Microfluidic platform and affinity analysis method for observing the depolymerization of organic supramolecular molecules

Abstract

The present invention discloses a microfluidic platform and affinity analysis method for observing the depolymerization of organic supramolecular. The microfluidic platform includes an optical observation platform and a microfluidic chip device, which can observe the depolymerization of organic supramolecular capable of photochemiluminescence in a weakly polar non-water-soluble solvent, collect images using the photoluminescence characteristics of organic supramolecular, observe the depolymerization of organic supramolecular in different degrees under different concentrations of guests, and analyze the fluorescence characteristics of different intensities, and analyze the affinity phenomenon in combination with mathematical statistics.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a microfluidic platform for observing organic supramolecular depolymerization and an affinity analysis method. Background Art

[0002] Microfluidic chips can be used in various analytical fields, such as biomedicine, synthesis and screening of new drugs, and other important application fields such as food and commodity inspection, environmental monitoring, criminal science, military science and aerospace science, among which biological analysis is a hot topic. At present, its applications are mainly concentrated in nucleic acid separation and quantification, DNA sequencing, gene mutation and gene differential expression analysis. In addition, protein screening has also been reported in microfluidic chips. For characteristic fragments of pathogenic microorganism genomes, sequence polymorphisms of chromosomal DNA, sites and characteristics of gene mutations, etc., suitable nucleic acid probes are designed and selected, and after PCR amplification, information such as pathogenic microorganism species, subtypes, virulence, drug resistance, pathogenicity, homology, polymorphism, variation and expression can be obtained, providing a good entry point for disease diagnosis and treatment. However, the material of conventional microfluidic chips is mostly polydimethylsiloxane (PDMS). Although PDMS itself has good biocompatibility, it can be used to simulate some biological environments. However, if some weakly polar and non-water-soluble solvents are used in the experiment, the PDMS microfluidic chip may swell and deform. It has been pointed out in the literature that the compatibility of PDMS microfluidic chips is affected by three factors: the swelling of PDMS in the solvent, the solute distribution between the solvent and PDMS, and the dissolution of PDMS oligomers in the solvent. Among these three factors, the swelling of PDMS has the greatest impact. The solvents that cause the least swelling of PDMS include water, nitromethane, dimethyl sulfoxide, ethylene glycol, perfluorotributylamine, perfluorodecalin, acetonitrile and propylene carbonate; the solvents that cause the most swelling of PDMS are weakly polar and non-water-soluble solvents such as diisopropylamine, triethylamine, pentane and xylene. Therefore, it is very necessary to build a microfluidic observation platform that can tolerate weakly polar and non-water-soluble solvents.

[0003] There are many reports on biological tropism. In 2015, Nature reported that using single-molecule fluorescence correlation spectroscopy, it was found that the diffusion coefficient of some enzymes would gradually increase during the exothermic catalysis process, and the diffusion rate was linearly related to the substrate concentration and the cumulative time. So can porphyrin aggregates carrying long aliphatic hydrocarbons also be regarded as enzymes, and when combined with guests that can depolymerize them, it can be regarded as a reaction. Angew.Chem. in 2021 reported that when a ligand with a longer branched chain is added, the "H"-type stacked porphyrin supramolecule will gradually change to a "J"-type stack. But if the guest is much smaller than the porphyrin supramolecule in a solution, will the "H"-type stacked porphyrin supramolecule gradually change to a "J"-type stack, or even form a single-molecule level structure with a ratio of 1:1? At present, there is no literature reporting the observation of the depolymerization, binding and directional migration of organic supramolecules under the action of guests in a microfluidic platform. Summary of the invention

[0004] The purpose of the present invention is to provide a microfluidic platform and an affinity analysis method for observing the depolymerization of organic supramolecular molecules in view of the deficiencies of the prior art.

[0005] The object of the present invention is achieved by the following technical solutions: a microfluidic platform for observing the depolymerization of organic supramolecular molecules, comprising a microfluidic chip device suitable for weakly polar and non-water-soluble materials, an optical detection platform, and a host and guest;

[0006] The microfluidic chip device suitable for weakly polar and non-water-soluble substances comprises a microfluidic chip and a chip-external flow injection pump system, which are connected via a PEEK base and a polytetrafluoroethylene microtube; the microfluidic chip is provided with three microchannel inlets, one reaction chamber and one microchannel outlet; the chip-external flow injection pump system comprises a three-channel flow injection pump, three 1 mL glass syringes, and polytetrafluoroethylene microtubes and PEEK adapters connected thereto;

[0007] The optical observation platform, the camera is connected to a computer via an external image acquisition card, and the acquired channel fluorescence images are transmitted to the computer for processing, presenting the fluorescence intensity distribution;

[0008] The host and guest solvents used in the microfluidic chip are all organic solvents.

[0009] Furthermore, the microfluidic chip is made of soda-lime glass, and the glass syringe, polytetrafluoroethylene microtube and PEEK adapter connected thereto are all resistant to organic solvents.

[0010] Furthermore, the organic solvent is a weakly polar water-insoluble solvent.

[0011] Furthermore, each microchannel inlet is injected with a main solution or a guest solution or a solvent through a flow injection pump. The order of the solution inlets is solvent, main solution, and guest solution. Each solution converges at the inlet confluence point, enters the reaction chamber, and finally flows out from the microchannel outlet.

[0012] Furthermore, the organic supramolecular host is a photochemiluminescent substance.

[0013] Furthermore, the photochemiluminescent substance is zinc porphyrin carrying a long aliphatic hydrocarbon side chain.

[0014] Furthermore, the guest is a N-containing organic heterocyclic substance capable of depolymerizing organic supramolecules.

[0015] Furthermore, the object is pyridine or imidazole.

[0016] Furthermore, after the host is dissolved in a weakly polar non-water-soluble solvent, it will form π-π stacking due to the intermolecular interaction force, so it presents a well-dispersed aggregation state; after the guest is added to the organic supramolecular host solution, the aggregation state of the host will be depolymerized to form a host-guest coordination structure.

[0017] An affinity analysis method for organic supramolecular depolymerization based on the above microfluidic platform comprises the following steps:

[0018] Step 1: preparing a main solution, a guest solution and a solvent, wherein the main solution and the guest solution are both prepared using a solvent;

[0019] Step 2: First, fully rinse the microfluidic channel with a solvent, and then pass the host solution, the guest solution and the solvent into the microchannel inlet of the microfluidic chip;

[0020] Step 3: Turn on the luminescent mercury lamp light source to excite the subject to produce photoluminescence. The fluorescence is transmitted to the external camera through the imaging objective to capture the fluorescence imaging of the channel, record the entrance confluence point of the microfluidic channel and the fluorescence images at different sites, and transmit the recorded images to the computer through the image acquisition card for processing and analysis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The microfluidic chip device constructed by the present invention can be used for experiments in a weakly polar, non-water-soluble organic solvent environment, and has high stability and is not prone to corrosion;

[0023] (2) The present invention uses zinc porphyrin with long aliphatic side chains and photoluminescent properties as the research object, allowing the concentration distribution of zinc porphyrin to be directly imaged by fluorescence microscopy when it is exposed to the guest that induces its depolymerization in the microfluidic channel;

[0024] (3) The microfluidic optical platform of the present invention is capable of observing the depolymerization and diffusion phenomena of organic supramolecules in an aggregated state under the action of a guest that induces their depolymerization, and has high feasibility;

[0025] (4) The Reynolds number of the fluid in the microchannel used in the present invention is small enough, and the flow state is laminar, mainly molecular diffusion, without turbulence, convection and other flow states. The diffusion and migration phenomena that occur when the organic supramolecules are acted upon by the guest that induces their depolymerization can be truly collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of a microfluidic platform for observing the depolymerization of organic supramolecular molecules; including a syringe pump 1, an inverted fluorescence microscope 2, a microfluidic chip 3, a long-life mercury lamp light source 4, and a computer 5;

[0027] Figure 2 Schematic diagram of the principle of the affinity analysis method of organic supramolecular depolymerization based on the microfluidic platform of the present invention;

[0028] Figure 3 This is the one-dimensional H-NMR spectrum of ZnTCPP-TOA synthesized using TCPP as raw material;

[0029] Figure 4 The UV absorption and visible spectra of ZnTCPP-TOA synthesized with TCPP as raw material and pyridine with different concentrations added;

[0030] Figure 5 The fluorescence emission spectra of ZnTCPP-TOA synthesized with TCPP as raw material and added with different concentrations of pyridine;

[0031] Figure 6 The SEM images of ZnTCPP-TOA synthesized with TCPP as raw material and added with different concentrations of pyridine; (A) is 0.1mM ZnTCPP-TOA blank control; (B) is 0.1mM ZnTCPP-TOA+10μM pyridine; (C) is 0.1mM ZnTCPP-TOA+5mM pyridine; (D) is 0.1mM ZnTCPP-TOA+1M pyridine;

[0032] Figure 7Schematic diagram of the depolymerization affinity phenomenon of 0.1mM ZnTCPP-TOA in response to 1M pyridine concentration gradient; wherein, (A) is a Ψ-type 3-inlet microfluidic channel used for the chemical trend study of the ZnTCPP-TOA and pyridine collection system; (B) and (C) are the chemical trends of the gradient changes in the host concentration caused by the guest-induced binding and depolymerization of the host; the (a) line in (B) and (C) represents the blank control, the (b) line in (B) represents the focusing phenomenon within the host, and the (b) line in (C) represents the directional migration;

[0033] Figure 8 Schematic diagram of the disaggregation and affinity-induced directional migration of 0.1 mM ZnTCPP-TOA in response to a 1 M pyridine concentration gradient, and the directional migration phenomenon that occurs when the channel configuration is different. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0035] like Figure 1 As shown, the present invention provides a microfluidic platform for observing the depolymerization of organic supramolecular molecules, including a microfluidic chip device suitable for weakly polar and water-insoluble materials and an optical detection platform.

[0036] The microfluidic chip device suitable for weakly polar and water-insoluble materials can observe the depolymerization of organic supramolecules capable of photochemiluminescence in weakly polar and water-insoluble solvents, utilize the photoluminescence characteristics of organic supramolecules to collect images, observe the organic supramolecules, and analyze the different degrees of depolymerization and fluorescence characteristics of different intensities under different concentrations of guests, and analyze the affinity phenomenon in combination with mathematical statistics.

[0037] The microfluidic chip device suitable for weakly polar non-water-soluble substances includes a microfluidic chip 3 and an external flow injection pump system. The microfluidic chip 3 is provided with 3 microchannel inlets, 1 reaction chamber and 1 microchannel outlet. The external flow injection pump system includes a three-channel flow injection pump 1, 3 1mL glass syringes, 3 polytetrafluoroethylene microtubes, and 3 PEEK adapters; wherein, a 1mL glass syringe, a polytetrafluoroethylene microtube, and a PEEK adapter are connected in sequence as a group. The microfluidic chip 3 and the external flow injection pump system are connected via a PEEK adapter and a polytetrafluoroethylene microtube.

[0038] The microfluidic chip 3 is made of soda-lime glass, and the glass syringe, polytetrafluoroethylene microtube and PEEK adapter connected thereto can all withstand weakly polar and non-water-soluble substances.

[0039] The optical observation platform includes an inverted fluorescence microscope 2, a camera and a long-life mercury lamp light source 4. The incident light passes through the fluorescence excitation block and is focused on the bottom of the microfluidic chip 3 via the objective lens, and the camera captures the fluorescence appearing in the microfluidic channel and images it.

[0040] In order to optimize the obtained fluorescence imaging, the fluorescence intensity distribution data of ROI (Region of Interest) of the obtained image was collected and then processed in Origin software.

[0041] The organic supramolecular host in the present invention is a zinc porphyrin carrying a long aliphatic hydrocarbon side chain, which has photochemiluminescence properties. For example, ZnTCPP-TOA is synthesized using meso-Tetra(4-carboxyphenyl)porphine (TCPP) and 3,4,5-tris(((S)-3,7-dimethyloctyl)oxy)aniline (TOA) as raw materials.

[0042] The guest in the present invention is a substance that can specifically bind to the host; for example, the ligand that can specifically bind to ZnTCPP-TOA is a N-containing organic heterocyclic substance, such as pyridine, imidazole, and the like.

[0043] The solvents of the host-guest solution in the present invention are all weakly polar water-insoluble solvents, such as methylcyclohexane (MCH), dichloromethane (DCM) and the like.

[0044] After being dissolved in a weakly polar water-insoluble solvent, the main body of the present invention will form π-π stacking due to the intermolecular interaction force, thus presenting a well-dispersed aggregation state.

[0045] After the guest in the present invention is added into the organic supramolecular host solution, the aggregation state of the host will be depolymerized to form a host-guest coordination structure.

[0046] Conventional PDMS microfluidic chips 3 have a certain degree of swelling in solvents of different polarities, which will cause large errors in precise measurements and analyses. Therefore, for weakly polar and water-insoluble solvents, microfluidic chips 3 made of other materials need to be used to ensure that in subsequent experiments, there will be no errors caused by material swelling. When TCPP is combined with long aliphatic hydrocarbon amines, ZnTCPP-TOA carrying long aliphatic hydrocarbons will be formed. At this time, when ZnTCPP-TOA is dissolved in a weakly polar and water-insoluble solvent, it will form π-π stacking due to its own action, thus forming an "H" type stacking state. Figure 2As shown, when organic amines are added, the π-π stacked ZnTCPP-TOA will be 2 The axial coordination between the hybrid zinc and the lone pair of electrons on the ligand N occurs in a strict 1:1 stoichiometric ratio, resulting in the gradual depolymerization of the "H" type stacked ZnTCPP-TOA to generate a new five-coordinated complex, which then changes to a single non-aggregated molecule in the solution. In addition, due to the fluorescence activity of the porphyrin itself, its optical stability in the above-mentioned interaction, and the change in fluorescence intensity before and after depolymerization, the depolymerization of the organic supramolecular can be keenly observed in the entire microfluidic system, thereby analyzing the depolymerization affinity.

[0047] The present invention provides an affinity analysis method for organic supramolecular depolymerization based on the above-mentioned microfluidic platform, comprising the following steps:

[0048] Step 1: Synthesize ZnTCPP-TOA using TCPP as raw material.

[0049] Step 1.1: Place a 50 mL round-bottom flask under nitrogen atmosphere and add purple-black TCPP (135 mg, 1.71 × 10 -4 mol) were suspended in 15 mL of chloroform (CHCl3) and added to a round-bottom flask. After the stirring was stable, oxalyl chloride (350 μL, 4.14×10 -3 mol), and two drops of anhydrous N,N-dimethylformamide (DMF) were added. The entire device was stirred overnight in a nitrogen atmosphere in the dark, and the solvent was blown dry with a nitrogen stream. Finally, the volatiles were removed in a vacuum drying oven at 65°C for 2 hours to obtain green TCPP chloride (TCPP-Cl).

[0050] Step 1.2: The TCPP-Cl obtained in step 1.1 was dissolved in 8.5 mL CHCl3, and 4 mL 3,4,5-tris(((S)-3,7-dimethyloctyl)oxy)aniline (TOA) (769 mg, 1.37 × 10 -3 mol) and N,N-diisopropylethylamine (700 μL, 4.02×10 -3mol) of CHCl3 solution, stirred in the dark under nitrogen protection for 48h. Then 60mL CHCl3 was added to dilute the reaction system, and the organic phase was extracted with 10% citric acid aqueous solution (10mL×3 times), 1M NaOH (10mL×2 times) and saturated NaCl (10mL) in sequence; the aqueous phase of each step was discarded, and the organic phase was finally dried with anhydrous Na2SO4 and placed in a 4℃ refrigerator overnight. Na2SO4 was separated by suction filtration, and the obtained product was purified by filling a silica gel column after rotary evaporation at 40℃, eluted with heptane / ethyl acetate (3:1) as the mobile phase, and the second retained component was collected to obtain a purple solid (TCPP-TOA).

[0051] Step 1.3: The TCPP-TOA obtained in step 1.2 was redissolved in dichloromethane (CH2Cl2), and an excess of zinc acetate (Zn(OAc)2, 500 mg, 2.73×10 -3 mol), placed in a dark nitrogen atmosphere and stirred overnight. The mixture was also filtered, rotary evaporated, and then passed through a silica gel column with heptane / chloroform / ethyl acetate (4:4:1) as the developing solvent to obtain ZnTCPP-TOA.

[0052] Step 2: Dissolve the ZnTCPP-TOA prepared in step 1 in deuterated chloride (CDCl3) and perform NMR characterization of ZnTCPP-TOA. Figure 3 As shown, by analyzing the one-dimensional H-NMR spectrum of ZnTCPP-TOA, the hydrogen position of ZnTCPP-TOA and the success of its synthesis can be determined.

[0053] Step 3: The ZnTCPP-TOA prepared in step 1 and pyridine of different concentrations were dissolved in methylcyclohexane, and characterized by UV-visible absorption spectrum and fluorescence emission spectrum.

[0054] Step 4: The ZnTCPP-TOA prepared in step 1 and pyridine of different concentrations were dissolved in methylcyclohexane and subjected to SEM morphology characterization.

[0055] Step 5: prepare a host solution and a guest solution; the solvents of the host solution and the guest solution are both methylcyclohexane.

[0056] Step 6: First, fully rinse the microfluidic channel with methylcyclohexane, and then introduce the host solution, guest solution and solvent into the microchannel inlet of the microfluidic chip 3 according to the experimental design.

[0057] Step 7: Turn on the long-life mercury lamp light source 4 to excite the receptor to produce photoluminescence. The fluorescence is transmitted to the camera through the imaging objective lens to capture the fluorescence imaging of the channel, record the entrance confluence point of the microfluidic channel and the fluorescence images at different sites, and transmit the recorded images to the computer 5 through the image acquisition card for processing and analysis.

[0058] In the following embodiments, the optical observation platform used includes an inverted fluorescence microscope 2 (Nikon EclipseTi2-U) and a long-life mercury lamp light source 4. The incident light passes through a green fluorescence excitation block (λ ex =538~580nm), and focused on the bottom of the microfluidic chip 3 through a 20x objective lens (numerical aperture: 0.75, Plan Apoλ∞ / 0.17, focal distance: 1.0). The fluorescence imaging of the channel was captured with a Nikon DS-Qi2 camera, and the (Timelapse) module of the NIS-Elements AR software was used to shoot in real time. Specific parameters: exposure interval 30s, cycle 15min, exposure time: 1s. The recorded images were transferred to the computer 5 through the image acquisition card for processing and analysis. The specific analysis method is as follows: select the fluorescence intensity of the region of interest (ROI), subtract the background and take the average value; then use the channel width as the function independent variable and use Origin 8.0 software for normalization.

[0059] Example 1

[0060] This example provides UV-visible absorption spectroscopy characterization of organic supramolecules under the action of a guest that induces their depolymerization, and the specific steps are as follows:

[0061] (1) Take 1.2 mg of ZnTCPP-TOA and dissolve it in 4 mL of methylcyclohexane to make 0.1 mM ZnTCPP-TOA as a stock solution.

[0062] (2) Dilute the stock solution 16 times to obtain 6×10 -6 M ZnTCPP-TOA solution, and then diluted step by step to 4.8×10 -6 M, 3.6×10 -6 M, 2.4×10 -6 M, 1.2×10 -6 M, 6×10 -7 M, 4.8×10 -7 M, 3.6×10 -7 M, 2.4×10 -7 M, 1.2×10 -7 M, 6×10 -8 M ZnTCPP-TOA solution, the solvent is MCH. Then 200 μL was added to a 96-well plate for UV-Vis spectrophotometric characterization.

[0063] (3) Dilute the stock solution 8 times to obtain 1.2×10 -5Then, 2M, 1M, 200mM, 100mM, 20mM, 10Mm, 2mM, 200μM, 20μM, and 2μM pyridine solutions were prepared respectively, and the solvent was MCH. After the selected ZnTCPP-TOA solution was mixed with the pyridine solution of different solutions in a ratio of 1:1, 6×10 -6 Mixed solutions of ZnTCPP-TOA and 1 M, 500 mM, 100 mM, 50 mM, 10 mM, 5 mM, 1 mM, 100 μM, 10 μM, and 1 μM pyridine were prepared. 200 μL of each solution was then added to a 96-well plate and characterized by UV-Vis spectrophotometry using an ELISA reader.

[0064] Depend on Figure 4 (A) It can be seen that for 6×10 -8 M to 4.8×10 -6 The UV absorption value of ZnTCPP-TOA solution increases with the increase of solubility. max =391nm shows the maximum value ( Belt), and according to Figure 4 (A) The inset shows that the absorbance value and concentration follow the Beer-Lambert law and are linearly related. Because ZnTCPP-TOA is less affected by hydrogen bonds in weakly polar non-water-soluble solvents, the force generated by π-π stacking is more obvious, so the overall morphology is stacked micelles, that is, "H" type stacking. When a high concentration of pyridine solution is added, Figure 4 As shown in (B), because the N on pyridine coordinates with the Zn of ZnTPPR, ZnTCPP-TOA gradually dissociates and gradually depolymerizes from the "H" type stacking to a single molecule complex. At this time, the overall UV absorption spectrum shows a large red shift.

[0065] Example 2

[0066] This example provides the fluorescence emission spectrum and SEM characterization of an organic supramolecule under the action of a guest that induces its depolymerization, and the specific steps are as follows:

[0067] (1) Take 2.4 mg of ZnTCPP-TOA and dissolve it in 4 mL of methylcyclohexane to make 0.2 mM ZnTCPP-TOA as a stock solution.

[0068] (2) Prepare 2M, 1M, 200mM, 100mM, 20mM, 10Mm, 2mM, 200μM, 20μM, and 2μM pyridine solutions respectively, and the solvent is MCH. After the ZnTCPP-TOA solution is mixed with the pyridine solutions of different solutions in a 1:1 ratio, a mixed solution of 0.1mM ZnTCPP-TOA and 1M, 500mM, 100mM, 50mM, 10mM, 5mM, 1mM, 100μM, 10μM, and 1μM pyridine is obtained. Then, 200μL is taken and dropped into a 96-well plate, and the fluorescence emission spectrum is characterized using an enzyme reader.

[0069] (3) The solutions prepared in the first two steps were configured into 0.1 mM ZnTCPP-TOA solution as a blank control group, and then 0.1 mM ZnTCPP-TOA + 10 μM pyridine solution, 0.1 mM ZnTCPP-TOA + 5 mM pyridine solution, and 0.1 mM ZnTCPP-TOA + 1 M pyridine solution were prepared in sequence. 10 μL of each solution was added dropwise onto the treated silicon wafer, and then dried and gold-sprayed for SEM characterization.

[0070] When characterizing fluorescence emission spectra, the excitation light used is set to λ ex =(λ ab ) max =391nm, the emission range is set to λ em = 560~750nm, use the "bottom reading" mode of the microplate reader to test, such as Figure 5 As shown in (A), the fluorescence emission spectra of ZnTCPP-TOA with different concentrations were obtained. The fluorescence emission has two maxima, which are at (λ em ) max =606nm and 660nm. Figure 5 As shown in the inset of (A), the fluorescence intensity area integral and concentration of ZnTCPP-TOA can be well fitted into an isothermal adsorption curve. Figure 5 As shown in (B), when different concentrations of pyridine were added, as the concentration of pyridine increased, compared with the fluorescence emission spectrum of ZnTCPP-TOA, (λ em ) max =606nm fluorescence peak gradually decreased, while (λ em ) max The fluorescence peak at =660nm also increases, which is the same as the conclusion obtained from the UV absorption spectrum. This is because when pyridine is added, the N on the pyridine coordinates with the Zn of the ZnTPPR, causing the ZnTCPP-TOA to gradually dissociate, resulting in a shift in the strongest peak of the fluorescence emission spectrum.

[0071] This can also be obtained from SEM characterization, such as Figure 6As shown in (A), the ZnTCPP-TOA solution without pyridine showed a large particle aggregation state after drying, and they were surrounded by each other. Figure 6 (B)(C)(D), it can be seen that with the increase of pyridine concentration, the aggregated state of ZnTCPP-TOA gradually disaggregates. When pyridine reaches the maximum concentration, the aggregated state of ZnTCPP-TOA has completely disaggregated.

[0072] Example 3

[0073] This embodiment provides an experiment for observing the chemical tendency behavior of organic supramolecules caused by depolymerization and combination of N-containing organic heterocyclic substances. The specific steps are as follows:

[0074] (1) 1.2 mg of ZnTCPP-TOA was dissolved in 4 mL of methylcyclohexane to prepare a 0.1 mM ZnTCPP-TOA solution, and a 1 M pyridine solution was prepared at the same time.

[0075] (2) Rinse thoroughly with methylcyclohexane. Figure 7 (A) Microfluidic channel shown.

[0076] (3) Figure 7 As shown in line (a) of (B) and (C), the following solutions were introduced into the three inlets of the microfluidic channel from left to right. This configuration was recorded as MCH / ZnTCPP-TOA / MCH, and the fluorescence intensity distribution data of the ROI at the inlet confluence and outlet of the microfluidic channel were recorded as a blank control.

[0077] (4) Figure 7 As shown in line (b) in (B), the three channels were respectively introduced with MCH / ZnTPPR+pyridine / MCH, and the fluorescence intensity distribution data of the ROI at the inlet confluence and outlet of the microfluidic channel were recorded as the internal focusing phenomenon of the organic supramolecule.

[0078] (5) Figure 7 As shown in line (b) in (C), MCH / ZnTPPR / pyridine were introduced into the three channels respectively, and the fluorescence intensity distribution data of the ROI at the inlet confluence and outlet of the microfluidic channel were recorded as the directional migration phenomenon occurring when the organic supramolecule binds to the guest.

[0079] Can be Figure 7 In (B), it is observed that the outlet has a cohesion of line (b) relative to line (a) compared to the inlet, that is, the chemical tendency of ZnTCPP-TOA to focus inward due to depolymerization after being affected by pyridine. Figure 7As shown in (C), line (b) relative to line (a), the symmetry of the lateral normal distribution of ZnTCPP-TOA was broken at the outlet compared with the inlet when the pyridine was introduced into the side flow, which proves that the addition of pyridine does cause the depolymerization of ZnTCPP-TOA, thereby causing the directional migration phenomenon.

[0080] Example 4

[0081] This embodiment provides a microfluidic optical analysis method for the chemical tendency behavior of directional migration caused by the depolymerization of ZnTCPP-TOA after the addition of pyridine, and the specific steps are as follows:

[0082] (1) Take 1.2 mg of ZnTCPP-TOA and dissolve it in 4 mL of methylcyclohexane to make a 0.1 mM ZnTCPP-TOA solution. At the same time, prepare 1 M, 500 mM, 100 mM, 50 mM, 10 mM, 5 mM, 1 mM, 100 μM, 10 μM, and 1 μM pyridine solutions.

[0083] (2) Use methylcyclohexane to thoroughly rinse the microfluidic channel.

[0084] (3) The microchannel was configured with three inlets from left to right, and MCH / ZnTCPP-TOA / MCH were introduced respectively, and the fluorescence intensity distribution data of the ROI at the outlet of the microfluidic channel was recorded as a blank control;

[0085] (4) The microchannel was configured from left to right with three inlets, and MCH / ZnTCPP-TOA / pyridine were introduced respectively. The fluorescence intensity distribution data of the ROI at the outlet of the microfluidic channel were recorded. All the obtained intensity distribution data were combined with the blank control data to obtain the directional migration of ZnTCPP-TOA after the addition of pyridine, as shown in FIG. Figure 8 (A);

[0086] (5) The fluorescence intensity distribution data obtained after adding pyridine of all concentration gradients were integrated by displacement unit intensity to obtain the depolymerization directional migration displacement data of ZnTCPP-TOA after adding pyridine, as shown in FIG. Figure 8 (B) shown.

[0087] from Figure 8 As can be seen from (A), with the increase of pyridine concentration, the directional migration distance of ZnTCPP-TOA becomes larger and larger. Figure 8It can be seen from (B) that the presented curve is almost consistent with the curve obtained by the fluorescence emission spectrum, which indicates that after the addition of pyridine flow, ZnTCPP-TOA will gradually migrate to the pyridine flow area due to binding depolymerization. This proves that pyridine can effectively depolymerize ZnTCPP-TOA, and ZnTCPP-TOA can migrate in the direction of the pyridine flow.

Claims

1. A microfluidic platform for observing organic supramolecular depolymerization, characterized in that: It includes microfluidic chip devices, optical detection platforms and hosts and guests suitable for weakly polar and water-insoluble substances; The microfluidic chip device suitable for weakly polar and non-water-soluble substances comprises a microfluidic chip and an external flow injection pump system, which are connected via a PEEK base and a polytetrafluoroethylene microtube; the microfluidic chip is provided with three microchannel inlets, one reaction chamber and one microchannel outlet; the external flow injection pump system comprises a three-channel flow injection pump, three 1 mL glass syringes, and polytetrafluoroethylene microtubes and PEEK adapters connected thereto; In the optical detection platform, the camera is connected to the computer via an external image acquisition card, and the acquired channel fluorescence images are transmitted to the computer for processing, presenting the fluorescence intensity distribution; The host and guest solvents used in the microfluidic chip are all organic solvents.

2. The microfluidic platform according to claim 1, characterized in that: The microfluidic chip is made of soda-lime glass, and the glass syringe, polytetrafluoroethylene microtube and PEEK adapter connected to it are all resistant to organic solvents.

3. The microfluidic platform according to claim 1, characterized in that: The organic solvent is a weakly polar water-insoluble solvent.

4. The microfluidic platform according to claim 1, characterized in that: The main solution, guest solution or solvent is injected into each microchannel inlet through a flow injection pump. The order of the solution inlets is solvent, main solution, guest solution. The solutions converge at the inlet confluence point, enter the reaction chamber, and finally flow out from the microchannel outlet.

5. The microfluidic platform according to claim 1, characterized in that: The organic supramolecular main body is a photochemiluminescent substance.

6. The microfluidic platform according to claim 5, characterized in that: The photochemiluminescent substance is zinc porphyrin carrying a long aliphatic hydrocarbon side chain.

7. The microfluidic platform according to claim 1, characterized in that: The object is a N-containing organic heterocyclic substance capable of depolymerizing organic supramolecules.

8. The microfluidic platform according to claim 7, characterized in that: The object is pyridine or imidazole.

9. The microfluidic platform according to claim 1, characterized in that: After being dissolved in a weakly polar non-water-soluble solvent, the host and guest will form π-π stacking due to the intermolecular interaction force, thus presenting a well-dispersed aggregation state; after the guest is added to the organic supramolecular host solution, the aggregation state of the host will be depolymerized to form a host-guest coordination structure.

10. An affinity analysis method for organic supramolecular depolymerization based on the microfluidic platform according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step 1: preparing a main solution, a guest solution and a solvent, wherein the main solution and the guest solution are both prepared using a solvent; Step 2: First, fully rinse the microfluidic channel with a solvent, and then pass the host solution, the guest solution and the solvent into the microchannel inlet of the microfluidic chip; Step 3: Turn on the luminescent mercury lamp light source to excite the subject to produce photoluminescence. The fluorescence is transmitted to the external camera through the imaging objective to capture the fluorescence imaging of the channel, record the entrance confluence point of the microfluidic channel and the fluorescence images at different sites, and transmit the recorded images to the computer through the image acquisition card for processing and analysis.

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