Multi-component droplet micro-reaction platform and method

Abstract

The invention belongs to the technical field of high-flux automatic reaction platforms, and particularly relates to a multi-component droplet micro-reaction platform and method. Different hydrophilic and hydrophobic or hydrophilic and oleophobic patterned printing polymer chips are designed and prepared according to different application scenes, and different stacking of multiphase immiscible solutions is carried out on the chips according to the surface wettability difference of the chips; according to the method, different chemical reactions are carried out in a liquid drop stacking system through machine-assisted liquid adding, in-situ real-time monitoring is carried out on the liquid drop stacking system, and volatilization of a low-boiling-point solvent in the reaction process is prevented. Therefore, the multi-component droplet micro-reaction platform and the method which are simple, universal, high in flux, good in compatibility and wide in applicable application scene are constructed, and the platform and the method have good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of high-throughput automatic reaction platforms, and in particular relates to a multi-component droplet micro-reaction platform and method. Background Art

[0002] The synthesis of complex organic molecules plays a central role in the discovery and preparation of functional compounds, and the development of systems that can support multi-step synthesis without manual handling of intermediates is a long-term goal of the chemical and pharmaceutical industries. Currently, most new synthesis platforms that are compatible with a variety of liquids are usually based on the integration of traditional batch reactors, which requires a lot of engineering investment and is limited by the technical bottlenecks of standardized software or multifunctional hardware, making it difficult to increase throughput and achieve rapid customization.

[0003] In order to cope with the above problems, the scientific and industrial communities have gradually expanded their demands for the design, processing technology and output of chip laboratories (microchips). Compared with traditional reaction platforms, microchips have significant advantages such as optimized mass transfer performance, sample miniaturization, low reagent usage, and less time and energy consumption. Despite the above advantages, there are still many challenges in the design and innovation of such reaction platforms in order to meet specific application requirements. For example, in the field of combinatorial chemistry, some reaction platforms face problems such as high cost, limited applicability to different reactions, and limited throughput of parallel processing. Based on this, researchers need a high-throughput method to realize diversified chemical reactions in multiphase droplets on a portable chip chemical synthesis platform. If such a platform can ensure the execution of high-throughput parallel experiments and the feedback of real-time spectral data, it is expected to obtain a large amount of experimental data while ensuring the accuracy of the results, thereby saving the economic and time consumption of exploring complex multi-step synthetic reactions.

[0004] To date, it has always been challenging to stack immiscible liquids in a multiphase liquid layer into a stable equilibrium system. This is because the surface tension between the multiphase liquids triggers the instability of the liquid system, resulting in the formation of a system that is destroyed by even a slight mechanical disturbance in a statically stacked liquid structure. One solution is to introduce surfactants. Such surfactants are commonly used in droplet microfluidics to create stable, multiphase-encapsulated droplet structures. In simple terms, droplet microfluidics uses a liquid pumping device to produce droplets dispersed in an immiscible continuous phase (for example, encapsulating aqueous droplets in a continuous oil phase), and surfactants can better ensure the monodispersity of the droplets. However, although the droplets produced by this method can be well dispersed and arranged, they can only be transported and subsequently screened in a one-dimensional channel or a two-dimensional plane, and more complex operations are required to obtain droplet arrangements on a three-dimensional structure. Therefore, although this strategy can increase the test throughput, it also complicates the chemical reaction system. In addition, the closed nature of the droplet structure also limits the study of products in the droplets, making it difficult to achieve in situ monitoring of droplets.

[0005] In order to overcome the surface tension between liquid layers, in a recent pioneering work, Cybulski et al. used a rotating reactor to generate centrifugal force, which dominated the surface tension between liquids, making the originally unstable multiphase liquid system balanced and determining the process sequence of the entire liquid phase system by the density of the liquid phase itself. This way of stacking liquid layers is novel and stable, but it also brings challenges to the in-situ monitoring of the introduced liquid layers.

[0006] In order to meet the needs of more application scenarios, this field still urgently needs to develop more high-throughput automated reaction platforms with novel design ideas. Summary of the invention

[0007] In view of the problems of the prior art, the present invention provides a multi-component droplet micro-reaction platform and method.

[0008] A multi-component droplet micro-reaction platform comprises a substrate, the surface of which is provided with an array consisting of at least one micro-reactor, wherein the micro-reactor is a patterned surface consisting of at least two regions with different wetting properties.

[0009] Preferably, the microreactor is a patterned surface consisting of at least one hydrophilic region and at least one hydrophobic region;

[0010] Alternatively, the microreactor is a patterned surface consisting of at least one oleophilic region and at least one oleophobic region.

[0011] Preferably, the hydrophilic region is a substrate surface modified with a hydrophilic acrylic monomer or a hydrophilic acrylate monomer.

[0012] Preferably, the hydrophilic acrylic monomer is selected from at least one of acrylic acid and methacrylic acid;

[0013] The hydrophilic acrylic ester monomer is at least one selected from methacryloylethyltrimethylammonium chloride, hydroxyethyl acrylate, and hydroxyethyl methacrylate.

[0014] Preferably, the hydrophobic region is a super hydrophobic region.

[0015] Preferably, the super hydrophobic region is a porous polymer film with perfluorooctanoyl chloride grafted on the surface;

[0016] Alternatively, the oleophobic region is a porous polymer film with polyperfluorooctylethyl methacrylate grafted on the surface.

[0017] Preferably, the porous polymer is selected from cross-linked acrylic polymers.

[0018] Preferably, the acrylic polymer is selected from a copolymer of hydroxyethyl methacrylate and ethylene glycol dimethacrylate.

[0019] Preferably, the patterned surface includes a closed area consisting of at least two hydrophobic areas or at least two oleophobic areas.

[0020] Preferably, the patterned surface includes a closed area consisting of two hydrophobic areas or two oleophobic areas, the widths of the closed areas are 0.5-10 mm and 0.5-10 mm respectively, and the spacing between the two closed areas is 0.5-10 mm;

[0021] Alternatively, the patterned surface includes a closed area consisting of three hydrophobic areas or three oleophobic areas, the widths of the closed areas from the inside to the outside are 0.5-10mm, 0.5-10mm and 0.5-10mm respectively, and the intervals between the closed areas are 0.5-10mm and 0.5-10mm respectively from the inside to the outside.

[0022] Preferably, the closed area is in the shape of a polygon or a ring.

[0023] Preferably, the material of the substrate is selected from at least one of glass, quartz and metal.

[0024] Preferably, a cover plate is arranged above the substrate, and a patterned surface which is mirror-symmetrical with the microreactor is arranged on the cover plate.

[0025] Preferably, a sealing device is provided between the base plate and the cover plate.

[0026] Preferably, a microelectronic printer for adding liquid to the microreactor is also included, and the liquid adding path, speed and air pressure of the microelectronic printer are controlled by computer software.

[0027] Preferably, it also includes a stirring device and / or a detection device.

[0028] Preferably, the detection device is selected from an ELISA reader.

[0029] The present invention also provides a method for preparing the multi-component droplet micro-reaction platform, comprising:

[0030] Step 1, using a DLP printer to print a porous polymer on the surface of the substrate to form a porous polymer film having a pattern of the hydrophobic region;

[0031] Step 2, grafting perfluorooctanoyl chloride on the surface of the printed porous polymer film; or, grafting polyperfluorooctylethyl methacrylate on the surface of the printed porous polymer film by photopolymerization using a DLP printer;

[0032] Step 3, performing hydrophilic modification on the surface of the substrate.

[0033] The present invention also provides a method for performing micro-reaction using the multi-component droplet micro-reaction platform, using regions with different wettability in the micro-reactor to respectively fix different liquid phases, so that different liquids contact and interact with each other.

[0034] Preferably, the liquid phase comprises at least one organic phase and at least one aqueous phase.

[0035] Preferably, the different liquid phases are contacted with each other in at least one of the following ways:

[0036] 1) The sides of different liquid phases are in contact with each other in the horizontal direction;

[0037] 2) One liquid phase covers another liquid phase in the vertical direction.

[0038] Preferably, the method of making one liquid phase cover another liquid phase in the vertical direction is: injecting the liquid phase with higher density below the liquid phase with lower density, or adding the liquid phase with lower density above the liquid phase with higher density.

[0039] Preferably, the interaction is a chemical reaction or an extraction.

[0040] Preferably, the liquid phase is added to the hydrophilic region or the hydrophobic region by a microelectronic printer.

[0041] Preferably, the micro-reaction is carried out under the saturated vapor pressure of the low-boiling point liquid.

[0042] Preferably, the liquid phase of the boiling point liquid is encapsulated within another liquid phase formed of a high boiling point solvent.

[0043] The present invention has developed a printed polymer chip with a super-hydrophobic (oleophobic)-hydrophilic patterned surface, the huge difference in wettability between different regions of which shows the selective fixation of different solvents. For example, the super-hydrophobic polymer film cannot be infiltrated by aqueous solutions, but can be infiltrated by organic solutions and anchor the organic solutions on it, while the oleophobic polymer film cannot be infiltrated by water and oil. This property gives the printed polymer chip the ability to manipulate different immiscible liquids (such as Figure 1 ), by combining the solubility and density differences of different solvents, a multiphase liquid stacking system can be formed on the printed polymer chip. And because the aqueous solution is designed to be anchored in the hydrophilic glass area, the printed chip has good acid and alkali corrosion resistance (such as Figure 2 This excellent corrosion resistance undoubtedly greatly expands the options for subsequent chemical synthesis on printed polymer chips. On this basis, vertical and lateral stacking of immiscible liquids (such as Figure 3 ) and successfully carried out extraction reactions in two-phase or three-phase vertically stacked droplets ( Figure 4 ), multi-step synthesis ( Figure 6 ) and high-throughput reactions ( Figure 7 Furthermore, by combining the transparent texture and small size of the printed polymer platform, the miniaturized droplet reaction on it can be monitored in situ for a long time using a commercial microplate reader. The experimental results can be transmitted and displayed on the computer in a timely manner, realizing real-time in situ monitoring of each step of the ongoing droplet reaction ( Figure 5 ). In addition, in order to solve the problem of high-throughput liquid addition steps being too cumbersome, an attempt was made to use a microelectronic printer to automate the initial liquid addition process. By designing the liquid addition program and site on the computer side, the syringe can be controlled to perform flexible liquid addition comparable to manual operation, which also ensures the accuracy of the reaction. Therefore, the machine-assisted multiphase droplet reaction truly achieves a closed process of computer-controlled automatic liquid addition—in-situ real-time monitoring—data transmission back to the computer side ( Figure 8 ), which will greatly promote the development of miniaturized automatic reaction platform. Therefore, the present invention has a good application prospect.

[0044] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0045] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .By printing capsule arrays with different wettability on the left and right sides, the oil and water are anchored in the left and right parts of the capsule respectively, where a is a schematic diagram of the model chip printing process, b is the actual printed product pattern of the model chip, and c is the area where water and octyl acetate are fixed on the surface of the printed chip;

[0047] Figure 2 .Long-term acid and alkali resistance test of printed chips to acid and alkali solutions.

[0048] Figure 3 .Multiple immiscible liquids were stacked on the printed hydrophilic and hydrophobic patterned chip, and the stability of the stacking system was characterized, where a is a two-phase vertical stack of water and toluene, b is the liquid-liquid interface of the two phases, c is a three-phase vertical (vertical) stack of water, toluene, and dichloromethane, d is a horizontal (lateral) stack of water, 1,3-dibromopropane, and octyl acetate, and e is a combined horizontal and vertical stack of water, n-hexadecane, and sodium bromide aqueous solution;

[0049] Figure 4 .Dye extraction is performed in a double-layer vertical liquid-liquid stacking system, and a stirring function is introduced during the process so that the extraction reaction can be carried out completely and quickly, and the reaction progress is judged by real-time monitoring of color changes, wherein a is a vertical stacking system of water and toluene for dye extraction, b is the color change of the water phase and the toluene phase over time, c is the absorbance change of the water phase under different stirring conditions, d is the color change of the water phase and the toluene phase over time during the multi-component dye extraction process, and e is the absorbance test of the water phase and the toluene phase after the extraction is completed;

[0050] Figure 5 .By combining with a commercial microplate reader, the extraction process in the double-layer vertical liquid-liquid stacking system was detected in real time in situ for a long time, where a is the detection image of vertically stacked droplets of water phase and n-hexadecane phase on the microplate reader, and b is the absorbance change in the n-hexadecane phase over time;

[0051] Figure 6.Multi-step reactions are carried out in a three-phase vertical liquid-liquid stacking system without manual handling of intermediates, and each step of the reaction is monitored in real time by a commercial microplate reader, wherein a is a schematic diagram of the multi-step reaction, b is a schematic diagram of the droplet reaction operation, c is the real-time monitoring of the absorbance of the first step reaction in the droplet, d is the real-time monitoring of the absorbance of the second step reaction in the droplet, e is the NMR spectrum of the purified product after the reaction is completed, and f is the NMR spectrum of the mixture of two different products;

[0052] Figure 7 .High-throughput reactions were carried out on a multi-component droplet micro-reaction platform, and real-time in-situ monitoring of each reaction unit was performed using a commercial microplate reader, wherein a is a schematic diagram of a multi-reactant system in a droplet, b is the filling of multi-component droplets using a microelectronic automatic droplet filling device, c is the color change of multiple droplets at different times, and d is a heat map of the characteristic absorption peak intensity of each droplet reaction unit;

[0053] Figure 8 .Automatic droplet printing is performed on a droplet micro-reaction platform by a microelectronic printer, and a droplet array with a concentration gradient can be printed, wherein a is the injection of multi-phase stacked droplets using a microelectronic automatic droplet injection device, b is the contact induced by multi-component droplets on a patterned surface, and c is the automatic construction of gradient concentration droplets;

[0054] Fig. 9 .Design and actual pictures of patterned chips for high-throughput reactions, where a is the printed patterned chip as the reaction platform base, b is the printed patterned chip as the reaction platform top cover, and c is the design of the flip-top metal sandwich aligner. DETAILED DESCRIPTION

[0055] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0056] Example 1 Multi-component droplet micro-reaction platform for vertical liquid-liquid stacking system

[0057] 1. Structure and preparation process of multi-component droplet micro-reaction platform

[0058] In order to achieve the effect of oil-water layer stacking, it is necessary to prepare a super-hydrophobic-hydrophilic patterned polymer chip. First, a DLP printer is used to print two concentric rings of hydroxyethyl methacrylate-ethylene glycol dimethacrylate polymer on a customized glass substrate at a time (as a preferred method, a number of grooves with a diameter of 8 mm are provided on the glass substrate, and the concentric rings use the center of the groove as the dot), the widths of the inner and outer rings are 4 mm and 3 mm respectively, and the spacing between the two rings is 4 mm.

[0059] The printing method of hydroxyethyl methacrylate-ethylene glycol dimethacrylate polymer is as follows: the polymerization solution used includes 3g HEMA, 2g EDMA, 100mg phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, 2g cyclohexanol and 3g decanol, and 0.2g Sudan1 and 0.3g p-methoxyphenol are added as UV absorbers. We selected a DLP printer to establish a maskless lithography system based on DMD. Before printing, 45μL of the polymerization solution was first dripped on the printing platform of the printer, and two polyimide films with a thickness of 6 microns were placed on both sides of the droplet to control the film thickness. Then, the pre-modified cover glass was covered on the printing platform, so that a liquid layer of uniform thickness was formed between the platform and the glass sheet. Finally, the printing pattern pre-designed on the PC side was imported into the DMD-based lithography system, and the pattern was projected onto the printing platform in the form of blue light. The illuminated area was UV-crosslinked to form a polymer film, and the exposed area was glass. After the exposure is completed, carefully lift the glass substrate with a knife, and thoroughly clean the surface with ethanol and dry it. (The reaction is carried out in a laboratory environment at room temperature and pressure)

[0060] In addition, the glass substrate must be modified in advance before the polymer film can be printed on it. Modification conditions: In order to make the porous polymer film adhere tightly to the surface of the substrate material, the surface of the material needs to be modified. First, dissolve 400μL of 3-(methacryloyloxy)propyltrimethoxysilane in 2mL of ethanol, mix well, and prepare a modified solution. Then, in order to expose the hydroxyl groups on the surface of the substrate, the substrate material needs to be specially treated. For the glass substrate, in order to expose the hydroxyl groups on the surface of the substrate, it is necessary to soak the cover glass in 1M HCl solution for 1h, and then soak it in 1M NaOH solution for 30min. After taking it out, wash it with deionized water and dry it with an air gun for use. Finally, take 50μL of the modified solution and drop it between the two treated materials. After standing for 6h, rinse it with ethanol and dry it with an air gun. The modification is complete.

[0061] Perfluorooctanoyl chloride was grafted onto the surface of the printed porous polymer film through esterification reaction (the conditions of the esterification reaction were: the printed sample was placed in a 50 ml centrifuge tube, 50 ml of anhydrous dichloromethane was added to immerse the sample, then 50 mg of 4-dimethylaminopyridine was added as an initiator, 80 microliters of triethylamine and 60 microliters of perfluorooctanoyl chloride were added, and the reaction was completed after stirring for 4-6 hours under an inert atmosphere) to make it super hydrophobic. The glass area not covered with porous polymer is hydrophilically modified with methacryloylethyltrimethylammonium chloride (the conditions for hydrophilic modification are: 50 mg of methacryloylethyltrimethylammonium chloride is dissolved in 500 mg of N,N-dimethylformamide, and 1 mg of phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide is added as an initiator. After mixing evenly, 40-60 microliters of the above reaction solution is added dropwise to the sample surface, and the glass surface to be grafted is exposed using a DLP printer to make it hydrophilic), thereby preparing a printed chip with a super hydrophobic-hydrophilic concentric circle pattern. Since the aqueous solution is designed to be anchored in the hydrophilic glass area, the printed chip has good acid and alkali corrosion resistance (the test results of resistance to 100mM HCl or 10mM NaOH are shown in Figure 2 shown).

[0062] The printed chip can form a multi-component droplet micro-reaction platform after further adding necessary components such as a cover plate and / or a sealing device and / or a microelectronic printer and / or a stirring device and / or a detection device.

[0063] 2. Formation of vertical liquid-liquid stacking system

[0064] When performing micro-reactions, different liquid phases need to be added to the super-hydrophobic-hydrophilic patterned polymer chip to form a stacking system of liquid phases for the reaction to proceed.

[0065] The stacking system of this embodiment can be constructed as follows:

[0066] Double-layer liquid-liquid stacking system: First, add 0.5-1mL of aqueous solution into the central super-hydrophobic polymer ring, and at the same time add 0.5-1mL of aqueous solution between the two outer super-hydrophobic polymer rings, so as to form two water circles on the substrate surface. Secondly, add 0.5-1mL of an organic solvent (such as toluene, n-hexadecane, octyl acetate, etc.) that is immiscible with water and has a lower density than the aqueous solution into the outermost water circle, so that it completely covers the inner water circle and penetrates the super-hydrophobic polymer film, thereby forming a double-layer liquid stacking system ( Figure 3 a).

[0067] Three-layer liquid-liquid stacking system: Different from the bottom-up liquid addition method of the two-layer liquid-liquid stacking system (i.e., first inject the bottom layer of liquid, then add the second layer), the top-down liquid addition method is used here. First, 0.5-1mL of aqueous solution is added to the middle area of ​​the two outer super-hydrophobic polymer rings to form the outermost water circle to achieve the purpose of limiting the organic solvent in the inner ring. Secondly, 0.7-1.3mL of organic solution with a density lower than that of aqueous solution (such as toluene, n-hexadecane, octyl acetate, etc.) is added to the water circle just formed, and then 0.2-0.7μL of aqueous solution is injected under toluene with a pipette. Due to the restriction of water by the internal super-hydrophobic polymer ring, the injected water will be confined to the super-hydrophobic polymer ring in the center and will not diffuse outward. Finally, 0.1-0.4 μL of organic solvent (with a density greater than that of water and immiscible with water, especially note that this embodiment uses a customized substrate, that is, a groove with a diameter of 8 mm is provided on the glass substrate in the middle of each concentric ring. Therefore, for the three-layer vertical stacking system, it is not necessary to print a super-hydrophobic ring in the inner circle, and the final organic solvent can be restricted by the groove. As a preferred method, a relatively narrow super-hydrophobic film with a width of 0.5 mm-1 mm can also be printed around the groove, so that the organic solvent can be effectively restricted while allowing the aqueous solution to cover it), thereby forming a three-layer vertical liquid-liquid stacking system ( Figure 3 c).

[0068] Example 2 Multi-component droplet micro-reaction platform for lateral liquid-liquid stacking system

[0069] 1. Structure and preparation process of multi-component droplet micro-reaction platform

[0070] In order to achieve lateral contact of multi-layer liquid, three concentric rings of hydroxyethyl methacrylate-ethylene glycol dimethacrylate polymer (the polymer used is the same as in Example 1) were first printed at one time on a customized glass substrate using a DLP 3D printer. The sizes of the polymer rings from the inside to the outside were 1.5 mm, 2.5 mm and 2.5 mm, respectively, and the intervals between the rings were 2 mm and 2.5 mm from the inside to the outside. Perfluorooctanoyl chloride was grafted on the surface of the printed ring polymer film through esterification reaction (the raw materials and preparation method of this step are the same as in Example 1) to make it super hydrophobic, while the glass area was hydrophilically modified with methacryloylethyl trimethylammonium chloride (the raw materials and preparation method of this step are the same as in Example 1), thereby preparing a printed chip with a super hydrophobic-hydrophilic concentric circle pattern, on which lateral liquids were stacked.

[0071] The printed chip can form a multi-component droplet micro-reaction platform after further adding necessary components such as a cover plate and / or a sealing device and / or a microelectronic printer and / or a stirring device and / or a detection device.

[0072] 2. Formation of vertical liquid-liquid stacking system

[0073] When performing micro-reactions, different liquid phases need to be added to the super-hydrophobic-hydrophilic patterned polymer chip to form a stacking system of liquid phases for the reaction to proceed.

[0074] The stacking system of this embodiment can be constructed as follows:

[0075] Formation of three-phase lateral liquid-liquid stacking system: Since the stacking of vertical multiphase liquids greatly depends on the sequential arrangement and combination of the densities of each phase of liquid, in order to break this limitation, a lateral liquid stacking system was developed ( Figure 3 d). First, 0.1-1 mL of aqueous solution is added to the glass region between the two outermost super-hydrophobic polymer rings to form the outermost water ring, and 0.1-1 mL of aqueous solution is added to the middle glass ring region to limit the 0.1-1 mL of organic solvent added later, and then 0.1-1 mL of organic solvent is added to the middle of the aqueous solution in the inner ring to form an organic phase in the middle, and then 0.1-1 mL of another organic solution is added to the super-hydrophobic polymer ring region between the two water rings, thereby forming a three-phase liquid-liquid lateral stacking system ( Figure 3 d), and so on to form a liquid-liquid lateral stacking system of four or more phases.

[0076] Formation of a four-phase liquid-liquid mixed stacking system: In the formed three-phase horizontal liquid-liquid stacking system, 50-250 μL of an aqueous solution (such as saturated sodium bromide) with a density greater than that of the organic phase is injected under the middle organic phase with a pipette, thereby adding a vertical liquid-liquid stacking system to the horizontal contact system to form a four-phase liquid-liquid mixed stacking system ( Figure 3 e).

[0077] Example 3 Droplet Microreactor for Combinatorial Chemistry

[0078] Based on Example 1 or 2, this example constructs a multi-component droplet microreaction platform (ie, droplet microreactor) for combinatorial chemistry.

[0079] In order to be suitable for high-throughput reactions, a flip-top metal sandwich aligner that matches the size of a commercial 96-well microplate was first customized to accommodate the pre-patterned chip. Subsequently, a microreactor array was prepared on a 75×100 mm substrate in the manner of Example 1 or Example 2, ensuring that the size and spacing of each circular microreactor matched the commercial 96-well microplate. At the same time, a set of superhydrophobic polymer rings that matched the circular microreactor size of the substrate were printed on another 75 mm×100 mm quartz slide (cover plate). This alignment ensures that when the aligner is closed, each unit of the substrate and cover plate is perfectly aligned, enabling the initiation of subsequent parallel chemical reactions ( Fig. 9 ).

[0080] Example 4: Using machine-assisted liquid addition and in-situ real-time monitoring of chemical reactions in a droplet stacking system

[0081] Based on Example 1 or Example 2, this example further improves the multi-component droplet micro-reaction platform and provides an application example.

[0082] 1. Machine-assisted liquid addition

[0083] The automatic liquid addition operation was completed using a micro-electronic printer. The path, speed and air pressure of the micro-electronic printer for liquid addition were pre-designed in the computer design software, so as to manipulate the injection needle to drip a certain amount of various solvents (such as octyl acetate, n-hexadecane, water, 1,3-dibromopropane, DMF, etc.) in a specific area. In order to test the stability and repeatability of the machine-assisted liquid addition, the droplet arrays of water and octyl acetate were printed on the oleophobic cover glass pre-modified with perfluorodecanethiol according to the set program. After the sample was weighed, the droplet array was printed again at the same position, keeping the printing conditions unchanged, and weighed again. The printing was repeated 9 times to study whether there was a linear relationship between the number of prints and the weight.

[0084] This strategy can also be used for printing liquids of different concentrations. A methylene blue-dyed aqueous solution (0.05 mg mL-1) is prepared in advance as a printing solution, and printed on a glass substrate (prepared according to the method of Example 1 and Example 2) with a 1×4 superhydrophobic circular polymer frame printed on the surface. First, the same amount of methylene blue-dyed water droplets are printed in four circular rings at a certain air pressure, and then the methylene blue-dyed aqueous solution is printed only in the last three circular rings under the same printing conditions, decreasing in sequence. After that, water droplets are printed in the four circular rings in the opposite order so that the total volume of water droplets in each circular ring is the same, thereby forming a droplet array with a concentration gradient, and finally the absorbance of the obtained droplet array is tested with a commercial microplate reader ( Figure 8 ).

[0085] 2. Machine-assisted printing droplet stacking system

[0086] Two-phase lateral stacking system: The circular liquid addition path is set in advance on the computer design software, and the injection end of the microelectronic printer is controlled to add water droplets to the hydrophilic glass area on the surface of the concentric circular printed polymer chip at a constant speed, thereby forming a closed water ring on the surface, and then the injection end is controlled to inject an organic solvent (such as octyl acetate) into the water ring according to a given program.

[0087] Vertical stacking system (liquid addition method: from top to bottom): The liquid addition sequence and conditions are set in advance on the computer design software, and the injection end of the microelectronic printer is controlled to first drip an organic solvent (such as octyl acetate) into the oleophobic polymer ring on the surface of the concentric circular printed polymer chip, and then the injection end is controlled to inject the aqueous solution under the octyl acetate phase according to a given program.

[0088] Vertical stacking system (liquid addition method: bottom-up): The liquid addition sequence and conditions are set in advance on the computer design software, and the injection end of the microelectronic printer is controlled to first drip the aqueous solution into the super-hydrophobic polymer ring in the central area of ​​the concentric circular printed polymer chip surface, and then the injection end is controlled to inject the organic solution (such as octyl acetate) above the aqueous phase according to the given program ( Figure 8 ).

[0089] 3. Extraction reaction in stacked droplets

[0090] In order to prove that the above-mentioned microreactor can effectively separate substances in droplets, a separation reaction of a mixed solution was designed. In order to introduce the stirring function into the extraction reaction system, a 50×50×4mm square glass substrate with a circular groove in the middle of the substrate (radius: 4mm, depth: 3mm) was specially customized. Two concentric polymer rings were printed on it and superhydrophobic modified, and the glass area was hydrophilic modified. Before the reaction started, 100-400μL of a mixed aqueous solution containing both amphiphilic dye 1 (0.5-2.5mg mL-1) and methylene blue (dye 2, 0.05-0.25mg mL-1) was added to the inner circle of the reactor, and then 0.8-1.5mL of an organic solvent (such as toluene) was added above the aqueous solution. The stirrer speed was maintained at 200-600rpm and the reaction was carried out at room temperature. When the colors of both the toluene and aqueous phases were stable, the reaction was considered complete, thereby terminating the extraction process ( Figure 4 ).

[0091] In order to enable in situ monitoring of the extraction process using a commercial microplate reader, a detection substrate was specially customized so that a 25×75×1mm cover glass could be embedded in it. Before the reaction started, 10-100μL of NaOH aqueous solution (pH=12) dyed with rhodamine B (0.001-0.005mgmL-1) was added to the inner ring of the printed polymer chip, followed by 200-500μL of n-hexadecane to cover the aqueous phase and start the extraction reaction. The computer program is designed to continuously monitor the system for 30h, while ensuring that an area containing only n-hexadecane is detected in situ every 2h, and the results are displayed in real time on the computer ( Figure 5 ).

[0092] 4. Multi-step reactions in stacked droplets

[0093] Three-phase vertical stacking droplets are constructed in the manner described in Example 2, and multi-step synthesis is carried out in three-phase vertical stacking droplets. In the first step, 65.5mg triphenylphosphine (0.25mmol) is dissolved in 750μL octyl acetate, and added to the reactor, 300μL deionized water is subsequently injected below the octyl acetate phase, and droplets are formed in the inner circle of the reactor, thereby forming a two-phase droplet vertical stacking system, followed by 23μL bromoacetate methyl esters (0.25mmol) dissolved in 250μL octyl acetate, and added to the octyl acetate phase in the reactor. The appearance of the white precipitate insoluble in octyl acetate marks the beginning of the first step of the reaction. The water phase is detected regularly using a commercial microplate reader, until the absorption peak at 268nm no longer increases, and the octyl acetate phase is clarified, indicating that the first step of the reaction is completed. In order to improve the yield, it is possible to choose to remove the upper octyl acetate phase with a pipette.

[0094] In the second step, 250 μL of terephthalaldehyde (33.5 mg, 0.25 mmol) dissolved in 1,3-dibromopropane was added to the bottom of the aqueous phase with a pipette to form droplets, and potassium carbonate (51.7 mg, 0.35 mmol) dissolved in 200 μL of water was injected into the aqueous phase, and a white precipitate was observed to be immediately produced in the water. At this time, the 1,3-dibromopropane phase was detected again using a commercial microplate reader until the absorption peak at 302 nm no longer increased and the aqueous phase became clear, marking the end of the entire reaction. Since the droplet reaction was small in scale and it was inconvenient to collect and characterize the final product, four parallel experiments were carried out under the same temperature, time and conditions, and the products were purified and combined. After washing several times with deionized water, saturated NaHCO3 solution and saturated sodium chloride solution, the organic phase was dried with anhydrous MgSO4. After removing MgSO4 and 1,3-dibromopropane by rotary evaporation, the resulting white powder was chromatographed on a silica gel column (DCM: hexane = 7:

[0095] 3) Further purification gave 28.4 mg of a white final product ( Figure 6 ).

[0096] 5. Machine-assisted high-throughput reactions

[0097] With the help of a microelectronic liquid dispenser, a 6×7 ternary droplet array containing each reactant and AuNP catalyst was dispensed onto the pre-patterned chip. For each subunit of the patterned array, 8 μL of a gradient concentration (ranging from 0 mM to 1.25 mM) of a resazurin solution and 8 μL of a gradient concentration (ranging from 0 mM to 1.5 mM) of an NH2OH solution were dispensed. The microelectronic dispenser uses software to adjust the nozzle position of the dispenser and then deposits the AuNP dispersion onto the central area of ​​the chip array, resulting in a total of 6×7 ternary droplet arrays of reactants and catalysts. After completing these steps, the cover chip fixed on the upper part of the aligner was flipped over to close it. The spacing between the cover layer and the substrate of the aligner was controlled at 0.8 mm to ensure precise alignment, allowing the reaction to be initiated by squeezing and mixing the three different solutions. The assembled device was then placed in a commercial microplate reader and the absorbance of each droplet unit was monitored during the reaction. Note that a sealing strip was used between the cover and the substrate to minimize droplet evaporation ( Figure 7 ).

[0098] Example 5: Strategy for preventing the volatilization of low boiling point solvents during the reaction

[0099] On the basis of Example 1 or Example 2, this example further improves the multi-component droplet micro-reaction platform and provides a strategy for preventing the volatilization of low-boiling point solvents during the reaction process.

[0100] 1. Packaging of high boiling point solvents

[0101] 100 μL of ionic liquid, octyl acetate and silicone oil were respectively added dropwise into the oleophobic ring (the preparation method of the oleophobic ring is to graft polyperfluorooctylethyl methacrylate on the surface of the porous polymer film printed in Example 1 by a DLP printer in a photopolymerization manner) shaped frame, exposed to air at room temperature and pressure for 96 hours, and weighed every 24 hours during this period to detect the volatility of the above high boiling point solvent in the air. The results show that the mass change of the high boiling point solution is not obvious during the 96 hours of being placed in the air, proving that no additional measures are required to prevent the high boiling point solvent from volatilizing.

[0102] 2. Packaging of low boiling point solvents

[0103] Water, which is more common in chemical reactions, was selected as a representative of low-boiling-point solvents to explore strategies to prevent the volatilization of low-boiling-point solvents. For a 10 μL water droplet microarray, a humidifier was placed in a transparent square glass cover in advance to create a saturated vapor pressure environment, and then the sample was placed in a transparent square glass cover to prevent the droplets from volatilizing.

[0104] For a 100μL water droplet, there are two encapsulation strategies to prevent the volatilization of the aqueous solution. The first is to encapsulate the water droplet with a high boiling point solvent, namely n-hexadecane. Specifically, a small microreactor chip is prepared so that water and n-hexadecane form a vertically stacked structure on the chip surface, that is, n-hexadecane covers the aqueous solution, thereby achieving the purpose of preventing the water droplet from volatilizing; the second method is to pre-print a resin frame with a 3D printer, solidify it on the printed chip, and encapsulate the water droplets on the chip surface, thereby reducing its volatilization.

[0105] Through the above examples, it can be seen that the present invention designs and prepares different hydrophilic and hydrophobic patterned printed polymer chips for different application scenarios, and utilizes the difference in wettability of the chip surface to stack different multiphase immiscible liquids on it, utilizes machine-assisted liquid addition to carry out different chemical reactions in the droplet stacking system and monitors them in situ in real time, and provides a strategy for preventing the volatilization of low-boiling point solvents during the reaction process. Therefore, the present invention provides a new multi-component droplet micro-reaction platform and method with good application prospects.

Claims

1. A multi-component droplet micro-reaction platform, characterized in that: The invention comprises a substrate, the surface of which is provided with an array composed of at least one microreactor, wherein the microreactor is a patterned surface composed of at least two regions with different wetting properties.

2. The multi-component droplet micro-reaction platform according to claim 1, characterized in that: The microreactor is a patterned surface consisting of at least one hydrophilic region and at least one hydrophobic region; Alternatively, the microreactor is a patterned surface consisting of at least one oleophilic region and at least one oleophobic region.

3. The multi-component droplet micro-reaction platform according to claim 2, characterized in that: The hydrophilic region is a substrate surface modified by a hydrophilic acrylic monomer or a hydrophilic acrylate monomer; and the hydrophobic region is a super-hydrophobic region.

4. The multi-component droplet micro-reaction platform according to claim 3, characterized in that: The hydrophilic acrylic monomer is selected from at least one of acrylic acid and methacrylic acid; the super hydrophobic region is a porous polymer film with perfluorooctanoyl chloride grafted on the surface, or the oleophobic region is a porous polymer film with polyperfluorooctylethyl methacrylate grafted on the surface.

5. The multi-component droplet micro-reaction platform according to claim 2, characterized in that: The patterned surface includes a closed area consisting of at least two hydrophobic areas or at least two oleophobic areas.

6. The multi-component droplet micro-reaction platform according to claim 1, characterized in that: The material of the substrate is selected from at least one of glass, quartz and metal.

7. The multi-component droplet micro-reaction platform according to claim 1, characterized in that: A sealing cover plate is arranged above the substrate, and a patterned surface which is mirror-symmetrical with the microreactor is arranged on the cover plate.

8. The multi-component droplet micro-reaction platform according to claim 1, characterized in that: The invention also comprises a microelectronic printer for adding liquid into the microreactor, wherein the liquid adding path, speed and gas pressure of the microelectronic printer are controlled by computer software.

9. The multi-component droplet micro-reaction platform according to claim 1, characterized in that: Also includes a stirring device and / or a detection device.

10. The method for preparing the multi-component droplet micro-reaction platform according to any one of claims 1 to 9, characterized in that: include: Step 1, using a DLP printer to print a porous polymer on the surface of the substrate to form a porous polymer film having a pattern of the hydrophobic region; Step 2, grafting perfluorooctanoyl chloride on the surface of the printed porous polymer film; or, grafting polyperfluorooctylethyl methacrylate on the surface of the printed porous polymer film by photopolymerization using a DLP printer; Step 3, performing hydrophilic modification on the surface of the substrate; Step 4, using regions with different wettability in the microreactor to fix different liquid phases respectively, so that different liquids contact and interact with each other for reaction and detection.

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