WO2023155453A1

WO2023155453A1 - Biomimetic membrane structure based on zinc protoporphyrin organic cage and use thereof

Info

Publication number: WO2023155453A1
Application number: PCT/CN2022/125334
Authority: WO
Inventors: 邓盛元; 万莹; 满天天; 朴禹滈; 陈嘉亮; 赵元章; 冯旭宇; 黄亚齐; 肖明
Original assignee: 南京理工大学
Priority date: 2022-02-21
Filing date: 2022-10-14
Publication date: 2023-08-24
Also published as: CN114705742A

Abstract

A biomimetic membrane structure based on a zinc protoporphyrin organic cage and a use thereof. The zinc protoporphyrin organic cage is prepared by enabling a tetraaldehyde phenyl porphyrin and (2,4,6-tri-butoxybenzene-1,3,5-tri)trimethylamine chloroform solution and a zinc acetate methanol solution to undergo a reaction; a zinc protoporphyrin organic cage, phospholipid and cholesterol chloroform solution of a certain concentration is added dropwise to the surface of an electrode, and a solvent is fully volatilized to obtain the biomimetic membrane structure. The calculated light-emitting intensity is associated with the concentration of a light-emitting body and membrane solution to obtain an optimal light-emitting condition. An electrochemical luminescence device is simple, the operation is simple and convenient, and a change trend of the light intensity along with the concentration of a reactant can be conveniently detected; by means of electrochemiluminescence detection, an initial reaction condition, a speed and a path can be controlled by adjusting the potential, so as to conveniently perform in-situ and on-site analysis; the present invention is applicable to melittin detection.

Description

A biomimetic membrane structure based on zinc porphyrin organic cage and its application

technical field

The invention belongs to the technical field of physical and chemical analysis, and in particular relates to a biomimetic membrane structure based on a zinc porphyrin organic cage and an application thereof.

Background technique

The cell is the basic unit that constitutes the structure and function of all life activities. The cell membrane is a lipid bilayer membrane formed by the self-assembly of phospholipid molecules, which plays an important role in maintaining the internal environment of the cell and transmitting physiological signals. Maintaining normal physiological activities of cells requires constant material exchange with the outside world, and membrane proteins with transmembrane transport function play a key role in this process. Natural membrane proteins have high transport efficiency and selectivity in the process of transmembrane transport, which is an important way for living cells to carry out metabolic activities and continuously exchange substances with the surrounding environment. With the development of molecular biology and patch clamp technology, people have a deeper understanding of the molecular structure and characteristics of ion channels, and found that the function and structure abnormalities of ion channels are related to the occurrence and development of many diseases, such as potassium ion , sodium channelopathy, etc., are the targets of many drugs. Therefore, researchers can use drugs that can affect the activity of ion channels to treat certain diseases, and further use relevant knowledge to guide the design and synthesis of related new drugs. Therefore, an in-depth understanding of the structure and function of ion channels is of great significance for in-depth exploration of the pathological mechanism of certain diseases, early diagnosis and discovery of specific therapeutic drugs or measures.

However, due to its complex structure and easy loss of activity once it leaves the biomembrane, the structural analysis of membrane proteins and the mechanism of material transmembrane transport are still not in-depth. In order to study the mechanism of transmembrane transport, in recent years, chemists have designed and synthesized a wide variety of artificial channel systems based on natural small molecule channels, and developed a series of optical sensors and electrochemical sensors based on ion channels. , and apply it to ion detection, protein, and DNA and RNA detection. Due to the characteristics of high sensitivity, low cost, simple operation and easy miniaturization, electrochemical sensing technology based on ion channels has become a research hotspot in the field of chemical sensing. Umezawa et al. first introduced ion channels into the field of electrochemical sensing, and realized the detection of various inorganic ions such as Ca ²⁺ , Mg ²⁺ and Ba ²⁺ . Gyurcsanyi et al. combined peptide-nucleic acid functionalized ion channels with ion-selective electrodes to realize the potential detection of miRNA.

Contents of the invention

The object of the present invention is to provide a biomimetic membrane structure based on a zinc porphyrin organic cage and its application against the deficiencies of the prior art. The invention can reliably and highly sensitively detect the melittin through the electrochemiluminescent method.

The purpose of the present invention is achieved by the following technical scheme: a kind of biomimetic membrane structure based on zinc porphyrin organic cage, its construction method comprises: by a certain concentration of zinc porphyrin organic cage, phospholipid and cholesterol chloroform solution, dropwise in electrode surface and evaporate the solvent.

Further, the zinc porphyrin organic cage concentration is 10-30 μM, and the solvent is chloroform.

Further, the zinc porphyrin organic cage is obtained by mixing 5 mM zinc acetate methanol solution with the porphyrin organic cage solution at a volume ratio of 1:1, stirring in a flask at room temperature for 2 days, extracting with prepared saturated saline for three times and drying.

Further, the porphyrin organic cage is added into chloroform by adding tetraaldehyde phenylporphyrin and (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine in a molar ratio of 3:4, Add 1-2 drops of trifluoroacetic acid dropwise, stir for a certain period of time under nitrogen atmosphere, and extract the reacted solution with saturated saline solution, potassium carbonate solution and saturated ammonium chloride solution in sequence.

Further, the concentration of phospholipids is 1-4 g/L; the range of cholesterol concentration is 0-2 g/L. The selected phospholipid is palmitoyl oleoyl phosphatidyl choline or dipalmitoyl phosphatidyl choline, or (2,3-dioleoyl-propyl)-trimethylammonium chloride; the solvent is chloroform.

A biomimetic membrane structure based on zinc porphyrin organic cage, applied to electrochemiluminescent detection of melittin, comprising:

With the glassy carbon electrode as the working electrode, the Ag/AgCl electrode as the reference electrode, and 10mM HEPES containing 0.3M potassium chloride with a pH of 7.4 as the electrolyte, 5-20 μL of the mixed solution of zinc porphyrin organic cage, phospholipid and cholesterol was added dropwise On the surface of the electrode, the solvent was evaporated to obtain a biomimetic membrane structure. The zinc porphyrin organic cage in the biomimetic membrane structure was used as the ECL light emitter, the electrochemiluminescence intensity in the electrolyte containing different concentrations of melittin was measured, and the relationship curve between the electrochemiluminescence intensity and melittin was calculated.

The electrochemiluminescence intensity of the sample to be tested is measured under the same conditions, and the corresponding melittin concentration is obtained through the relationship curve between the electrochemiluminescence intensity and the melittin concentration.

The beneficial effects of the present invention are as follows:

(1) The construction method based on the zinc porphyrin organic cage provided by the present invention is simple and easy, and the prepared zinc porphyrin organic cage has good electrochemiluminescence performance, and the biomimetic film is easy to interact with the detection object;

(2) The device of electrochemiluminescence of the present invention is simple, easy and simple to operate, can detect light intensity change trend with the concentration of reactant easily;

(3) The present invention utilizes electrochemiluminescence detection, can control the initial conditions, speed and course of the reaction by adjusting the potential, and conveniently perform in-situ analysis and on-site analysis;

(4) The present invention is applicable to the detection of melittin.

Description of drawings

Fig. 1 is the synthesizing schematic diagram of zinc porphyrin organic cage; Wherein, between porphyrin and (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine is a Schiff base;

Figure 2 is the ultraviolet and fluorescence spectra of porphyrin organic cage, zinc porphyrin organic cage, tetraaldehyde phenyl porphyrin, (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine Figure; Wherein, (A) is ultraviolet spectrogram, (B) is fluorescence spectrogram;

Fig. 3 is the proton nuclear magnetic spectrum of tetraaldehyde phenylporphyrin, (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine, porphyrin organic cage; Wherein, (A) is Tetraformyl phenylporphyrin, (B) is (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine, and (C) is a porphyrin organic cage;

Fig. 4 is the CV and light intensity potential diagram of the biomimetic membrane structure based on zinc porphyrin organic cage; Wherein, (A) is CV curve, (B) is light intensity potential diagram;

Fig. 5 is the light intensity time diagram based on the biomimetic film structure of zinc porphyrin organic cage;

Figure 6 is a schematic diagram of the relationship curve fitted by adding melittin to the buffer solution and changing the concentration of melittin from 1 to 100 μM in luminescence intensity; the fitting equation is y=a+bx, where a=9828.36963±299.84413, b=-95.12459±9.61116, ^R2 =0.961.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments and accompanying drawings.

The invention relates to a biomimetic membrane structure based on zinc porphyrin organic cages and its application, in particular to the components of the biomimetic membrane and the use of tetraaldehyde phenyl porphyrin (5,10,15,20-tetraphenyl-21H,23H-porphine, Abbreviated as TFPP), (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine and zinc acetate to prepare zinc porphyrin organic cage. The electrochemiluminescence intensity of the sample to be tested is measured under the same conditions, and the corresponding melittin concentration is obtained through the relationship curve between the electrochemiluminescence intensity and the melittin concentration.

The chemical formula of tetraaldehyde phenylporphyrin is as follows:

The chemical formula of (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine is as follows:

In the following examples, the biomimetic membrane structure is obtained by adding a certain concentration of zinc porphyrin organic cage, phospholipid and cholesterol chloroform solution on the electrode surface and evaporating the solvent. The zinc porphyrin organic cage is prepared by Schiff base reaction . Add tetraaldehyde phenylporphyrin and (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine into chloroform, stir for 14 hours under nitrogen atmosphere, and obtain porphyrin by extraction Organic cage solution; subsequently, react the porphyrin organic cage solution with zinc acetate for 2 days, and extract to obtain the zinc porphyrin organic cage.

In the following examples, the detection system used is an ECL luminometer, and the parameters of the ECL luminometer include that the bias voltage of the ECL photomultiplier tube (PMT) is -900V and -800V, the number of amplification stages is 3, and the scan rate is 0.1 V/s, the scanning potential is -1.5~0V. The recorded data was transferred to the computer for processing and analysis. The specific analysis method was as follows: select the luminous intensity of the stable area, subtract the background and take the average value; then take the concentration of melittin as the independent variable and process it with OriginPro 8.5 software.

Embodiment 1: the synthesis of zinc porphyrin organic cage

As shown in Figure 1, add 27mg tetraaldehyde phenylporphyrin, 18mg (2,4,6-tributoxybenzene-1,3,5-tri)trimethylamine in 50ml flask and dissolve in 20ml chloroform solution 1-2 drops of trifluoroacetic acid were added dropwise, stirred at room temperature for 14 h under nitrogen atmosphere, and the reacted solution was transferred into a separatory funnel. The prepared saturated saline solution, potassium carbonate solution and saturated ammonium chloride solution are sequentially extracted to obtain a porphyrin organic cage solution. The relevant NMR characterization of the product is shown in Figure 3. The reduction of the aldehyde group H at the d position and the retention and splitting of the H at the e and f positions in the figure prove that the porphyrin organic cage was successfully synthesized. Prepare 5mM zinc acetate methanol solution, mix it with the porphyrin organic cage solution at a volume ratio of 1:1, stir in the flask at room temperature for 2 days, extract three times with the prepared saturated saline and dry the solvent zinc porphyrin organic cage. The shift of the peak in Figure 2 (A) and the shift of the peak position near 425 nm and the change in signal intensity in Figure 2 (B) prove that the zinc porphyrin organic cage was successfully synthesized.

Example 2: Electrochemiluminescence of biomimetic membrane structures based on zinc porphyrin organic cages

(1) Polish the glassy carbon electrode with a diameter of 2.5 mm with Al ₂ O ₃ polishing powder, ultrasonically clean it six times with ultrapure water and ethanol in turn, and dry the electrode surface with nitrogen.

(2) Use chloroform to prepare a mixed solution containing 10-30 μM zinc porphyrin organic cage solution, 1-4 g/L palmitoyl oleoyl phosphatidylcholine solution and 0-2 g/L cholesterol, and use a 20 μL pipette to draw 15 μL of the solution Drop it on the surface of GCE (Glassy Carbon Electrode), and bake it slowly under an infrared baking lamp to form a uniform film.

(3) Using Ag/AgCl as the reference electrode, platinum wire as the counter electrode, and the modified glassy carbon electrode as the working electrode are correctly connected in the cassette of the chemiluminescence detector, and the electrochemical workstation and the weak light detection system (ECL luminescence instrument) were connected together, and the high voltage of the photomultiplier tube was set to -900V for electrochemiluminescence detection. It can be seen from Figure 4 that the luminescent site is around 1.45; as shown in Figure 5, the light intensity time diagram shows that the biomimetic film has stable electrochemiluminescence performance.

Example 3: Electrochemiluminescence of biomimetic membrane structures based on zinc porphyrin organic cages

(2) Use chloroform to prepare a mixed solution containing 10-30 μM zinc porphyrin organic cage solution, 1-4 g/L dipalmitoylphosphatidylcholine solution, and 0-2 g/L cholesterol, and use a 20 μL pipette to draw 15 μL solution drops Add it to the surface of GCE (Glassy Carbon Electrode), and slowly bake it under an infrared baking lamp to form a uniform film.

(3) The Ag/AgCl is used as the reference electrode, the platinum wire is used as the counter electrode, and the modified glassy carbon electrode is used as the working electrode and correctly connected in the cassette of the chemiluminescence detector, and the electrochemical workstation and the weak light detection system are connected together , the high voltage of the photomultiplier tube was set to -900V, and the electrochemiluminescence detection was carried out. The results showed that the biomimetic film had stable electrochemiluminescence performance.

Example 4: Electrochemiluminescence of biomimetic membrane structures based on zinc porphyrin organic cages

(1) Polish the glassy carbon electrode with a diameter of 2.5 mm with Al ₂ O ₃ polishing powder, ultrasonically clean it with ultrapure water and ethanol six times in turn, and dry the electrode surface with nitrogen;

(2) Use chloroform to prepare zinc porphyrin organic cage solution containing 10-30 μM, 1-4 g/L (2,3-dioleoyl-propyl)-trimethylammonium chloride and 0-2 g/L cholesterol solution, use a 20 μL pipette gun to draw 15 μL of the solution and drop it on the surface of the GCE (glassy carbon electrode), and slowly bake it under an infrared baking lamp to form a uniform film.

Embodiment 5: detection of melittin

(2) Use chloroform to prepare a mixed solution containing 10-30 μM zinc porphyrin organic cage solution, 1-4 g/L palmitoyl oleoyl phosphatidylcholine, and 0-2 g/L cholesterol, and use a 20 μL pipette to draw 15 μL solution drops Add it to the surface of GCE (Glassy Carbon Electrode), and slowly bake it under an infrared baking lamp to form a uniform film.

(3) The Ag/AgCl is used as the reference electrode, the platinum wire is used as the counter electrode, and the modified glassy carbon electrode is used as the working electrode and correctly connected in the cassette of the chemiluminescence detector, and the electrochemical workstation and the weak light detection system are connected together , the high voltage of the photomultiplier tube was set to -800V, and the electrochemiluminescence was detected in the 10mM HEPES (containing 0.3M KCL) electrolyte at pH 7.5. The results showed that the biomimetic film had stable electrochemiluminescence performance.

(4) Observe the electrochemiluminescence intensity of melittin added to the electrolyte, then record the relationship between the value of the electrochemiluminescence intensity and the concentration of melittin, and draw the working curve as shown in Figure 6.

(5) Measure the electrochemiluminescence intensity of the sample to be tested under the same conditions, and calculate the corresponding melittin concentration through the fitting equation in FIG. 6 .

Claims

A biomimetic membrane structure based on zinc porphyrin organic cage is characterized in that it is obtained by dripping zinc porphyrin organic cage solution, phospholipid solution and cholesterol solution on the surface of an electrode and evaporating the solvent.
The biomimetic membrane structure according to claim 1, characterized in that the concentration of the zinc porphyrin organic cage solution is 10-30 μM.
The biomimetic membrane structure according to claim 1, wherein the solvents of the zinc porphyrin organic cage solution, the phospholipid solution and the cholesterol solution are all chloroform.
The biomimetic membrane structure according to claim 1, characterized in that the preparation method of the zinc porphyrin organic cage comprises: configuring 5mM zinc acetate methanol solution, mixing with the porphyrin organic cage solution at a volume ratio of 1:1, and stirring at room temperature≥ After 2 days, extract with saturated saline three times or more, and dry to obtain the zinc porphyrin organic cage.
According to the described biomimetic membrane structure of claim 4, it is characterized in that, the preparation method of described porphyrin organic cage solution comprises: tetraaldehyde group phenylporphyrin and (2,4,6-tributoxybenzene-1, 3,5-tri)trimethylamine, add chloroform at a molar ratio of 3:4, add 1 to 2 drops of trifluoroacetic acid dropwise, stir under nitrogen atmosphere, and wash the reacted solution with saturated saline solution, potassium carbonate solution, Saturated ammonium chloride solution for extraction, each solution is extracted once or more to obtain a porphyrin organic cage solution.
The biomimetic membrane structure according to claim 1, characterized in that the concentration of the phospholipid solution is 1-4 g/L.
The biomimetic membrane structure according to claim 1, characterized in that the concentration of the cholesterol solution is 0-2 g/L.
According to the described biomimetic membrane structure of claim 1, it is characterized in that, selected phospholipid is selected from palmitoyl oleoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, (2,3-dioleoyl-propyl)-three Methyl ammonium chloride.
An application of the biomimetic membrane structure according to any one of claims 1 to 8, characterized in that the concentration of melittin is detected by electrochemiluminescence.
The application according to claim 9, characterized in that it comprises:

With the glassy carbon electrode as the working electrode, the Ag/AgCl electrode as the reference electrode, and 10mM HEPES containing 0.3M potassium chloride with a pH of 7.4 as the electrolyte, 5-20 μL of the mixed solution of zinc porphyrin organic cage, phospholipid and cholesterol was added dropwise On the surface of the electrode, the solvent was evaporated to obtain a biomimetic membrane structure; the zinc porphyrin organic cage in the biomimetic membrane structure was used as an ECL light emitter, and the electrochemiluminescence intensity in the electrolyte containing different concentrations of melittin was measured, and the electrochemiluminescent intensity was calculated. The relationship curve between chemiluminescence intensity and melittin;

The electrochemiluminescence intensity of the sample to be tested is measured under the same conditions, and the corresponding melittin concentration is obtained through the relationship curve between the electrochemiluminescence intensity and the melittin concentration.