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Multi-element environment-responsive polysaccharide microspheres as well as preparation and application methods thereof

An environmentally responsive and environmentally responsive technology, applied in the fields of polymer materials and biomedicine, can solve the problems of uncontrollable strict orthogonality of required enzymes, easy mutual interference, and reduced reaction efficiency, so as to avoid interference and cross-contamination, The effect of low cost and simple synthesis process

Active Publication Date: 2022-03-22
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the multi-step enzymatic reactions are easily interfered with each other, side reactions may occur between the reactants of the next step before the end of the previous step, and the strict orthogonality of the enzymes required for each step cannot be controlled, so that The chain reaction cannot proceed smoothly
Therefore, additional separation and purification steps need to be added between reactions, which greatly reduces the reaction efficiency and increases the reaction cost

Method used

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  • Multi-element environment-responsive polysaccharide microspheres as well as preparation and application methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of photoresponsive polysaccharide microspheres embedded with nucleic acid polymerase

[0061] (1) Dissolve the modified agarose grafted with melanin molecules (Tm=100°C) and high-temperature stable DNA polymerase in the buffer solution, and heat at 100°C to prepare the water phase W.

[0062] (2) Heating isooctyl alcohol to 100°C to obtain oil phase O.

[0063] (3) The water phase W was quickly transferred to the oil phase O, and vigorously shaken by hand for 30 seconds to obtain a water-in-oil emulsion.

[0064] (4) Quickly transfer the emulsion to an ice-water bath, and solidify at low temperature for 1 hour.

[0065] (5) Centrifuge the emulsion at a speed of 1500 rpm for 5 minutes, and wash with absolute ethanol, absolute ethanol and pure water in sequence to remove excess oil phase on the surface of the microspheres.

Embodiment 2

[0066] Example 2: Preparation of magnetic field-responsive polysaccharide microspheres embedded with endonucleases

[0067] (1) Dissolve modified hyaluronic acid (Tm=60° C.), iron ferric oxide particles, and endonuclease in a buffer solution, and heat at 60° C. to prepare a water phase W.

[0068] (2) Mix petroleum ether and liquid paraffin at a volume ratio of 1:1, and heat to 60°C to obtain oil phase O.

[0069] (3) Using a microfluidic device to inject the water phase into the oil phase to obtain a water-in-oil emulsion.

[0070] (4) Quickly transfer the emulsion to liquid nitrogen, and wait for the emulsion to return to room temperature after curing at low temperature for 20 seconds.

[0071] (5) Centrifuge the emulsion at a speed of 1000rpm for 3 minutes, wash with absolute ethanol, absolute ethanol and pure water in sequence to remove excess oil phase on the surface of the microspheres.

Embodiment 3

[0072] Example 3: Preparation of electric field-responsive polysaccharide microspheres for embedding nucleic acid molecules

[0073] (1) Dissolve the modified positively charged aminoalginic acid (Tm=50°C), negatively charged silicon carbide particles and guiding DNA molecules in buffer solution, and heat at 50°C to prepare the aqueous phase W.

[0074] (2) Heating soybean oil to 50° C. to obtain oil phase O.

[0075] (3) The water phase W was quickly transferred to the oil phase O, and stirred at a speed of 1000 rpm for 3 minutes to obtain a water-in-oil emulsion.

[0076] (4) Quickly transfer the emulsion to an ice-water bath, and solidify at low temperature for 3 hours.

[0077] (5) Centrifuge the emulsion at a speed of 1000 rpm for 5 minutes, and wash with absolute ethanol, absolute ethanol and pure water in sequence to remove excess oil phase on the surface of the microspheres.

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Abstract

The invention discloses a multi-element environment-responsive polysaccharide microsphere and a preparation and application method thereof, and relates to the field of high polymer materials and biological medicine, the structure of the multi-element environment-responsive polysaccharide microsphere comprises a water phase and an oil phase, and the oil phase wraps the water phase; the water phase contains biomolecules, thermo-sensitive polysaccharide and an environment-responsive material; the preparation method comprises the following steps: adding biomolecules into a water solution mixed solution of temperature-sensitive polysaccharide and an environment-responsive material, and mixing with a proper oil-phase solvent under the assistance of common emulsion preparation processes such as stirring, homogenizing or extruding; the multi-element environment-responsive polysaccharide microspheres embedded with consecutive reactants are obtained through the processes of cooling solidification and centrifugal cleaning after emulsification; the invention also discloses application of the method in enzymatic tandem reaction. Through simple environmental stimulation of illumination, an electric field and a magnetic field, step-by-step dissolution of the environmental responsive microspheres is triggered, so that the internally embedded biomolecules are accurately released, and the enzymatic serial reaction in the same system is realized.

Description

technical field [0001] The invention relates to the fields of polymer materials and biomedicine, in particular to a multi-element environment-responsive polysaccharide microsphere and a preparation and application method thereof. Background technique [0002] Enzyme-catalyzed cascade reactions are one of the most fundamental complex biochemical reactions. As a biocatalyst, the enzyme catalyzes the reaction, and the product of the previous reaction can be further catalyzed by the enzyme to generate other products. Enzyme-catalyzed cascade reactions are widely used in the field of biomedicine, such as nucleic acid detection and amplification, gene editing, drug design and synthesis, research on pathogenic mechanisms, etc., all involve multi-step enzymatic cascade reactions. However, because the multi-step enzymatic reactions are easily interfered with each other, side reactions may occur between the reactants of the next step before the end of the previous step, and the stric...

Claims

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Application Information

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IPC IPC(8): B01J13/02C12N11/04C12N11/10C07K17/04C07K17/10
CPCB01J13/02C12N11/10C12N11/04C07K17/10C07K17/04
Inventor 窦红静于小蕾
Owner SHANGHAI JIAO TONG UNIV
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