Photoresponsive gel microspheres for dPCR-method nucleic acid detection and application of photoresponsive gel microspheres in Escherichia coli detection

A photoresponsive, gel microsphere technology, applied in the field of molecular biology and microfluidics, can solve the problems of limited promotion, and achieve the effect of overcoming easy evaporation, good biocompatibility and dispersion, and simple process

Pending Publication Date: 2022-01-04
WENZHOU INST UNIV OF CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, this technology needs to rely on complex temperature control modules, which has become a barrier to its promotion; in addition to heat conduction, there are few reports on thermal cycle modes driven by other heat generation or heat transfer methods

Method used

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  • Photoresponsive gel microspheres for dPCR-method nucleic acid detection and application of photoresponsive gel microspheres in Escherichia coli detection
  • Photoresponsive gel microspheres for dPCR-method nucleic acid detection and application of photoresponsive gel microspheres in Escherichia coli detection
  • Photoresponsive gel microspheres for dPCR-method nucleic acid detection and application of photoresponsive gel microspheres in Escherichia coli detection

Examples

Experimental program
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Embodiment 1

[0039] This embodiment provides a light-responsive phase-changeable composite gel microsphere for nucleic acid detection by dPCR method, which is prepared according to the following method:

[0040] (1) Preparation of SiO 2 @MXene@SiO 2 composite nanomaterials

[0041] The mother liquor (75mL ultrapure water, 480mL absolute ethanol, 45mL ammonia water, 6mL seed crystal) was mixed in a three-necked flask with mechanical stirring, and A was added simultaneously by a syringe pump at a rate of 6 seconds / drop and 4 seconds / drop. liquid (1200mL of anhydrous ethanol, 960mL of tetraethyl orthosilicate) and liquid B (360mL of ultrapure water, 1560mL of anhydrous ethanol, 240mL of ammonia water), and finally centrifugal cleaning to obtain silica nanoparticles;

[0042] Then as figure 1As shown, through the oxidation reaction of dopamine hydrochloride, several particles were adhered together, and a polydopamine layer was modified on the surface; then placed in a 1 mg / mL MXene aqueous ...

Embodiment 2

[0048] This embodiment provides a light-responsive phase-changeable composite gel microsphere for nucleic acid detection by dPCR method, which is prepared according to the following method:

[0049] (1) Preparation of SiO 2 @MXene@SiO 2 composite nanomaterials

[0050] The mother liquor (75mL ultrapure water, 480mL absolute ethanol, 45mL ammonia water, 6mL seed crystal) was mixed in a three-necked flask and mechanically stirred, and A liquid (1200 mL of anhydrous ethanol, 960 mL of tetraethyl orthosilicate) and B solution (360 mL of ultrapure water, 1560 mL of anhydrous ethanol, 240 mL of ammonia water), and finally centrifugally washed to obtain silica nanoparticles.

[0051] Then as figure 1 As shown, several particles were adhered together through the oxidation reaction of dopamine hydrochloride, and the polydopamine layer was modified on the surface; then it was placed in a 1 mg / mL MXene aqueous solution for mechanical stirring for 6 hours, and the surface coated with M...

Embodiment 3

[0057] The light-responsive phase-changeable composite gel microspheres prepared in Example 1 were used to detect Escherichia coli.

[0058] Through programmed illumination, 95°C for 1min, (95°C for 30s, 55°C for 60s, 68°C for 60s)*35 cycles, 68°C for 3min. Finally, the fluorescence signal of the microspheres is read by a fluorescence microscope, and the fluorescence ratio is counted. Estimate the content of initial E. coli nucleic acid molecules.

[0059] Figure 4 (a) the photo-responsive phase-changeable gel microspheres prepared in Example 1 of the present invention, Figure 4 (b) is the result of the fluorescence signal of the microspheres after photothermal dPCR, in which the concentration of E. coli DNA template is 0.5 copies / droplet (consistent with the theoretical DNA concentration of the sample to be tested), and the composite nanomaterials converge in the sol to form cluster. Figure 5 The results of Sanger sequencing of the amplified products validated the exper...

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Abstract

The invention provides photoresponsive gel microspheres for dPCR-method nucleic acid detection and application of the photoresponsive gel microspheres in Escherichia coli detection. A composite nanomaterial with efficient photothermal conversion characteristics and good biocompatibility is prepared through coating each silica microsphere sequentially with an MXene layer and a silica coating; and a hydrogel mixed solution doped with the composite nanomaterial is emulsified into composite gel microspheres through a micro-fluidic chip, so that a gel-sol-controllable-conversion and temperature-control microcarrier with photoresponsiveness is obtained. The prepared composite gel microspheres can generate heat under near-infrared light to be converted into sol micro-droplets so as to ensure diffused contact of a system, a PCR temperature cycle process can be completed under programmed light stimulation, and the composite gel microspheres are solidified into spheres after reaction so as to stably store monoclonal information. The novel method does not depend on complex and expensive equipment, is wide in application range, can be used for effectively detecting the most common pathogen Escherichia coli infecting a human body, and has a huge value in multiple aspects such as public health.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology and microfluidics, and in particular relates to a light-responsive gel microsphere for nucleic acid detection by dPCR method and its application in the detection of Escherichia coli. Background technique: [0002] Escherichia coli is one of the most common pathogens that infect the body and cause diseases, and can infect many organs and parts such as blood, urinary tract, and skin. Digital polymerase chain reaction (dPCR) has brought a turning point to the detection status because of its high sensitivity and absolute quantification. Nucleic acid detection based on it is a frontier hotspot in the field of molecular biology. At present, this technology needs to rely on complex temperature control modules, which has become a barrier to its promotion; in addition to thermal conduction, other thermal cycle modes driven by heat generation or heat transfer are rarely reported. [0003] Nanoma...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686G01N21/64C12R1/19
CPCC12Q1/689C12Q1/686G01N21/6486C12Q2563/159
Inventor 叶方富张乐翔赵远锦王月桐余筠如
Owner WENZHOU INST UNIV OF CHINESE ACAD OF SCI
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