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Soluble hydrogel microsphere and preparation method and application thereof in single cell detection

A hydrogel microsphere, soluble technology, applied in biochemical equipment and methods, immobilized on/in organic carriers, determination/inspection of microorganisms, etc., can solve the problem of reducing single cell capture rate, channel blockage, plastic It can solve the problems of poor plasticity of beads, and achieve the effect of solving difficult operation, preventing channel blockage and improving utilization rate.

Active Publication Date: 2019-06-07
SUZHOU GENO TRUTH BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem with this method is the poor plasticity of plastic beads, which leads to channel blockage during droplet formation, thereby reducing the capture rate of single cells.
Most of the hydrogel microspheres in the prior art are insoluble, cDNA amplification is carried out on the gel microspheres, and there are certain limitations in the amount and effect of cDNA amplification

Method used

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  • Soluble hydrogel microsphere and preparation method and application thereof in single cell detection
  • Soluble hydrogel microsphere and preparation method and application thereof in single cell detection
  • Soluble hydrogel microsphere and preparation method and application thereof in single cell detection

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preparation example Construction

[0034] One aspect of the embodiments of the present invention provides a method for preparing soluble hydrogel microspheres, comprising:

[0035] mixing acrylamide, N,N-bis(acryloyl)amide, acetic acid and 2-hydroxy-2-methyl-1-phenyl-1-propanone uniformly to form a mixed mother liquor;

[0036] Mix the mixed mother liquor and sodium hydroxide evenly to obtain a prepolymer solution;

[0037] Using a single emulsion device, the prepolymer solution is used as the inner phase and the oil phase substance is used as the outer phase to prepare microdroplets, and then online polymerization at 30° C. to 65° C. for 10 to 14 hours to obtain soluble hydrogel microspheres.

[0038] In some embodiments, the flow rate of the prepolymer solution is 200-400 μL / h, the flow rate of the oil phase substance is 400-600 μL / h, and an appropriate flow rate is selected according to the required microsphere size.

[0039] Further, the diameter of the soluble hydrogel microsphere is 20-120 μm. By adjust...

Embodiment 1

[0076] (1) Prepare the microfluidic chip

[0077] (2) Preparation of soluble hydrogel microspheres:

[0078] 1) Weigh 0.29g of acrylamide, 0.01g of N,N-bis(acryloyl)amide, dissolve in 1ml of 10% acetic acid, and add 3μL of 2-hydroxy-2-methyl-1-phenyl- 1-Acetone to give a total volume of 1 mL of 30% mother liquor.

[0079] 2) Mix 300 μL of the mother liquor with 600 μL of 1 mol / L sodium hydroxide to obtain a prepolymer solution with a concentration of 10%.

[0080] 3) Using the above-mentioned prepolymer solution as the internal phase, silicone oil as the external phase, the flow rate of the prepolymer solution is 250 μL / h, and the flow rate of the oil phase substance is 450 μL / h, and micro-droplets are prepared by a single emulsion device.

[0081] 4) Polymerization at 65°C for 12 hours.

[0082] 5) Wash with n-hexane several times to remove excess silicone oil. Then use ethanol aqueous solution (100%, 90%,

[0083] 80%, 75%, 50%) gradient replacement, and finally collect...

Embodiment 2

[0091] (1) Prepare the microfluidic chip

[0092] (2) Preparation of soluble hydrogel microspheres:

[0093] 1) Weigh 0.22g of acrylamide, 0.01g of N,N-bis(acryloyl)amide, dissolve in 1ml of 10% acetic acid, and add 3μL of 2-hydroxy-2-methyl-1-phenyl- 1-Acetone to give a total volume of 1 mL of 30% mother liquor.

[0094] 2) Mix 300 μL of the mother liquor with 600 μL of 1 mol / L sodium hydroxide to obtain a prepolymer solution with a concentration of 10%.

[0095] 3) The above-mentioned prepolymer solution is used as the internal phase, the silicone oil is used as the external phase, the flow rate of the prepolymer solution is 300 μL / h, and the flow rate of the oil phase substance is 500 μL / h, and micro-droplets are prepared by a single emulsion device.

[0096] 4) Polymerization at 60°C for 12 hours.

[0097] 5) Wash with n-hexane several times to remove excess silicone oil. Afterwards, gradient replacement with ethanol aqueous solution (100%, 90%, 80%, 75%, 50%) was carr...

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Abstract

The invention discloses a soluble hydrogel microsphere and a preparation method and an application thereof in single cell detection. The preparation method comprises following steps: evenly mixing acrylamide, N,N-bis(acrylamide)amide, acetic acid, and 2-hydroxyl-2-methyl-1-phenyl-1-acetone, adding sodium hydroxide to obtain a prepolymer solution; adopting a single emulsion device, taking the prepolymer solution as an inner phase and an oil phase substance as an outer phase to obtain micro droplets, and carrying out online polymerization to obtain the microsphere. The single cell detection method comprises following steps: marking a bar code on the soluble hydrogel microsphere; injecting the soluble hydrogel microsphere, single cell suspension, and lysate of reverse transcriptase into a droplet micro reactor to form oil-in-water droplets, breaking the droplets, dissolving the soluble hydrogel microsphere, carrying out specific catalytic reverse transcriptional reactions and cDNA amplification; and differentiating single cells through the bar codes. The provided single cell detection method can improve the utilization rate of reverse transcriptase and solves the problem of channel obstruction.

Description

technical field [0001] The present invention relates to a single-cell detection method, in particular to a soluble barcode-labeled hydrogel microsphere and a preparation method thereof, and a method for single-cell detection and detection of single-cell-related life information using droplet microfluidic chip technology , belongs to the technical field of nucleic acid detection analysis and detection. Background technique [0002] Single-cell sequencing technology aims to amplify and sequence the genome or transcriptome at the level of a single cell, reflecting the genetic information of a single cell, because even a single cell from the same source, due to biological processes and environmental perturbations, is closely related to each other in many There are also differences in aspects, which is what we call cellular heterogeneity. Conventional genome sequencing technology cannot avoid the influence of cell heterogeneity. This phenomenon is particularly important in tumor...

Claims

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Application Information

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IPC IPC(8): C08F220/56C08F222/38C08J3/075C08L33/26C08L83/04C12Q1/6806
CPCC08F220/56C08F222/38C08J3/075C08L33/26C08L83/04C12N11/04C12Q1/6806
Inventor 张惠丹吴佳梅李莹玉
Owner SUZHOU GENO TRUTH BIOTECHNOLOGY CO LTD
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