the porous anodized aluminum film is connected and fixed on the porous anodized aluminum film through an amide bond, and specifically comprises the following steps:
step S1, adopting a solvothermal synthesis method, taking TCPP as a ligand to generate an MOF structure on a porous anodic alumina film in situ, and preparing an AlTCPP@MOF material;
step S2, soaking the AlTCPP@MOF material obtained in the step S1 in EDC/NHS mixed solution for reaction to obtain a carboxyl activated AlTCPP@MOF material;
and S3, bonding and fixing the carboxyl-activated AlTCPP@MOF material obtained in the step S2 and an amino-modified nucleic acid aptamer on the AlTCPP@MOF material through an amide bond to obtain an AlTCPP@MOF film.
Further, in step S1, the porous anodic alumina membrane includes a plurality of nanochannels, and the average pore diameter of the nanochannels is 80-110 nm, and the average density is 10 -11 ～10 -12 Individual/cm 2 The thickness of the porous anodic aluminum oxide film is 20-25 mm.
Further, in step S1, the process for preparing the altcpp@mof material is as follows: dissolving TCPP in DMF water solution, mixing to obtain mixed solution, adding porous anodic alumina film into the mixed solution, ultrasonic treating for 15-20 min, transferring to a reaction kettle, reacting at 120-140 deg.C for 2-4 h to obtain synthetic product, washing the synthetic product with DMF and water, vacuum drying at room temperature to obtain AlTCPP@MOF material.
Further, in the step S2, the process of preparing the carboxyl activated AlTCPP@MOF material is to prepare EDC/NHS solution with the volume ratio of 2:1-3:1 by using PBS buffer solution with the pH value of 7.4+/-0.1, soak the AlTCPP@MOF material in the EDC/NHS solution, and react for 0.5-1 h, so that the carboxyl exposed on the inner surface and the outer surface of the AlTCPP@MOF material is activated.
Further, in the step S3, the AlTCPP@MOF is soaked in a modified amino aptamer solution, and the reaction time is 10-12 hours.
The second object of the invention is to provide an AlTCPP@MOF film which is prepared by the preparation method.
The third object of the invention is to provide the application of the AlTCPP@MOF film, wherein the AlTCPP@MOF film is used for qualitative and quantitative analysis of microcystins.
Further, the aptamer of the nucleic acid aptamer on the AlTCPP@MOF film is an aptamer of which the 5' -end is modified with an amino group, and the sequence is as follows: 5' -NH 2 -C 6 -GGCGCCAAACAGGACCACCATGACAATTACCCATACCACCTCATTATGCCCCATCTCCGC-3’。
The fourth object of the present invention is to provide a method for detecting microcystins, wherein the AlTCPP@MOF membrane is soaked in a microcystin solution, and the reaction is carried out for 1-4 hours, and the microcystins are combined with a nucleic acid aptamer specific structure, so that the microcystins are detected.
Further, the feeding ratio of the microcystin solution to the AlTCPP@MOF film is 100-150 mu L and is 0.3-0.5cm 2 The concentration of the microcystin solution is 0.1-1000 ppb.
The invention has the following beneficial effects:
the invention provides an AlTCPP@MOF film based on a porous alumina nano channel, and a preparation method and application thereof. According to the invention, aluminum ions are provided through the AAO film, the aluminum ions are bonded with the organic ligand to form the MOF structure, the MOF structure is uniformly filled in the nano channel reaching the AAO film, and more grafting sites are provided. According to the method for detecting microcystin, disclosed by the invention, the effect of target specificity of MOF is improved through surface modification/functionalization of the aptamer, so that the sensitivity of microcystin detection is improved. The nucleic acid aptamer reacts with the carboxyl of AlTCPP@MOF by virtue of the amino modified at the tail end to form an amide bond, and is covalently fixed on the MOF; the target binds to the aptamer-specific structure, resulting in a change in MOF transmembrane current. The method is simple to operate, has small sample quantity, can detect the specificity of microcystin in the environment, has strong specificity and has sensitivity as high as 0.004ng/mL.
Drawings
FIG. 1 is a schematic illustration of the surface of an AlTCPP@MOF film according to an embodiment of the invention;
FIG. 2 is a schematic diagram of a cross section of an AlTCPP@MOF film according to an embodiment of the invention;
FIG. 3 is a graph showing the limit of detection of microcystins using AlTCPP@MOF membranes in an embodiment of the invention;
FIG. 4 is a graph showing the specificity of AlTCPP@MOF for other toxins in an example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described below.
Example 1
The embodiment is to prepare an AlTCPP@MOF film based on a porous alumina nano channel, which comprises the following steps:
step S1, synthesizing AlTCPP@MOF material by using solvothermal synthesis method
S11, taking a round anodic aluminum oxide film (AAO) with the diameter of 25mm and the diameter of a pore canal on the film of 80-110 nm;
step S12, weighing 14-16 mg meso-tetra (4-carboxyphenyl) porphine (TCPP) dissolved in 10mL Dimethylformamide (DMF) aqueous solution (V DMF :V Water =3:1), then adding the AAO film of the step (1) to the above mixed solution, and performing ultrasonic treatment on the solution for 15-20 min. Placing the mixture into a reaction kettle to react for 2-4 hours at 120-140 ℃; washing the synthesized product with DMF and water respectively, and vacuum drying at room temperature to obtain an AlTCPP@MOF material;
step S2, activation of carboxyl groups of AlTCPP@MOF material
Preparing EDC/NHS solution with a volume ratio of 2:1 by using PBS with pH=7.4 as a buffer solution, soaking the AlTCPP@MOF in the solution, and reacting for 0.5-1 h to activate exposed carboxyl groups on the inner and outer surfaces of the AlTCPP@MOF;
step S3, using a chemical modification method to modify the microcystin aptamer
Soaking the carboxyl-activated AlTCPP@MOF material obtained in the step S2 in an amino-modified nucleic acid aptamer solution for 10-12 hours, so that the nucleic acid aptamer reacts with the carboxyl of the AlTCPP@MOF material through the amino modified at the tail end to form an amide bond, and covalently fixing the amide bond on the MOF to obtain the AlTCPP@MOF film.
The structure of the AlTCPP@MOF film prepared in example 1 is characterized, and the result is shown in fig. 1 and 2, and it can be seen that the nano sheets are closely packed and limited in an AAO nano channel, so that sufficient grafting sites can be provided for the nucleic acid aptamer, and the nucleic acid aptamer with the terminal modified amino group is covalently bound with the AlTCPP@MOF with a large number of exposed carboxyl groups.
Comparative example 1
This comparative example is a porous alumina-based nanochannel membrane prepared without organic ligands, specifically comprising the following steps:
step S1, taking a round anodic aluminum oxide film (AAO) with the diameter of 25mm and the pore diameter of 80-110 nm, respectively carrying out ultrasonic treatment on the circular anodic aluminum oxide film in ultrapure water and ethanol solution for 5-10 min, and cleaning surface impurities;
step S2, activation of hydroxyl groups on the AAO surface
Soaking the AAO film in the step S1 in an APTES (3-aminopropyl triethoxysilane) isopropanol (or acetone) solution with the volume fraction of 5%, sealing by using a sealing film, and soaking for 10-12 hours in a dark place to activate hydroxyl groups on the AAO surface;
step S3, attachment of AAO surface aldehyde groups
Soaking the AAO film obtained in the step S2 in glutaraldehyde water solution with the volume fraction of 5%, so that hydroxyl groups and aldehyde groups on the surface of the nano pore canal film fully react under the light-shielding condition for 10-12 h, and connecting aldehyde groups with relatively high activity on the AAO surface;
step S4, modifying the microcystin aptamer by using a chemical modification method
Soaking the AAO film obtained in the step S3 in a microcystin aptamer solution, and reacting for 10-12 h to enable the AAO film with one end connected with aldehyde group to carry out covalent reaction with the aptamer with the end group being amino; the porous anodic alumina film modified with the aptamer is obtained.
The applicant analyzed the sensitivity of the AlTCPP@MOF film prepared in example 1 and the aptamer-modified porous anodic alumina film prepared in comparative example 1 to detect microcystin, specifically:
respectively soaking the AlTCPP@MOF film and the porous anodic aluminum oxide film modified with the aptamer in a microcystin solution for reaction for 1-4 hours, and combining the microcystin with a nucleic acid aptamer specific structure to finish detection of the microcystin, wherein the feeding ratio of the microcystin solution to the AlTCPP@MOF film (or the porous anodic aluminum oxide film modified with the aptamer) is 100-150 mu L and 0.3-0.5cm 2 The concentration of the microcystin solution is in the range of 0.1 to 1000ppb.
The transmembrane current is detected by cyclic voltammetry, and is specifically as follows:
adopts self-made double electricityThe device for testing the transmembrane current of the polar system consists of two separated organic glass electrolytic tanks, a pair of self-made Ag/AgCl electrodes, electrolyte and an external electrochemical workstation. An opening is arranged in the middle of the electrolytic bath, and the effective test area is 0.5-1mm 2 The electrolyte was a 0.1M Tris buffer solution. During the test, an AlTCPP@MOF film (or a porous anodic aluminum oxide film modified with an aptamer) is added between two electrolytic tanks, and ions can be transported along the electric field direction in a transmembrane mode under the action of an external electric field.
Under an applied electric field, a current-voltage curve (I-V) was recorded. And (3) exploring the influence rule of targets with different concentrations on the nanopore ion detection signal by analyzing the ion current change before and after the reaction of the aptamer and the target.
The results are shown in FIG. 3, wherein the abscissa represents the microcystin solution concentration and the ordinate represents the limit of quantification, and it can be intuitively seen that the AlTCPP@MOF membrane prepared in example 1 has a stronger current signal, the signal is enhanced with the increase of concentration, the linear range of coverage is 0.1ng/mL-1000ng/mL, and the limit of detection is about 0.004ng/mL; whereas the aptamer-modified porous anodized aluminum film prepared in comparative example 1 showed little change in signal with increasing concentration.
To demonstrate the specificity of the AlTCPP@MOF membrane of the present application for detecting microcystin MC-LR, the applicant detected LY, RR and YR using the AlTCPP@MOF membrane prepared in example 1, MC being a monocyclic heptapeptide substance with significant hepatotoxicity. Due to the difference in the composition of the two variable amino acids in polypeptides, there are multiple isomers. Among the most common and most abundant are the 3 microcystins MC-LR, MC-RR, MC-YR (L, R, Y stands for leucine, arginine and tyrosine, respectively).
As shown in FIG. 4, the detection signals of the AlTCPP@MOF film prepared in example 1 on MC-LR, MC-LY, MC-RR and MC-YR are far stronger than those of the detection signals of the AlTCPP@MOF film on MC-LR, so that the AlTCPP@MOF film has a specific recognition effect on MC-LR.
The above is not relevant and is applicable to the prior art.
While certain specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the foregoing examples are provided for the purpose of illustration only and are not intended to limit the scope of the invention, and that various modifications or additions and substitutions to the described specific embodiments may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the invention as defined in the accompanying claims. It should be understood by those skilled in the art that any modification, equivalent substitution, improvement, etc. made to the above embodiments according to the technical substance of the present invention should be included in the scope of protection of the present invention.
Claims (9)
1. The preparation method of the AlTCPP@MOF film based on the porous alumina nano channel is characterized by comprising the following steps of:
s1, adopting a solvothermal synthesis method, taking TCPP as a ligand to generate an MOF structure on a porous anodic alumina film in situ, and preparing an AlTCPP@MOF material;
s2, soaking the AlTCPP@MOF material obtained in the step S1 in an EDC/NHS mixed solution for reaction to obtain a carboxyl activated AlTCPP@MOF material;
s3, bonding and fixing the carboxyl-activated AlTCPP@MOF material obtained in the step S2 and an amino-modified nucleic acid aptamer on the AlTCPP@MOF material through an amide bond to obtain an AlTCPP@MOF film, wherein the amino-modified nucleic acid aptamer has the nucleotide sequence as follows:
5’-NH 2 -C 6 -GGCGCCAAACAGGACCACCATGACAATTACCCATACC
ACCTCATTATGCCCCATCTCCGC-3’。
2. the method according to claim 1, wherein in step S1, the porous anodic alumina membrane comprises a plurality of nanochannels having an average pore diameter of 80 to 110nm and an average density of 10 -11 ~10 -12 Individual/cm 2 The thickness of the porous anodic aluminum oxide film is 20-25 mm.
3. The method according to claim 1, wherein in step S1, the process for preparing the altcpp@mof material is as follows: dissolving TCPP in DMF aqueous solution, uniformly mixing to obtain mixed solution, adding a porous anodic aluminum oxide film into the mixed solution, carrying out ultrasonic treatment for 15-20 min, transferring into a reaction kettle, reacting for 2-4 h at 120-140 ℃ to obtain a synthetic product, washing the synthetic product with DMF and water respectively, and vacuum drying at room temperature to obtain the AlTCPP@MOF material.
4. The preparation method of claim 1, wherein in the step S2, the process of preparing the carboxyl-activated altcpp@mof material is to prepare an EDC/NHS solution with a volume ratio of 2:1-3:1 by using a PBS buffer solution with a pH of 7.4±0.1, soak the altcpp@mof material in the EDC/NHS solution, and react for 0.5-1 h to activate carboxyl groups exposed on the inner and outer surfaces of the altcpp@mof material.
5. The preparation method of claim 1, wherein in step S3, the altcpp@mof is soaked in a solution of an amino-modified aptamer for a reaction time of 10-12 hours.
6. An altcpp@mof film prepared by the method of any one of claims 1-5.
7. The use of the altcpp@mof film according to claim 6, wherein said altcpp@mof film is used for qualitative and quantitative analysis of microcystins.
8. A method for detecting microcystins is characterized in that an AlTCPP@MOF film in claim 6 is soaked in microcystins solution and reacts for 1-4 hours, and microcystins are combined with a nucleic acid aptamer specific structure, so that microcystins are detected.
9. The method for detecting microcystins according to claim 8, wherein the feeding ratio of the microcystins solution to the AlTCPP@MOF film is 100-150 mu L, 0.3-0.5cm 2 The concentration of the microcystin solution is 0.1-1000 ppb.
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| CN108178120A (en) * | 2017-12-27 | 2018-06-19 | 温州大学 | The pattern of anodic alumina films surface nanometer array and controlled in nanometer confinement space its pattern grow method, application |
| CN109696430B (en) * | 2019-03-01 | 2021-07-27 | 长江师范学院 | Method for measuring concentration of microcystin |
| CN114561451B (en) * | 2022-02-23 | 2023-12-05 | 中国地质大学（武汉） | Precise modified nano pore canal membrane and preparation method and application thereof |
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