A method for preparing water-water microspheres based on strong charge interaction

Abstract

The invention discloses a method for preparing water-water microspheres based on strong charge action, including: dissolving a polyanionic compound containing a strong negative charge in water, filtering, and obtaining a dispersed phase; mixing an emulsifier, a polycationic compound containing a strong positive charge, and a solvent, ultrasonicating, filtering, and obtaining a continuous phase; the dispersed phase arrives at an intersection along a dispersed phase channel; the continuous phase arrives at an intersection along two symmetrical continuous phase channels, and the continuous phase contacts the dispersed phase at the intersection, and the dispersed phase is wrapped by the continuous phase through shear force, and the polyanionic compound with a negative charge on the surface and the polycationic compound with a positive charge undergo an ionic crosslinking reaction at the contact interface of the dispersed phase and the continuous phase to form a water-water microsphere with a strong charge action as a template; washing and further crosslinking of the water-water microsphere with a strong charge action as a template to obtain a water-water microsphere product. The invention also discloses the application of water-water microspheres based on strong charge action in chemical analysis detection.

Description

Technical Field

[0001] The invention belongs to the fields of microfluidic technology, material chemistry, etc., and relates to a preparation method of water-water microspheres based on strong charge action. Background Art

[0002] Aqueous two-phase systems, also called fully aqueous systems, are formed by the phase separation of an aqueous mixture containing two incompatible additives, such as two incompatible polymers or a polymer and other salts, above a critical concentration. In the 1950s, Per-Ake Albertsson utilized aqueous two-phase systems to concentrate and separate different types of biological materials. Since then, aqueous two-phase systems have gained great attention in various fields such as food industry, chemical synthesis, and biomedical engineering.

[0003] Microspheres refer to a microparticle dispersion system formed by drug dispersion or adsorption in a macromolecular or polymer matrix. They can be used for loading and delivery of drugs, cells, etc. Currently, most microspheres are composed of an aqueous phase and an oil phase. The inert oil and surfactant in the oil phase will affect cells and drugs, resulting in reduced activity of the loaded material. Chemical substances in the aqueous phase are also prone to leakage and destroy the stability of the surfactant, affecting the stability of the microspheres. Microspheres containing an oil phase have low biocompatibility and cause great damage to the loaded material, which is not conducive to the widespread application of microspheres.

[0004] Therefore, in an all-water environment, water-in-water microspheres produced by introducing an aqueous two-phase system have attracted more and more interest. The formation of two-phase aqueous microspheres can be achieved by changing the interfacial tension, viscosity and flow rate of the two aqueous phases. The preparation methods include passive method, piezoelectric disk method, pulse pressure control method, capillary method, etc. The preparation method is too complicated and involves a variety of instruments, which greatly limits the wide application of two-phase aqueous microspheres. Summary of the invention

[0005] The purpose of the present invention is to provide a simple method for preparing water-water microspheres based on strong charge interaction. By using a microfluidic chip and a two-phase aqueous system, water-water microspheres with controllable morphology and high yield are prepared in a one-step manner within the chip, providing early technical support for the development of pharmacy, medicine, biology, etc.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing water-water microspheres based on strong charge interaction comprises the following steps:

[0008] Step (1), microfluidic chip: the microfluidic chip comprises a continuous phase inlet, a dispersed phase inlet, a continuous phase channel, a dispersed phase channel, an intersection, a microsphere forming channel, and a microsphere outlet; the continuous phase inlet is communicated with two symmetrically arranged continuous phase channels, the dispersed phase inlet is communicated with the dispersed phase channel, the outlet ends of the two continuous phase channels, the outlet end of the dispersed phase channel, and the inlet end of the microsphere forming channel are communicated through a "cross" shaped intersection, and the outlet end of the continuous phase channel is vertically distributed with the outlet end of the dispersed phase channel, and the outlet end of the microsphere forming channel is connected to the microsphere outlet;

[0009] Step (2), preparation of dispersed phase and continuous phase: dissolving a polyanionic compound containing a strong negative charge in water, filtering to obtain a dispersed phase; mixing an emulsifier, a polycationic compound containing a strong positive charge and a solvent, ultrasonicating, filtering to obtain a continuous phase;

[0010] Step (3), formation of water-water microspheres with strong charge as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet and reaches the intersection along the dispersed phase channel; the continuous phase enters the microfluidic chip through the continuous phase inlet and reaches the intersection along two symmetrical continuous phase channels, the continuous phase contacts the dispersed phase at the intersection, and the formation of microspheres is promoted by shear force, the dispersed phase (as the inner phase) is wrapped by the continuous phase (as the outer phase), and the polyanionic compound with negative charge on the surface and the polycationic compound with positive charge undergo ion crosslinking reaction at the contact interface between the dispersed phase and the continuous phase, thereby forming water-water microspheres with strong charge as template;

[0011] Step (4), washing and further cross-linking of water-water microspheres with strong charge as template: collecting microspheres dispersed in the continuous phase, the microspheres are oil-in-water-in-water, washing with acetone and then centrifuging to remove the supernatant to obtain water-in-water microspheres, then adding a polycationic compound solution containing a strong positive charge for further cross-linking, centrifuging to remove the supernatant, and then washing with anhydrous ethanol and then centrifuging to remove the supernatant to obtain a water-water microsphere product, which is stored in PBS buffer.

[0012] In step (1), a microfluidic chip is prepared by using a silicon wafer as a base template and a soft etching method. The silicon wafer template preparation process includes designing a chip structure, printing a photomask, laying a photosensitive resin, ultraviolet exposure, developing and etching, etc. A microfluidic chip is prepared using polydimethylsiloxane (PDMS) as a material, PDMS is mixed and poured on a silicon wafer template, solidified, and peeled off to obtain a transparent PDMS layer with microchannels, and holes are punched at the inlet and outlet positions of the PDMS layer to form a continuous phase inlet, a dispersed phase inlet, and a microsphere outlet. The PDMS layer and the blank PDMS base layer are dusted with tape and placed in a plasma cleaning machine for 60 seconds. Under high-speed and high-energy plasma bombardment, the PDMS surface structure is maximized, and at the same time, the blank PDMS base layer and the chip surface containing microchannels form an active layer. The blank base layer is covered on the chip surface to form a bond between the PDMS layer and the blank PDMS base layer, and the PDMS layer is cured at 75°C for 2 hours to obtain a microfluidic chip.

[0013] The width of the dispersed phase channel is 30-50 μm, the width of the continuous phase channel is 80-100 μm, and the width of the microsphere forming channel is 80-100 μm; the height of the dispersed phase channel is 30-50 μm, and the dispersed phase channel, continuous phase channel and microsphere forming channel have the same height.

[0014] In step (2), preferably, the dispersed phase is prepared by dissolving a polyanionic compound containing a strong negative charge in water and filtering with a 0.22 μm filter to obtain a dispersed phase.

[0015] The polyanion compound containing strong negative charge is at least one of alginate or its derivatives, hyaluronic acid or its derivatives.

[0016] The concentration of the polyanion compound in the dispersed phase is 1-2% (g / ml).

[0017] Preferably, the continuous phase is prepared by mixing an emulsifier, a polycationic compound containing a strong positive charge and a solvent, ultrasonicating for 1.5 to 5 hours at an ultrasonic power of 400 W, and then filtering through a 0.45 μm organic phase filter membrane to obtain a continuous phase, which is a solution.

[0018] The polycationic compound containing strong positive charge is at least one of α-polylysine, ε-polylysine, polyornithine, poly-L-arginine, chitosan, tetramethylammonium oxalate, tetramethylammonium bromide and tetramethylammonium chloride.

[0019] The emulsifier is at least one of emulsifier EM90 and Span80 (sorbitan oleate). The continuous phase is an oil phase, and the microfluidic chip is hydrophilic. The emulsifier is added to improve the hydrophilicity of the continuous phase, so that the continuous phase can flow according to the channel shape. If the emulsifier is not added, there is no way to prepare microspheres.

[0020] The solvent is undecanol, paraffin oil, soybean oil, C 12 -C 18 At least one of alkanes and silicone oils.

[0021] The concentration of the polycationic compound with strong positive charge in the continuous phase is saturated concentration, and the concentration of the emulsifier is 1-5% (ml / ml).

[0022] In step (3), the flow rate of the dispersed phase is 10-35 μL / h; the flow rate of the continuous phase is 300-1800 μL / h. The dispersed phase and the continuous phase are in contact at the cross-intersection, the flow rate of the continuous phase is large, the flow rate of the dispersed phase is small, the force generated by the continuous phase is perpendicular to the dispersed phase, and a force is generated in the adjacent part of the dispersed phase fluid, so that the dispersed phase is relatively displaced and deformed along the direction of the continuous phase force, and due to the push of the pump, the microspheres formed by shearing can move forward. And within the dispersed phase flow rate range of the present invention, as the dispersed phase flow rate increases, the microsphere diameter increases; within the continuous phase flow rate range of the present invention, as the continuous phase flow rate increases, the microsphere diameter decreases.

[0023] In step (4), the acetone washing time is 0.5 to 2 minutes; the centrifugal speed is 1000 to 10000 rpm / min, and the centrifugal time is 3 to 5 minutes.

[0024] The concentration of the polycationic compound solution containing strong positive charge is 1-5% (g / ml), and the polycationic compound solution containing strong positive charge is prepared with water as solvent; the polycationic compound containing strong positive charge is at least one of α-polylysine, ε-polylysine, polyornithine, polyarginine, chitosan, tetramethylammonium oxalate, tetramethylammonium bromide, and tetramethylammonium chloride. Further, the polycationic compound containing strong positive charge is the same as step (2).

[0025] The cross-linking time is 20 to 60 seconds. The present invention avoids the situation that there are almost no microspheres after washing by further cross-linking. After cross-linking, centrifuge at a speed of 1000 rpm / min for 3 minutes to remove the supernatant.

[0026] The washing time with anhydrous ethanol is 0.5 to 2 minutes; the centrifugal speed is 1000 to 10000 rpm / min, and the centrifugal time is 3 to 5 minutes.

[0027] Another object of the present invention is to provide the use of the water-water microspheres based on strong charge interaction in chemical analysis detection, preferably in the detection of hydrogen peroxide and dopamine.

[0028] Specifically, a method for detecting hydrogen peroxide includes: dispersing a platinum disulfide solution in a dispersed phase so that the concentration of platinum disulfide in the dispersed phase is 0.2 mg / mL, and preparing microspheres according to the preparation method of the microspheres of the present invention; taking the microspheres and placing them in 50 μL of NaH2PO4 (pH5.0) buffer solution; adding 5 μL of a sample to be detected and 1 μL of a 50 mM TMB solution to 50 μL of the NaH2PO4 (pH5.0) buffer solution containing the microspheres, and adding 20 mM NaH2PO4 to keep the total volume at 100 μL; observing under a microscope, if the microspheres change from colorless to blue, it indicates that the sample to be detected contains hydrogen peroxide.

[0029] Specifically, a method for detecting dopamine includes: dispersing a platinum disulfide solution in a dispersed phase so that the concentration of platinum disulfide in the dispersed phase is 0.2 mg / mL, and preparing microspheres according to the preparation method of the microspheres of the present invention; taking the microspheres and placing them in 50 μL of NaH2PO4 (pH5.0) buffer solution; adding 5 μL of a 9.8M hydrogen peroxide solution and 1 μL of a 50mM TMB solution to 50 μL of the NaH2PO4 (pH5.0) buffer solution containing the microspheres, adding 20 mM NaH2PO4 to keep the total volume at 100 μL, and after the color of the microspheres turns blue and no longer changes, adding 5 μL of a sample to be detected; observing under a microscope, if the color of the microspheres becomes lighter in blue, it indicates that the sample to be detected contains dopamine.

[0030] Beneficial effects of the present invention:

[0031] The present invention uses a microfluidic chip as a platform and a two-phase aqueous system as an integrated template to prepare composite microspheres containing oppositely charged polyanionic compounds such as sodium alginate and polycationic compounds such as ε-polylysine. The microspheres can be used for chemical analysis and detection, and play a huge role in the fields of biology, medicine, pharmacy, chemistry, etc.

[0032] The present invention generates shear force by the continuous phase, promotes the formation of microspheres by the shear force, and avoids "the width of the dispersed phase channel is large, and a large shear force needs to be applied to form microspheres. At this time, a large continuous phase flow rate and a small dispersed phase flow rate are required to form microspheres, but the microsphere diameter is large" by controlling the flow rates of the dispersed phase and the continuous phase, and avoids "the continuous phase flow rate is too high to cause the dispersed phase to reflux, and the dispersed phase flow rate is too small to flow out of the dispersed phase channel, which affects the formation of microspheres". BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the structure of a microfluidic chip; wherein, 1-continuous phase inlet, 2-dispersed phase inlet, 3-microsphere outlet, 4-continuous phase channel, 5-dispersed phase channel, 6-microsphere formation channel, 7-intersection.

[0034] Figure 2 This is an optical microscope characterization image of the microspheres in Example 1 (scale: 500 μm).

[0035] Figure 3 This is an optical microscope characterization image of the microspheres in Example 2 (scale: 500 μm).

[0036] Figure 4 This is an optical microscope characterization image of the microspheres in Example 3 (scale: 500 μm).

[0037] Figure 5 This is an optical microscope characterization image of hydrogen peroxide detection using microspheres (scale: 500 μm).

[0038] Figure 6 This is an optical microscope characterization image of the microspheres in Example 5 (scale: 500 μm).

[0039] Figure 7 This is an optical microscope characterization image of the microspheres in Example 6 (scale: 500 μm). DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below through specific implementation methods.

[0041] The microspheres were observed using an optical microscope.

[0042] Example 1

[0043] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0044] Step (1), preparation of microfluidic chip: use silicon wafer as base template and soft etching method to make microfluidic chip, the silicon wafer template preparation process includes designing chip structure, photomask printing, photosensitive resin laying, ultraviolet exposure, development and etching, etc. Use polydimethylsiloxane (PDMS) as material to prepare microfluidic chip, mix PDMS and pour it on the silicon wafer template, solidify, peel off, and obtain a transparent PDMS layer printed with microchannels; before chip assembly, first punch holes at the inlet and outlet positions of the PDMS layer to form a continuous phase inlet, dispersed phase inlet, and microsphere outlet respectively, remove dust from the surface of the PDMS layer and the blank PDMS base layer with tape, place them in a plasma cleaner for 60 seconds for bonding, and solidify at 75°C for 2 hours to obtain a microfluidic chip. Figure 1As shown, the microfluidic chip includes a continuous phase inlet 1, a dispersed phase inlet 2, a continuous phase channel 4, a dispersed phase channel 5, an intersection 7, a microsphere forming channel 6, and a microsphere outlet 3; the continuous phase inlet 1 is connected to two symmetrically arranged continuous phase channels 4, the dispersed phase inlet 2 is connected to the dispersed phase channel 5, the outlet ends of the two continuous phase channels 4, the outlet end of the dispersed phase channel 5 and the inlet end of the microsphere forming channel 6 are connected through a "cross"-shaped intersection, and the outlet end of the continuous phase channel 4 is vertically distributed with the outlet end of the dispersed phase channel 5, and the outlet end of the microsphere forming channel 6 is connected with the microsphere outlet 3; wherein the channel cross-sections of the continuous phase channel 4, the dispersed phase channel 5 and the microsphere forming channel 6 are all rectangular, the continuous phase channel 4 is 90 μm wide and 30 μm high, the dispersed phase channel 5 is 40 μm wide and 30 μm high, and the microsphere forming channel 6 is 90 μm wide and 30 μm high.

[0045] Step (2), preparation of dispersed phase: dissolving sodium alginate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium alginate concentration of 2% (g / ml);

[0046] Preparation of continuous phase: emulsifier EM90, ε-polylysine and 1-undecanol were mixed, ultrasonicated for 3 h at an ultrasonic power of 400 W, and filtered through a 0.45 μm organic phase filter to obtain a continuous phase with an EM90 concentration of 5% (ml / ml) and an ε-polylysine concentration of saturated concentration. The continuous phase was a solution;

[0047] Step (3), formation of water-water microspheres with strong charge as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 20 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 900 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the shear force promotes the formation of microspheres. The sodium alginate with anions on the surface and the ε-polylysine with cations undergo ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase, forming sodium alginate@ε-polylysine microspheres with the dispersed phase droplets containing sodium alginate as templates;

[0048] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect the sodium alginate-ε-polylysine microspheres dispersed in the continuous phase, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 3% (g / ml) ε-polylysine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0049] The optical microscopy characterization of the microspheres prepared in this example is as follows Figure 2 The prepared microspheres have a diameter of 65-75 μm, a size deviation of less than 5%, and a uniform morphology.

[0050] Example 2

[0051] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0052] Step (1), preparation of the microfluidic chip is the same as step (1) of Example 1;

[0053] Step (2), preparation of dispersed phase: dissolving sodium alginate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium alginate concentration of 2% (g / ml);

[0054] Preparation of continuous phase: emulsifier EM90, ε-polylysine and 1-undecanol were mixed, ultrasonicated for 3 h at an ultrasonic power of 400 W, and filtered through a 0.45 μm organic phase filter to obtain a continuous phase with an EM90 concentration of 5% (ml / ml) and an ε-polylysine concentration of saturated concentration;

[0055] Step (3), formation of water-water microspheres using strong charge as a template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 20 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 900 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the shear force promotes the formation of microspheres. The sodium alginate with anions on the surface and the ε-polylysine with cations undergo an ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase to form sodium alginate@ε-polylysine microspheres;

[0056] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect the sodium alginate-ε-polylysine microspheres dispersed in the continuous phase, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 1% (g / ml) ε-polylysine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0057] The optical microscopy characterization of the microspheres prepared in this example is as follows Figure 3The diameter of the prepared microspheres is 70-75 μm, the size deviation is less than 5%, and the morphology is uniform. Compared with Example 1, the concentration of the ε-polylysine solution is changed in this example, so that the surface of the microspheres is more dense.

[0058] Example 3

[0059] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0060] Step (1), preparation of the microfluidic chip is the same as step (1) of Example 1;

[0061] Step (2), preparation of dispersed phase: dissolving sodium alginate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium alginate concentration of 2% (g / ml);

[0062] Preparation of continuous phase: emulsifier EM90, ε-polylysine and 1-undecanol were mixed, ultrasonicated at an ultrasonic power of 400 W for 3 h, filtered through a 0.45 μm organic phase filter to obtain a continuous phase with an EM90 concentration of 5% (ml / ml) and an ε-polylysine concentration of saturated concentration;

[0063] Step (3), formation of water-water microspheres using strong charge as a template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 25 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 1200 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the shear force promotes the formation of microspheres. The sodium alginate with anions on the surface and the ε-polylysine with cations undergo an ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase to form sodium alginate@ε-polylysine microspheres;

[0064] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect the sodium alginate-ε-polylysine microspheres dispersed in the continuous phase, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 3% (g / ml) ε-polylysine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0065] The optical microscopy characterization of the microspheres prepared in this example is as follows Figure 4The diameter of the prepared microspheres is 60-70 μm, and the size deviation is less than 5%. Compared with Example 1, the flow rates of the dispersed phase and the continuous phase are changed in this example, so that the diameter of the microspheres is changed.

[0066] Example 4

[0067] The water-water microspheres based on strong charge interaction of Example 3 were used to detect hydrogen peroxide and dopamine respectively.

[0068] The detection method of hydrogen peroxide is as follows:

[0069] The platinum disulfide solution was dispersed in the dispersed phase of step (2) of Example 3, and the concentration of platinum disulfide in the dispersed phase was 0.2 mg / mL. The rest was the same as in Example 3 to prepare microspheres. 1000 microspheres were placed in 50 μL of NaH2PO4 (pH 5.0) buffer solution and observed under a microscope. The microspheres were colorless.

[0070] 5 μL of 9.9 M hydrogen peroxide solution and 1 μL of 50 mM TMB solution were quickly added to 50 μL of NaH2PO4 (pH 5.0) buffer solution containing 1000 microspheres, and 20 mM NaH2PO4 was added to keep the total volume at 100 μL.

[0071] The microspheres were observed to change from colorless to blue ( Figure 5 ), indicating that the microspheres of the present invention can be successfully used for the detection of hydrogen peroxide.

[0072] Dopamine is detected as follows:

[0073] The platinum disulfide solution was dispersed in the dispersed phase of step (2) of Example 3, and the concentration of platinum disulfide in the dispersed phase was 0.2 mg / mL. The rest was the same as in Example 3 to obtain microspheres. 1000 microspheres were placed in 50 μL of NaH2PO4 (pH 5.0) buffer solution and observed under a microscope. The microspheres were colorless.

[0074] 5 μL of 9.9M hydrogen peroxide solution and 1 μL of 50mM TMB solution were quickly added to 50 μL of NaH2PO4 (pH5.0) buffer solution containing 1000 microspheres, and 20mM NaH2PO4 was added to keep the total volume at 100 μL. After the color of the microspheres turned blue and no longer changed, 5 μL of 1mM dopamine solution was added to the above solution. It was observed under a microscope that the blue color of the microspheres gradually became lighter and finally faded completely, indicating that the microspheres of the present invention can be successfully used for the detection of dopamine.

[0075] Example 5

[0076] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0077] Step (1), preparation of the microfluidic chip is the same as step (1) of Example 1;

[0078] Step (2), preparation of dispersed phase: dissolving sodium hyaluronate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium hyaluronate concentration of 2% (g / ml);

[0079] Preparation of continuous phase: emulsifier EM90, ε-polylysine and 1-undecanol were mixed, ultrasonicated at an ultrasonic power of 400 W for 3 h, filtered through a 0.45 μm organic phase filter to obtain a continuous phase with a concentration of EM90 of 5% (ml / ml) and ε-polylysine as a saturated concentration;

[0080] Step (3), formation of water-water microspheres with strong charge as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 25 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 1200 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the shear force promotes the formation of microspheres. The sodium hyaluronate with anions on the surface and the ε-polylysine with cations undergo ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase, forming sodium hyaluronate@ε-polylysine microspheres with the dispersed phase droplets containing sodium hyaluronate as templates;

[0081] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect the sodium hyaluronate@ε-polylysine microspheres dispersed in the continuous phase, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 3% (g / ml) ε-polylysine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0082] The optical microscopy characterization of the microspheres prepared in this example is as follows Figure 6 The diameter of the prepared microspheres is 55-60 μm, and the size deviation is less than 5%.

[0083] Example 6

[0084] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0085] Step (1), preparation of the microfluidic chip is the same as step (1) of Example 1;

[0086] Step (2), preparation of dispersed phase: dissolving sodium alginate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium alginate concentration of 2% (g / ml);

[0087] Preparation of continuous phase: emulsifier Span 80, ε-polylysine and soybean oil were mixed, ultrasonicated at an ultrasonic power of 400 W for 3 h, filtered through a 0.45 μm organic phase filter membrane to obtain a continuous phase with a Span 80 concentration of 5% (ml / ml) and a saturated ε-polylysine concentration;

[0088] Step (3), formation of water-water microspheres with strong charge effect as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 25 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 1200 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the formation of microspheres is promoted by shear force. The sodium alginate with anions on the surface and the ε-polylysine with cations undergo ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase, forming sodium alginate@ε-polylysine microspheres with the sodium alginate dispersed phase as a template;

[0089] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect the sodium alginate@ε-polylysine microspheres dispersed in the continuous phase, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 3% (g / ml) ε-polylysine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0090] The optical microscopy characterization of the microspheres prepared in this example is as follows Figure 7 The diameter of the prepared microspheres is 60-70 μm, and the size deviation is less than 5%.

[0091] Example 7

[0092] A method for preparing water-water microspheres based on strong charge interaction, the steps are as follows:

[0093] Step (1), preparation of the microfluidic chip is the same as step (1) of Example 1;

[0094] Step (2), preparation of dispersed phase: dissolving sodium alginate in water and filtering through a 0.22 μm filter to obtain a dispersed phase with a sodium alginate concentration of 2% (g / ml);

[0095] Preparation of continuous phase: emulsifier EM90, poly-L-arginine and 1-undecanol were mixed, ultrasonicated at an ultrasonic power of 400 W for 3 h, filtered through a 0.45 μm organic phase filter to obtain a continuous phase with an EM90 concentration of 5% (ml / ml) and a polyarginine concentration of saturated concentration;

[0096] Step (3), formation of water-water microspheres with strong charge effect as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet 2, and reaches the intersection 7 along the dispersed phase channel 5 at a flow rate of 20 μL / h; the continuous phase enters the microfluidic chip through the continuous phase inlet 1, and reaches the intersection 7 along the continuous phase channel 4 at a flow rate of 900 μL / h. The continuous phase contacts the dispersed phase at the intersection 7, and the formation of microspheres is promoted by shear force. The sodium alginate with anions on the surface and the polyarginine with cations undergo ionic crosslinking reaction at the contact surface of the dispersed phase and the continuous phase, forming sodium alginate@polyarginine microspheres with the sodium alginate dispersed phase as a template;

[0097] Step (4), washing and further cross-linking of water-water microspheres with strong charge interaction as template: collect sodium alginate@polyarginine microspheres dispersed in undecanol, add acetone to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, then add 3% (g / ml) polyarginine solution (prepared with water as solvent) for further cross-linking for 20 s, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, add anhydrous ethanol to wash for 1 min, centrifuge at 1000 rpm / min for 3 min to remove the supernatant, and add the obtained microspheres to PBS buffer for storage.

[0098] The diameter of the microspheres prepared in this embodiment is 60-70 μm, and the size deviation is less than 5%.

Claims

1. A method for preparing water-water microspheres based on strong charge interaction, characterized in that: The steps include: Step (1), microfluidic chip: the microfluidic chip comprises a continuous phase inlet, a dispersed phase inlet, a continuous phase channel, a dispersed phase channel, an intersection, a microsphere forming channel, and a microsphere outlet; the continuous phase inlet is communicated with two symmetrically arranged continuous phase channels, the dispersed phase inlet is communicated with the dispersed phase channel, the outlet ends of the two continuous phase channels, the outlet end of the dispersed phase channel, and the inlet end of the microsphere forming channel are communicated through a "cross"-shaped intersection, and the outlet end of the continuous phase channel is vertically distributed with the outlet end of the dispersed phase channel, and the outlet end of the microsphere forming channel is connected to the microsphere outlet; the width of the dispersed phase channel is 30-50 μm, the width of the continuous phase channel is 80-100 μm, and the width of the microsphere forming channel is 80-100 μm; the height of the dispersed phase channel is 30-50 μm, and the heights of the dispersed phase channel, the continuous phase channel, and the microsphere forming channel are the same; Step (2), preparation of dispersed phase and continuous phase: dissolving a polyanionic compound containing a strong negative charge in water, filtering to obtain a dispersed phase, wherein the concentration of the polyanionic compound in the dispersed phase is 1-2% g / mL; mixing an emulsifier, a polycationic compound containing a strong positive charge and a solvent, ultrasonicating, filtering to obtain a continuous phase, wherein the concentration of the polycationic compound in the continuous phase is a saturated concentration, and the concentration of the emulsifier is 1-5% mL / mL; The polyanionic compound containing strong negative charge is at least one of alginate or its derivatives, hyaluronic acid or its derivatives; the polycationic compound containing strong positive charge is at least one of α-polylysine, ε-polylysine, polyornithine, poly-L-arginine, chitosan, tetramethylammonium oxalate, tetramethylammonium bromide, and tetramethylammonium chloride; the emulsifier is at least one of emulsifier EM90 and Span80; the solvent is undecyl alcohol, paraffin oil, soybean oil, C 12 -C 18 At least one of alkanes and silicone oils; Step (3), formation of water-water microspheres with strong charge as template: the dispersed phase enters the microfluidic chip through the dispersed phase inlet and reaches the intersection along the dispersed phase channel; the continuous phase enters the microfluidic chip through the continuous phase inlet and reaches the intersection along two symmetrical continuous phase channels, the continuous phase contacts the dispersed phase at the intersection, the dispersed phase is wrapped by the continuous phase through shear force, the polyanionic compound with negative charge on the surface and the polycationic compound with positive charge undergo ion crosslinking reaction at the contact interface between the dispersed phase and the continuous phase, forming water-water microspheres with strong charge as template; wherein the flow rate of the dispersed phase is 10-35 μL / h; the flow rate of the continuous phase is 300-1800 μL / h; Step (4), washing and further cross-linking of water-water microspheres with strong charge as template: collecting microspheres dispersed in the continuous phase, washing with acetone and centrifuging to remove the supernatant to obtain water-in-water microspheres, adding a polycationic compound solution containing strong positive charge to cross-link, centrifuging to remove the supernatant, and then washing with anhydrous ethanol and centrifuging to remove the supernatant to obtain a water-water microsphere product; wherein the concentration of the polycationic compound solution containing strong positive charge is 1-5% g / mL; the polycationic compound containing strong positive charge is at least one of α-polylysine, ε-polylysine, polyornithine, polyarginine, chitosan, tetramethylammonium oxalate, tetramethylammonium bromide, and tetramethylammonium chloride; and the cross-linking time is 20-60s.

2. The method for preparing water-water microspheres based on strong charge interaction according to claim 1, characterized in that: In step (2), the dispersed phase is prepared by dissolving a polyanionic compound containing a strong negative charge in water and filtering with a 0.22 μm filter to obtain a dispersed phase; The continuous phase is prepared by mixing an emulsifier, a polycationic compound containing a strong positive charge and a solvent, ultrasonicating for 1.5 to 5 hours at an ultrasonic power of 400 W, and then filtering through a 0.45 μm organic phase filter membrane to obtain a continuous phase.

3. The method for preparing water-water microspheres based on strong charge interaction according to claim 1, characterized in that: In step (4), the acetone washing time is 0.5 to 2 minutes; the centrifugal speed is 1000 to 10000 rpm / min, and the centrifugal time is 3 to 5 minutes; The washing time with anhydrous ethanol is 0.5 to 2 minutes; the centrifugal speed is 1000 to 10000 rpm / min, and the centrifugal time is 3 to 5 minutes.

4. Application of the water-water microspheres based on strong charge interaction obtained by the method of claim 1 in chemical analysis detection.

5. The use according to claim 4, characterized in that: The application is the application of water-water microspheres based on strong charge interaction in the detection of hydrogen peroxide and dopamine.

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