CN108845014B - Molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode and construction method and application thereof - Google Patents
Molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode and a construction method and application thereof, the method comprises the steps of firstly reconstructing and combining a cholesterol-chitosan carbonate/histamine mixed component monomolecular membrane at an air/water interface by using L angmuir technology through the interaction force of functional groups between amphiphilic cholesterol-chitosan carbonate and histamine, then transferring the mixed component monomolecular membrane to an ITO glass electrode by using L B technology, then eluting the histamine from the mixed component L B membrane by using a molecular imprinting method to prepare a molecularly imprinted L B membrane electrode for selectively identifying the histamine, and finally measuring that the molecularly imprinted L B membrane electrode has high electrochemical responsiveness to the histamine, wide linear histamine detection range, low detection limit, good selectivity, repeatability and stability by combining an electrochemical method, and realizing rapid and sensitive detection of the histamine in trace amounts in beer and wine.
Description
Technical Field
The invention belongs to the field of functional polymer ultrathin membranes, and particularly relates to a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode and a construction method and application thereof.
Background
Histamine (HA), 2- (4-imidazole) -ethylamine, is produced in humans by the catalytic decarboxylation of histidine by Histidine Decarboxylase (HDC), is one of the widely distributed and powerful bioactive substances in human tissue cells. Histamine is involved in the regulation of various physiological functions in vivo, such as cardiovascular circulation, and the pathological processes of diseases, such as tumors, through the mediation of G-protein coupled receptors. With the aid of modern genetic engineering techniques, the structure of the histamine receptor and its interaction with histamine have already been investigated at the genetic level. To date, there are many methods for measuring histamine content in living organisms, such as fluorescence analysis, high performance liquid chromatography, etc. widely used in blood plasma and tissue detection. But the defects of the method cause that the method is difficult to meet the requirements of people on simplicity and high efficiency of the test analysis process, such as a complicated extraction process of a sample is required before the test by a fluorescence analysis method; the test sample of the high performance liquid chromatography consumes long time and large amount. Therefore, the development of a sensor that can be tested directly and give results quickly and accurately is highly desirable.
L angmuir membrane technology is to prepare ordered and dense amphiphilic monolayer (i.e. L angmuir membrane) on air/liquid interface, forming L angmuir membrane with uniform and precisely controllable thickness and highly ordered arrangement of molecules in the membrane, Britt research group using L angmuir monolayer surface imprinting method, using cationic and non-ionic liposomes to form L angmuir monolayer at the air-water interface, liposome end groups will undergo structural reorganization with the added protein molecules, allowing part of their reorganized end group structure to complement the protein molecules in shape and charge, thus achieving western blotting the molecularly imprinted membrane prepared by L B method, with uniform distribution, easy and simple operation, controllable membrane thickness, etc. this method has the advantage of good mass transfer and ability to combine with sensor platform, so far the work related to imprinting with non-liposomal amphiphilic compounds has not been reported, L angmuir-Blodgett (L B) membrane technology is to prepare the above mentioned monolayer-Blodgett (L) membrane technology and to achieve the important molecular transfer of the above mentioned technology to the air/liquid interface and to the monolayer, thus achieving the precise molecular assembly of the membrane.
Chitosan is a derivative of natural macromolecule chitin through deacetylation, and is characterized in that a large number of reactive amino and hydroxyl groups are contained at the C2, C3 and C6 sites on a molecular chain, so that functional groups with covalent bond combination, electrostatic interaction and coordination can be introduced through structural modification（Tong Y, et al. Synthesis ofO,O' -dipalmitoyl chitosans and itsamphio phenolic properties and capacity of cholestrol adsorption. Carbohydr. Polym., 2005, 60(2): 229-233. Studies show that amphiphilic chitosan derivatives can be used as raw material for L angmuir membranes that adsorb and recognize perfluorooctanesulfonate (Zhanli. Studies on the amphiphilicity of chitosan derivatives at the air-water interface and their interaction with histamine. Master thesis, 2016, 40-50).
The amphiphilic molecularly imprinted membrane material is based on amphiphilic cholesterol-chitosan carbonate formed by introducing a cholesterol hydrophobic chain end on a chitosan molecular chain hydroxyl and reserving an amino hydrophilic end on the chitosan molecular chain. The molecular chain of the imprinted molecular histamine of the invention has amino and imidazolyl functional groups. The cholesterol-chitosan carbonate and the histamine can interact with each other by means of various forms of interaction force such as hydrogen bond, electrostatic interaction, van der waals force and the like, so that the interaction between the chitosan derivative and the histamine is realized.
The method comprises the steps of firstly forming a L angmuir membrane at an air/water interface by utilizing cholesterol-chitosan carbonate with amphipathy and reactive amino groups on molecular chains through L angmuir technology, forming a cholesterol-chitosan carbonate/histamine mixed component L angmuir membrane by the combination of the amphipathy cholesterol-chitosan carbonate with histamine in an aqueous phase through the interaction reconstruction of the amphipathy cholesterol-chitosan carbonate and the histamine on the surface of a L angmuir tank, then transferring the mixed component monomolecular membrane onto an Indium Tin Oxide (ITO) glass electrode by adopting L B technology, then preparing a histamine serving as a target molecule from a mixed component L B membrane through elution by utilizing molecular imprinting technology to form a histamine selective histamine-recognizing molecular imprinting L B membrane electrode matched with cavities of the structure and the size of histamine molecules, thereby realizing the interaction between the histamine derivative and the histamine specificity, finally combining an electrochemical method, researching the electrochemical response and recognition performance of the molecular imprinting L B membrane electrode for recognizing the histamine, and applying the molecular imprinting electrode to the detection of beer and grape modified actual samples, realizing the quick sensitive detection of the histamine, the electrochemical detection of the amphipathic alcohol and the high-trace-quantity of the working electrode and the electrochemical stability of the ITO and the wide-based beer.
Disclosure of Invention
The method utilizes a plurality of reactive amino groups on a cholesterol-chitosan carbonate molecular chain, and histamine molecules have amino and imidazolyl functional groups, and can realize the interaction between the chitosan derivative and histamine through various forms of interaction forces of hydrogen bonds, electrostatic interaction and van der Waals force.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode comprises the following steps of (1) preparation of amphiphilic cholesterol-chitosan carbonate and histamine solution
Dissolving cholesterol-chitosan carbonate in chloroform to prepare a series of cholesterol-chitosan carbonate solutions with different concentrations, fixing the volume in a volumetric flask for later use, dissolving histamine in ultrapure water to prepare a histamine solution with the concentration of 1.0 mg/m L, and storing the solution at low temperature for later use;
(2) cholesterol-chitosan carbonate/histamine mixed component L angmuir film formation
After the earlier stage work is completed according to the requirements of L angmuir tank operation method, adding ultrapure water (the resistivity is not less than or equal to)
18.25 Ω · m) as subphase, adding 100 μ L aqueous histamine solution (diluted from the aqueous histamine solution prepared in step (1) with concentration of 0.2 mg/m L dropwise with a microsyringe, mixing well, adding cholesterol-chitosan carbonate solution prepared in step (1) dropwise with a microsyringe, slowly adding the solution to the subphase water surface, spreading the solution on the water surface, returning the pressure sensor to zero when the chloroform is completely volatilized, and setting the barrier velocity to 50cm2(ii)/min, start compression of the A and B barriers to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), record the monolayer surface pressure-average monolayer area isotherm curve and collapse pressure of the mixed component film to determine;
(3) transfer of Cholesterol-Chitosan carbonate/Histamine Mixed component L angmuir film
According to the operation method requirement of L angmumir tank, putting the pretreated ITO electrode into L angmumir tank, respectively dripping histamine and cholesterol-chitosan carbonate solution into subphase according to the step (2), after forming cholesterol-chitosan carbonate/histamine mixed monolayer film, using L angmumir-Blodgett (L B) technology to pull the mixed component monomolecular film at the speed of 5 mm/s to finish the film transfer, depositing the film on the ITO substrate, and forming the ITO glass electrode modified by the cholesterol-chitosan carbonate/histamine mixed component L B film;
(4) preparation of molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode
Washing the ITO glass electrode surface modified by the cholesterol-chitosan carbonate/histamine mixed L B membrane formed in the step (3) with a certain amount of ultrapure water for a certain time, and removing histamine template molecules to obtain a histamine-recognized molecularly imprinted cholesterol-chitosan carbonate L B membrane modified electrode.
(5) Experiment for identifying histamine by molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode
An electrochemical three-electrode system is utilized, the potential range of cyclic voltammetry is-0.4-0.8V, and the scanning speed is 50 mV/s; with ethanol and 0.2M KCl and 1mM K3Fe(CN)6As a water mixed solution ofA test solution system of an oxidation-reduction probe is adopted to probe the adsorption performance of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode (called a molecularly imprinted L B membrane for short) on histamine, the electrochemical responsiveness of the molecularly imprinted L B membrane modified electrode in the presence or absence of histamine (3 ml 1 mu M) is respectively tested, the result shows that under the optimized adsorption condition, the molecularly imprinted L B membrane can recognize histamine molecules, while the non-molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode (called a non-molecularly imprinted L B membrane for short, and the process of imprinting without adding the histamine molecules) prepared by the same method has almost no response to histamine, namely has higher sensitivity in the aspect of detecting the target molecule of the histamine, the peak current response (i) of a linear scanning voltammetry curve is in a linear relation with the histamine concentration within the range of 0.01-1.5 mu M, the detection limit (S/N =3) is 0.09 mu M, and the relative standard deviation of the response peak current of the same molecularly imprinted L B membrane to the histamine is 2.4% (N = 6.4%).
(6) Construction of non-molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode and electrochemical responsiveness thereof in histamine solution
① Cholesterol-chitosan carbonate single layer film is formed by adding ultrapure water as subphase into L angmuir tank after the previous work is completed according to the requirement of operation method of the tank, transferring 100 mu L of the cholesterol-chitosan carbonate chloroform solution prepared in the step (1) by a microsyringe, slowly dripping the chloroform solution on the subphase water surface to spread the chloroform on the water surface, after the chloroform is completely volatilized, returning the pressure sensor to zero, and fixing the sliding barrier speed to 50cm2Min, starting to compress the sliding barriers A and B to obtain an orderly and densely arranged cholesterol-chitosan carbonate L angmuir film, recording the surface pressure-average monomolecular film area isothermal curve and the collapse pressure of L angmuir film, repeating each isothermal curve at least three times to ensure reproducibility, ② transferring the cholesterol-chitosan carbonate L angmuir film, putting the pretreated ITO electrode into L angmuir cell according to the operation method of L angmuir cell, dripping the cholesterol-chitosan carbonate solution into subphase according to the step ①, stopping compression when the sliding barriers A and B are compressed to the state that the cholesterol-chitosan carbonate monolayer film corresponds to the surface pressure at about half of the solid isothermal curve section, and then utilizing L angmuiruir-Blodgett (L B) technology, wherein a monomolecular film is lifted at a speed of 5 mm/s to complete film transfer, so that the film is deposited on an ITO substrate to form 4 layers of cholesterol-chitosan carbonate L B film modified ITO glass electrodes, ③ non-molecularly imprinted amphiphilic chitosan derivatives L B film modified electrodes are manufactured, i.e. the ITO glass electrode surface modified by the cholesterol-chitosan carbonate L B film formed in the step ② is rinsed with a certain amount of ultrapure water for a certain time, histamine template molecules are removed to obtain a non-histamine-identified molecularly imprinted cholesterol-chitosan carbonate L B film modified electrode (marked as non-ITO A), ④ non-ITO A has electrochemical responsiveness in a histamine solution, i.e. the non-histamine-identified molecularly imprinted cholesterol-chitosan carbonate L B film modified electrode is tested by a cyclic voltammetry method under the condition that the electrochemical responsiveness of histamine is almost consistent with the histamine concentration of 3 ml 1 mu M or is not existed in the condition that histamine is contained in the condition that a redox probe test solution system is adopted, and the cyclic voltammetry method shows that the electrochemical response of the histamine is almost consistent with the histamine-containing histamine, and the cyclic voltammetry method shows that the electrochemical response of the group of the;
(7) identification selectivity experiment of molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode
5 groups of solutions including 1 mu M histamine solution, a solution containing 5 mu M histidine and 1 mu M histamine, a solution containing 5 mu M dopamine and 1 mu M histamine, and a solution containing 5 mu M histidine, dopamine and 1 mu M histamine are respectively prepared, the same molecular imprinting L B membrane modified electrode is respectively identified and adsorbed, and after 8-16 min, the electrochemical response signal is measured to find that the peak current difference is very small, because cavities left by the histamine imprinting on the surface of the electrode are only matched with the structure of the histamine, and the histidine and the dopamine cannot be matched with the structure, therefore, the histidine and the dopamine have no interference with the identification of the histamine by the molecular imprinting L B membrane modified electrode.
In the method of the present invention, the cholesterol-chitosan carbonate is added in the amount of 25-300. mu. L (concentration of 1.0 mg/m L) in steps (2) and (3).
In the method of the present invention, the membrane of cholesterol-chitosan carbonate/histamine mixed component L B described in step (3) may have 1 to 30 layers, and the membrane of cholesterol-chitosan carbonate L B described in step (6) may have 1 to 30 layers.
In the method, the mode for washing the ITO glass electrode surface modified by the cholesterol-chitosan carbonate/histamine mixed L B membrane in the step (4) comprises soaking washing and rinsing, wherein the soaking washing water amount is 5-100 m L, the washing time is 8-20 min, the rinsing water amount is 5-20 m L, and the washing time is 30-100 s.
In the method, the adsorption time of the molecular engram L B membrane modified electrode in the step (5) for adsorbing histamine is 2-20 min, the linear sweep voltammetry in the step (5) is used for measuring the relation between the peak current response (Δ i) and the histamine concentration, and the histamine concentration is observed to be 0.01-500 mu M.
In the method of the present invention, the structural analogs mentioned in step (7) are histidine and dopamine in an amount of 0.5-35. mu.M.
The molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode prepared by the construction method is provided by the invention.
The molecular imprinting amphiphilic chitosan derivative L B membrane modified electrode prepared by the construction method is used for detection, and the specific steps are that a molecular imprinting amphiphilic chitosan derivative L B membrane is introduced to the surface of an ITO glass conductive electrode, so that the modified electrode is endowed with selective identification on histamine, the modified electrode is used for detecting histamine in beer and wine actual samples by a standard sample adding method, the average recovery rates of the beer and wine samples are respectively 98.3-105.9% and 99.8-105.2%, and the relative standard deviation of electrochemical response peak current is less than 4.5% (n = 3).
The invention relates to a corresponding non-molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode, which is obtained by washing and histamine removing steps of a cholesterol-chitosan carbonate single-component L B membrane modified electrode.
The molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode provided by the invention and the construction method and the application thereof have the advantages that:
(1) the cholesterol-chitosan carbonate serving as the molecular imprinting ultrathin film base material is formed by combining natural high-molecular chitin and cholesterol in a covalent form after deacetylation, and has the advantages of biocompatibility, biodegradability, bioactivity, low immunogenicity and the like;
(2) the invention takes a cholesterol-chitosan carbonate and histamine mixed component monomolecular layer L B multilayer film as a molecular engram ultrathin film precursor, L B has thin film, uniform thickness and controllable film thickness, so that the elution of template molecules is faster and more complete in the process of preparing the molecular engram film, and the elution efficiency of the template molecules directly influences the recognition and separation capability of the molecular engram film;
(3) the invention utilizes the characteristics of thin film thickness, controllable, uniform and compact film thickness and the like of the L B film, the characteristics of predetermination, identification and practicability of a molecular imprinting technology, and the advantages of rapidness, convenience in detection and the like of an electrochemical method, and combines the three methods to assemble the histamine molecular imprinting L B film on the surface of the glass electrode.
(4) The molecular imprinting amphiphilic cholesterol-chitosan carbonate L B film imprinting method adopted by the invention is a novel and exquisite surface molecular imprinting technology at present, and the novel L B film surface molecular imprinting method not only provides an effective method for environmental monitoring, but also provides a theoretical basis for researching other high molecular material surface functionalized molecular imprinting polymers.
The invention is used for the structural design of the molecularly imprinted polymer ultrathin film sensitive element, the construction of the modified conductive electrode and the specific detection of target molecules.
Drawings
FIG. 1 is a Surface pressure-average monolayer Area (Area) isotherm curve of a monomolecular film of an amphiphilic cholesterol-chitosan carbonate monocomponent (a) and its mixed component with histamine (b) prepared in the present invention.
FIG. 2 is a transfer curve of an amphiphilic cholesterol-chitosan carbonate/histamine mixed component monomolecular film prepared in the present invention.
FIG. 3 is a graph comparing electrochemical responses of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode (I) and a non-molecularly imprinted L B membrane modified electrode (II) constructed according to the invention.
FIG. 4 is a selectivity diagram of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode constructed by the invention.
Detailed Description
The present invention will be described in detail with reference to the following examples and accompanying drawings
The invention utilizes the structural characteristics and the interaction mechanism of an amphiphilic polymer and a template molecule histamine to form an amphiphilic chitosan derivative/histamine mixed component monomolecular layer at an L angmuir groove air/subphase interface, transfers the mixed component monomolecular layer to an ITO surface by means of a L B membrane technology to further construct a L B membrane and a molecular imprinting L B membrane, compared with the traditional molecular imprinting material, the constructed molecular imprinting polymer L B membrane is a novel molecular imprinting material, has the characteristics of good biocompatibility, biodegradability, low toxicity, cell compatibility and the like, can simulate cell membranes and serve as a drug delivery carrier, compared with the traditional molecular imprinting method, the molecular imprinting method adopted by the invention is a novel exquisite surface imprinting technology, not only overcomes the defect that the imprinted template molecules in the conventional molecular imprinting method are incompletely eluted from a bulk molecular imprinting material, but also accelerates the recombination of the molecular imprinting ultrathin membrane material and a target molecule through an imprinting site, and the molecular imprinting ultrathin surface imprinting technology must become the development field of the molecular imprinting material.
Example 1
Synthesis of amphiphilic cholesterol-chitosan carbonate. The method comprises the following steps: first, 5.49 g of cholesteryl chloroformate was dissolved in a reaction vesselN, N-dimethylformamide in the presence of 2.4 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and with stirring for 5 h (50 ℃) to give an activated cholesteryl chloroformate solution; then in another container add 1.0g, stirring the chitosan and the methanesulfonic acid until the chitosan is completely dissolved, dropwise adding the activated cholesteryl chloroformate solution obtained in the step under the protection of nitrogen, and reacting for 60 hours at 50 ℃. And precipitating, centrifuging, washing and freeze-drying the product mixed system to obtain the cholesterol chitosan carbonate solid. The chemical structure of the product is verified by infrared spectroscopy and nuclear magnetic carbon spectroscopy.
Example 2
(1) Preparation of amphiphilic cholesterol-chitosan carbonate and histamine solution
Dissolving histamine in ultrapure water to prepare a histamine solution with the concentration of 1.0 mg/m L, and storing the solution at low temperature for later use;
(2) cholesterol-chitosan carbonate/histamine mixed component L angmuir film formation
After the earlier stage work is completed according to the requirements of L angmuir tank operation method, adding ultrapure water (the resistivity is not less than or equal to)
18.25. omega. m) as a subphase, an aqueous histamine solution having a concentration of 0.2 mg/m L (diluted with the aqueous histamine solution prepared in step (1) of example 2) of 100. mu. L was added dropwise thereto using a microsyringe]After mixing, 25 μ L of the cholesterol-chitosan carbonate solution prepared in step (1) of example 2 was transferred by a microsyringe, slowly added dropwise to the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor of the L angmuir cell was set to zero at a fixed barrier velocity of 50cm2Permin, compression of the A and B dams in L angmuir cells was started to obtain an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), and the surface pressure-average monolayer area isotherm curve of the monolayer and the collapse pressure of the mixed component film were recorded to determine the surface pressure for monolayer transfer the operation of step (2) in this example 2 was repeated at least three times to ensure reproducibility.
Example 3
Cholesterol-chitosan carbonate/histamine mixture component L ANGMuir film formation following the procedure of L ANGMuir cellAfter the completion of the previous work, ultrapure water (resistivity: 18.25. omega. m or more) was added as a subphase to the tank, and a 100. mu. L aqueous histamine solution (diluted from the aqueous histamine solution prepared in step (1) of example 2) having a concentration of 0.2 mg/m L was added dropwise thereto using a microsyringe]After mixing, 50 μ L of the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was transferred by a microsyringe, slowly dropped onto the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was returned to zero at a fixed barrier velocity of 50cm2Permin, compression of the A and B barriers was started to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), and the surface pressure-average monolayer area isotherm curve of the monolayer and the collapse pressure of the mixed component film were recorded to determine the surface pressure for monolayer transfer the procedure of this example 3 was repeated at least three times to ensure reproducibility.
Example 4
Cholesterol-chitosan carbonate/histamine mixture component L angmuir film formation after completion of the preceding work as required by the procedure for operation of L angmuir cell, ultrapure water (resistivity 18.25. omega. m or more) was added to the cell as a subphase, and an aqueous histamine solution of 100. mu. L at a concentration of 0.2 mg/m L (diluted from the aqueous histamine solution prepared in step (1) of example 2) was added dropwise thereto using a microsyringe]After mixing, the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was sampled at 75 μ L using a microsyringe, slowly dropped onto the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was returned to zero at a fixed barrier velocity of 50cm2Permin, compression of the A and B barriers was started to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), and the surface pressure-average monolayer area isotherm curve of the monolayer and the collapse pressure of the mixed component film were recorded to determine the surface pressure for monolayer transfer the procedure of this example 4 was repeated at least three times to ensure reproducibility.
Example 5
Cholesterol-chitosan carbonate/histamine mixture component L angmuir film formation, according to L aAfter the preceding operation of the ngmuir cell was required, ultrapure water (resistivity. gtoreq.18.25. omega. m) was added to the cell as a subphase, and an aqueous histamine solution (diluted from the aqueous histamine solution prepared in step (1) of example 2) of 100. mu. L concentration of 0.2 mg/m L was added dropwise thereto using a microsyringe]After mixing, 100. mu. L of the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was transferred by a microsyringe, slowly dropped onto the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was reset to zero, and the fixed barrier velocity was 50cm2At/min, compression of the a and B slipbarriers was started, resulting in an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), recording the surface pressure-average monolayer area isotherm curve of the monolayer (as shown in fig. 1B) and the collapse pressure of the mixed component film to determine the surface pressure for monolayer transfer, while for comparison with mixed component L angmuir film, a cholesterol-chitosan carbonate monocomponent L angmuir film was made and the surface pressure-average monolayer area isotherm curve of the monolayer (as shown in fig. 1 a) was recorded, each isotherm curve was repeated at least three times to ensure reproducibility.
Example 6
Cholesterol-chitosan carbonate/histamine mixture component L angmuir film formation after completion of the preceding work as required by the procedure for operation of L angmuir cell, ultrapure water (resistivity 18.25. omega. m or more) was added to the cell as a subphase, and an aqueous histamine solution of 100. mu. L at a concentration of 0.2 mg/m L (diluted from the aqueous histamine solution prepared in step (1) of example 2) was added dropwise thereto using a microsyringe]After mixing, 150 μ L of the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was transferred by a microsyringe, slowly dropped onto the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was returned to zero at a fixed barrier velocity of 50cm2Min, compression of the A and B barriers was started to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), and the surface pressure-average monolayer area isotherm curves of the monolayer and collapse pressure of the mixed component film were recorded to determine the monolayerSurface pressure of transfer. The procedure of example 6 was repeated at least three times to ensure reproducibility.
Example 7
Cholesterol-chitosan carbonate/histamine mixture component L angmuir film formation after completion of the preceding work as required by the procedure for operation of L angmuir cell, ultrapure water (resistivity 18.25. omega. m or more) was added to the cell as a subphase, and an aqueous histamine solution of 100. mu. L at a concentration of 0.2 mg/m L (diluted from the aqueous histamine solution prepared in step (1) of example 2) was added dropwise thereto using a microsyringe]After mixing, the cholesterol-chitosan carbonate chloroform solution 200 μ L prepared in step (1) of example 2 was transferred by a microsyringe, slowly dropped on the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was reset to zero and the fixed barrier velocity was 50cm2Permin, compression of the A and B barriers was started to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), and the surface pressure-average monolayer area isotherm curve of the monolayer and the collapse pressure of the mixed component film were recorded to determine the surface pressure for monolayer transfer the procedure of this example 7 was repeated at least three times to ensure reproducibility.
Example 8
Cholesterol-chitosan carbonate/histamine mixture component L angmuir film formation after completion of the preceding work as required by the procedure for operation of L angmuir cell, ultrapure water (resistivity 18.25. omega. m or more) was added to the cell as a subphase, and an aqueous histamine solution of 100. mu. L at a concentration of 0.2 mg/m L (diluted from the aqueous histamine solution prepared in step (1) of example 2) was added dropwise thereto using a microsyringe]After mixing, the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was transferred by a microsyringe at 300. mu. L, slowly dropped on the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was reset to zero, and the fixed barrier velocity was 50cm2Min, compression of the A and B barriers was started to give an ordered dense array of cholesterol-chitosan carbonate/histamine mixed component L angmuir film (monolayer), the surface pressure-average monolayer area isotherm curve of the monolayer and collapse of the mixed component film were recordedThe pressure was applied to determine the surface pressure for monolayer transfer. The procedure of this example 8 was repeated at least three times to ensure reproducibility.
Example 9
Transferring a cholesterol-chitosan carbonate/histamine mixed component L angmuir film according to the operation method of L angmuir tank, respectively performing ultrasonic treatment on acetone, ethanol, 0.1M sodium hydroxide and isopropanol mixture (volume ratio is 1.5: 1) and deionized water for 30 min, and drying the pretreated ITO glass electrode [ namely Indium Tin Oxide (ITO) glass electrode which is a commercially available product]Put into an L angmumir tank, and an aqueous histamine solution of 100. mu. L concentration of 0.2 mg/m L (diluted with the aqueous histamine solution prepared in step (1) of example 2) was added dropwise thereto using a microsyringe]After mixing, 100. mu. L of the cholesterol-chitosan carbonate chloroform solution prepared in step (1) of example 2 was transferred by a microsyringe, slowly dropped onto the subphase water surface to spread it on the water surface, after the chloroform was completely volatilized, the pressure sensor was reset to zero, and the fixed barrier velocity was 50cm2And/min, starting to compress the A and B slide barriers, when the surface pressure of the formed cholesterol chitosan carbonate/histamine mixed component L angmuir film (monomolecular layer) in a dense order is 41.00 mN/m, using L angmuir-Blodgett (L B) technology, pulling the mixed component monomolecular film at the speed of 5 mm/s to complete the film transfer, and depositing 4 films alternately on an ITO substrate to form a cholesterol-chitosan carbonate/histamine mixed component L B film modified ITO glass electrode (the film transfer curve is shown in figure 2), and under the same conditions, constructing 50 modified electrodes, and simultaneously manufacturing the cholesterol-chitosan carbonate component L B film modified ITO glass electrode for comparison with the mixed component L angmuir film modified ITO glass electrode.
Example 10
Preparing a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode, washing the surface of the ITO glass electrode modified by the cholesterol-chitosan carbonate/histamine mixed L B membrane constructed in the embodiment 8 with 10 m L of ultrapure water for 1 min, removing histamine template molecules to obtain a histamine-recognized molecularly imprinted cholesterol-chitosan carbonate L B membrane modified electrode (marked as ITO A), constructing 10 pieces of molecularly imprinted L B membrane modified electrodes respectively under the same conditions, washing the surface of the cholesterol-chitosan carbonate single-component L B membrane modified ITO glass electrode prepared in the embodiment 8 with 10 m L of ultrapure water for 1 min under the same conditions, and preparing a non-molecularly imprinted L B membrane modified electrode (marked as non-ITO A).
Example 11
Preparation of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode the surface of the cholesterol-chitosan carbonate/histamine mixed L B membrane modified ITO glass electrode constructed in example 8 was washed with 50 m L ultrapure water for 10 min, and histamine template molecules were removed to obtain a histamine-recognized molecularly imprinted cholesterol-chitosan carbonate L B membrane modified electrode (labeled as ITO B). under the same conditions, 10 pieces of molecularly imprinted L B membrane modified electrodes were constructed.
Example 12
The molecular imprinting L B film modified electrode ITO A constructed in the example 9 adsorbs histamine by adopting an electrochemical three-electrode system, the potential range of cyclic voltammetry is-0.4-0.8V, the scanning speed is 50 mV/s, and ethanol, 0.2M KCl and 1mMK are adopted3Fe(CN)6The cyclic voltammetry is used for measuring the electrochemical responsiveness of the molecularly imprinted L B membrane modified electrode in the presence or absence of histamine (3 ml of 1 mu M), and the result shows that under the optimized adsorption condition, the molecularly imprinted L B membrane modified electrode rapidly responds to electrochemical signals of histamine [ as shown in figure 3(I), wherein a curve a represents a bare ITO electrode, B represents ITO A, c represents ITO A and binds with histamine molecule HA and is labeled as ITO A-HA [ ], and]whereas the non-molecularly imprinted L B membrane-modified electrode prepared in the same manner as in example 8 (without imprinting with histamine as a template molecule) showed almost no electrochemical response signal to histamine [ as shown in FIG. 3(II), in which curve a represents a bare ITO electrode, B represents non-ITO A, c represents non-ITO A and HA is labeled non-ITO A-HA in combination with histamine]The peak current response (Δ i) of a linear scanning voltammetry curve is in a linear relation with the histamine concentration in the range of 0.01-1.50 μ M, the detection limit (S/N =3) is 0.09 μ M, and the relative standard deviation of the peak current response of the same molecular imprinting L B membrane modified electrode to the histamine is 2.4% (N = 6).
Example 13
The molecular imprinting L B film modified electrode ITO B constructed in the example 9 adsorbs histamine by adopting an electrochemical three-electrode system, the potential range of cyclic voltammetry is-0.4-0.8V, the scanning speed is 50 mV/s, and ethanol, 0.2M KCl and 1mMK are adopted3Fe(CN)6The cyclic voltammetry was used to measure the electrochemical responsiveness of the molecularly imprinted L B membrane modified electrode in the presence or absence of histamine (3 ml 1. mu.M), and the results showed that under optimized adsorption conditions, the molecularly imprinted L B membrane modified electrode rapidly responds to the electrochemical signal of histamine, while the non-molecularly imprinted L B membrane modified electrode prepared by the same method as example 8 (no imprinting process with addition of the template molecule histamine) had almost no electrochemical response signal to histamine, i.e., the molecularly imprinted L B membrane modified electrode had higher sensitivity in detecting the target molecule of histamine. the peak current response of the linear scanning curve (voltammetry i) was linear with the histamine concentration in the range of 0.01-1.45. mu.M, with a detection limit (S/N =3) of 0.10. mu.M, and the relative standard deviation of the response peak current of the same molecularly imprinted L B membrane modified electrode to histamine was 2.5% (N = 6).
Example 14
A1 mu M histamine solution (marked as HA), a solution containing 5 mu M histidine and 1 mu M histamine (marked as HA-HD), a solution containing 5 mu M dopamine and 1 mu M histamine (marked as HA-DM), a solution containing 5 mu M histidine, dopamine and 1 mu M histamine (marked as HA-HD-DM) are respectively prepared, the molecular imprinting L B membrane modified electrode A or B constructed in example 9 is respectively inserted into the solution, and after 8 min, the electrochemical response signals of the molecular imprinting electrode A or B show very small difference of peak current values, because cavities left by the histamine imprinting on the surface of the electrode are only matched with histamine molecules, and the histidine and the dopamine serving as histamine structural analogs cannot be matched with the histamine molecules, so the histidine and the dopamine have no interference with the ITO A of the molecular imprinting L B membrane modified electrode (shown in figure 4).
Example 15
The histamine in the beer actual sample was measured by the standard sample addition method, 0.5, 1.0 and 1.5. mu.M histamine solutions were added to the test solutions, respectively, and the average recovery rates of histamine were 105.9, 103.0 and 98.3%, respectively, and the standard deviations were 3.5, 2.6 and 2.7%, respectively, as detected by the molecular imprinting L B membrane-modified electrode (ITO A) constructed in example 9, thereby realizing rapid and sensitive detection of trace amounts of histamine in beer and wine.
Example 16
The histamine in the actual sample of wine was measured by the standard sample addition method, 0.5, 1.0 and 1.5. mu.M histamine solutions were added to the test solutions, respectively, and the average recovery rates of histamine were 104.4, 99.8 and 105.2%, respectively, and the standard deviations were 1.9, 3.5 and 4.3%, respectively, as detected by the molecularly imprinted L B membrane-modified electrode (ITO B) constructed in example 9, thereby realizing rapid and sensitive detection of trace amounts of histamine in beer and wine.
The above-described embodiments are further detailed descriptions of the present invention, but the scope of the claims of the present invention is not limited to the scope described in the embodiments, and all technical solutions using equivalent substitutions or equivalent modifications fall within the scope of the claims of the present invention.
Claims (10)
1. A construction method of a molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode is characterized by comprising the following steps:
(1) preparation of amphiphilic cholesterol-chitosan carbonate and histamine solution
Dissolving cholesterol-chitosan carbonate in chloroform to prepare a series of cholesterol-chitosan carbonate solutions with different concentrations, fixing the volume in a volumetric flask for later use, dissolving histamine in ultrapure water to prepare a histamine solution with the concentration of 1.0 mg/m L, and storing the solution at low temperature for later use;
(2) cholesterol-chitosan carbonate/histamine mixed component L angmuir film formation
Adding ultrapure water as a subphase into an L angmuir tank, and adding 100 μ L diluted histamine solution prepared in step (1) and having a concentration of 0.2 mg/ml by a microsyringem L, uniformly mixing, transferring the cholesterol-chitosan carbonate chloroform solution prepared in the step (1) by using a micro-sampler, slowly dripping the chloroform solution on a subphase water surface to spread the chloroform solution on the water surface, and after the chloroform is completely volatilized, returning a pressure sensor to zero, wherein the fixed sliding barrier speed is 50cm2The slide barrier is compressed at min, a cholesterol-chitosan carbonate/histamine mixed component L angmuir film which is orderly and densely arranged is obtained, the surface pressure-average monomolecular film area isothermal curve and the collapse pressure of the mixed component film are recorded, and each isothermal curve is repeated at least three times to ensure the reproducibility;
(3) transfer of Cholesterol-Chitosan carbonate/Histamine Mixed component L angmuir film
Putting the pretreated ITO glass electrode into a L angmuir tank, adding ultrapure water into an L angmuir tank to serve as a subphase, dropwise adding a 100 mu L histamine water solution prepared in the step (1) into a histamine water solution with the concentration of 0.2 mg/m L by using a microsyringe, uniformly mixing, transferring the cholesterol-chitosan carbonate chloroform solution prepared in the step (1) by using a microsyringe, slowly dropwise adding the cholesterol-chitosan carbonate chloroform solution onto the subphase water surface, spreading the cholesterol-chitosan carbonate chloroform solution on the water surface, completely volatilizing the chloroform, returning a pressure sensor to zero, and fixing the sliding barrier speed to 50cm2And/min, starting to compress the sliding barrier, stopping compressing when the sliding barrier is compressed to the surface pressure of the corresponding monomolecular film when the cholesterol-chitosan carbonate/histamine mixed component L angmuir film in the step (2) is half of the surface pressure of the monomolecular film-average monomolecular film area isothermal curve solid section, and then using L angmuir-Blodgett (L B) technology to pull the mixed component monomolecular film at the speed of 5 mm/s to finish the film transfer so that the film is deposited on an ITO substrate to form an ITO glass electrode modified by the cholesterol-chitosan carbonate/histamine mixed component L B film;
(4) preparation of molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode
Washing the ITO glass electrode surface modified by the cholesterol-chitosan carbonate/histamine mixed L B membrane formed in the step (3) with a certain amount of ultrapure water for a certain time, and removing histamine template molecules to obtain a molecular imprinting cholesterol-chitosan carbonate L B membrane modified electrode for identifying histamine;
(5) recognition performance of molecular imprinting amphiphilic chitosan derivative L B membrane modified electrode on histamine
An electrochemical three-electrode system is adopted, the potential range of cyclic voltammetry is-0.4-0.8V, and the scanning speed is 50 mV/s; with ethanol and 0.2M KCl and 1mM K3Fe(CN)6The electrochemical responsiveness of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode in the presence or absence of histamine with the concentration of 3M L of 1 mu M is respectively tested by adopting a cyclic voltammetry to test the adsorption performance of the molecularly imprinted L B membrane modified electrode on histamine, and the result shows that under the optimized adsorption condition, the molecularly imprinted L B membrane modified electrode can identify histamine molecules, namely, the sensitivity in detecting the target molecule of histamine is high, the peak current response (Δ i) of a linear scanning voltammetry curve is in a linear relation with the histamine concentration within the range of 0.01-1.50 mu M, the detection limit S/N =3 is 0.09 mu M, and the relative standard deviation of the same imprinted L B membrane modified electrode to the histamine after 6 cycles of adsorption-desorption on the peak current N =6 of the histamine is 2.4%.
2. The method for constructing the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the non-molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode is constructed and the electrochemical responsiveness thereof in histamine solution
① Cholesterol-chitosan carbonate single layer film is formed by adding ultrapure water as a subphase into a L angmuir tank, transferring 100 mu L of the cholesterol-chitosan carbonate chloroform solution prepared in the step (1) by using a microsyringe, slowly dripping the cholesterol-chitosan carbonate chloroform solution on the subphase water surface to spread the subphase water surface, returning a pressure sensor to zero when the chloroform is completely volatilized, and fixing the sliding barrier speed to 50cm2At min, starting to compress the sliding barrier to obtain an orderly and densely arranged cholesterol-chitosan carbonate L angmuir film, recording the surface pressure-average monomolecular film area isothermal curve and the collapse pressure of L angmuir film, and repeating each isothermal curve at least three times to ensure the reproducibility;
② transferring the cholesterol-chitosan carbonate L angmuir film, namely putting the pretreated ITO glass electrode into a L angmuir groove, dropwise adding a cholesterol-chitosan carbonate solution into a sub-phase according to a step ①, stopping compression when a sliding barrier is compressed to the surface pressure of the corresponding monomolecular film when the surface pressure of the cholesterol-chitosan carbonate L angmuir film is half of the solid section of the isothermal curve of the surface pressure-average monomolecular film area, and then lifting the monomolecular film at the speed of 5 mm/s by using L angmuir-Blodgett (L B) technology to finish the transfer of the film, so that the film is deposited on an ITO substrate to form the ITO glass electrode modified by the cholesterol-chitosan carbonate L B film;
③ preparing non-molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode, namely rinsing the surface of the ITO glass electrode modified by the cholesterol-chitosan carbonate L B membrane formed in the step ② with a certain amount of ultrapure water for a certain time, removing histamine template molecules to obtain the non-histamine-recognized molecularly imprinted cholesterol-chitosan carbonate L B membrane modified electrode which is marked as non-ITO A;
④ electrochemical responsiveness of non-ITO A in histamine solution the electrochemical responsiveness of non-ITO A in the presence or absence of histamine at a concentration of 3 ml at a concentration of 1 μ M was tested by cyclic voltammetry using the same electrochemical three-electrode system and redox probe test solution system as in step (5), and the results showed that the cyclic voltammetry curves were almost consistent with or without histamine, meaning that the non-ITO A produced almost no electrochemical response to histamine.
3. The method for constructing the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode has the recognition selectivity:
the method comprises the specific steps of respectively preparing 4 groups of solutions including 1 mu M histamine solution, 5 mu M histidine and 1 mu M histamine solution, 5 mu M dopamine and 1 mu M histamine solution and 5 mu M histidine, dopamine and 1 mu M histamine solution, respectively identifying and adsorbing the solutions by using the same molecular imprinting L B membrane modified electrode, and detecting electrochemical response signals of the electrodes after 8-16 min to find that the peak current difference is very small, wherein cavities left by histamine blots on the surface of the electrode are only matched with the histamine structure, and histidine and dopamine cannot be matched with the histamine structure, so that the histidine and dopamine have no interference with histamine identification of the molecular imprinting amphiphilic chitosan derivative L B membrane modified electrode.
4. The method for constructing the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the concentration of the cholesterol-chitosan carbonate in the steps (2) and (3) is 1.0 mg/m L, and the addition amount is 25-300 μ L.
5. The method for constructing the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 2, wherein the cholesterol-chitosan carbonate/histamine mixed component L B membrane in the step (3) is 1-30 layers, and the cholesterol-chitosan carbonate L B membrane is 1-30 layers.
6. The construction method of the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the washing manner for washing the cholesterol-chitosan carbonate/histamine mixed L B membrane modified ITO glass electrode surface in the step (4) comprises soaking washing and rinsing, the soaking washing water amount is 5-100 m L, the washing time is 8-20 min, the rinsing water amount is 5-30 m L, and the washing time is 30-100 s.
7. The method for constructing the molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the adsorption time of histamine adsorbed by the molecularly imprinted L B membrane modified electrode in the step (5) is 2-20 min.
8. The method for constructing a molecularly imprinted amphiphilic chitosan derivative L angmuir-Blodgett membrane (L B membrane) modified electrode according to claim 1, wherein the linear sweep voltammetry in step (5) is used to determine the relationship between peak current response (Δ i) and histamine concentration, and the histamine concentration is observed to be 0.01-500. mu.M.
9. A molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode prepared by the construction method of any one of claims 1 to 8.
10. The use of a molecularly imprinted amphiphilic chitosan derivative L B membrane modified electrode of claim 9, wherein the molecularly imprinted amphiphilic chitosan derivative L B membrane is introduced on the surface of the glass conductive electrode, so that the modified electrode is endowed with selective recognition of histamine, and is used for detecting histamine in beer and wine actual samples by a standard sample adding method, the average recovery rates of histamine in beer and wine standard samples are respectively 98.3-105.9% and 99.8-105.2%, and the relative standard deviation of electrochemical response peak current of the glass conductive electrode after 3 adsorption-desorption cycles is less than 4.5%.
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