Nucleic acid aptamer-suspension chip magnetic detection microspheres based on single-walled carbon nano-tubes, preparation method and detection method thereof

A technology of single-walled carbon nanotubes and nucleic acid aptamers, which can be used in measurement devices, instruments, fluorescence/phosphorescence, etc. The effect of detection sensitivity

Inactive Publication Date: 2017-06-30
河南大学深圳研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the traditional antigen-antibody is not strong in binding to the characteristic groups of small molecules, and there are certain limitations in the recognition of small molecule chemicals

Method used

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  • Nucleic acid aptamer-suspension chip magnetic detection microspheres based on single-walled carbon nano-tubes, preparation method and detection method thereof
  • Nucleic acid aptamer-suspension chip magnetic detection microspheres based on single-walled carbon nano-tubes, preparation method and detection method thereof
  • Nucleic acid aptamer-suspension chip magnetic detection microspheres based on single-walled carbon nano-tubes, preparation method and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Preparation of single-walled carbon nanotubes modified by surface amination

[0061] Carbon nanotubes were treated by a two-step acidification method: 100 mL of 98% concentrated H 2 S0 4 with 68% concentrated HN0 3 Mix according to the volume ratio of 3:1, add 200mg SWNT to the mixed acid, and reflux for 4h under the condition of ultrasonic vibration at 38°C. After cooling to room temperature, add deionized water to dilute, filter through a cellulose filter, and wash with deionized water until the pH of the filtrate is neutral. The reaction product was placed in an electric blast drying oven, and dried under vacuum at 60° C. overnight.

[0062] 100mL 98% concentrated H 2 S0 4 with 30% H 2 0 2 Mix according to the volume ratio of 4:1, put the mixed acid acidified product in 1 in H 2 S0 4 with H 2 0 2 In the mixed solution, reflux at 70°C for 2h. After cooling to room temperature, add deionized water to dilute, filter with a membrane filter, and was...

Embodiment 2

[0063] Example 2. Coupling of amino-modified single-walled carbon nanotubes and magnetically encoded microspheres on a suspension chip

[0064] Take 3mg of amino-modified SWNT, add 3mL dH 2 In O, ultrasound was performed for 5 minutes, and the cumulative time was repeated 6 times to reach 30 minutes, and ice water was used to cool down halfway. Take 800 μL and disperse into 1.5 mL of distilled water. Take out the brown bottle (52#) of the blank carboxylated fluorescence-encoded magnetic microsphere suspension in a light-proof environment, let it air to room temperature, ultrasonicate in a water bath for 30 seconds, and vortex mix for 1 minute. Take out 120 μL of the suspension into a 1.5 mL centrifuge tube wrapped with aluminum foil, place it on a magnetic separator for 5 min, and suck off the supernatant by adhering to the wall. Add 120 μL of 0.1M MES (pH=4.5) to the microspheres and mix thoroughly to wash the microspheres. Magnetically separate, discard the supernatant, a...

Embodiment 3

[0067] Example 3. Preparation of biotinylated nucleic acid aptamer-coupled fluorescent reporter molecule-streptavidin phycoerythrin

[0068] Take the biotinylated nucleic acid aptamer, centrifuge at 12000rpm for 60s before opening the cap, slowly open the tube cap, add 34 μL of PBS (containing 0.1M MgCl 2 ) buffer solution, prepared as a 100 μM stock solution, covered with a lid and shaken to mix well, and dispensed (2 μL / PCR tube, 10 tubes) for later use, and the rest were stored at -20°C; take 1 tube of 2 μL biotinylated nucleic acid adapter body, treated in a water bath at 95°C for 5 min, and cooled to room temperature. Then make 100-fold dilution to get 1μM, that is, add 198μL Tris-HCl (20mM Tris-HCl, pH=7.4, 100mM NaCl, 2mM MgCl 2 ) Coupling buffer, a total of 200 μL for use; take 2 μL of SA-PE in the dark, dilute 100 times with PBS buffer to 200 μL for use;

[0069] Take 1 μM biotinylated nucleic acid aptamer and 100× diluted SA-PE and mix them at a volume ratio of 3:2...

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Abstract

The invention belongs to the technical field of nanometer biology, and specifically discloses a nucleic acid aptamer-suspension chip based on single-walled carbon nano-tubes, a preparation method and a detection method thereof. According to the present invention, with the application of the nucleic acid aptamer-suspension chip based on single-walled carbon nano-tubes to detect a target substance, the detected median fluorescence signal intensity is negatively correlated with the concentration of the substance to be detected, and the concentration of the substance to be detected in the sample to be detected can be obtained by establishing the standard curve between the concentration of the substance to be detected and the fluorescence signal; and the detection method has advantages of wide detection range, high detection sensitivity and high specificity, and has great advantages compared to other detection techniques.

Description

technical field [0001] The invention relates to the field of nanobiology technology, in particular to a single-walled carbon nanotube nucleic acid aptamer-suspension chip detection method. Background technique [0002] The suspension chip uses polystyrene with a diameter of 5.6 μm or doped magnetic fluorescent microspheres as a solid phase carrier, which can exist in a suspended and free state in the detection system, and flow through the fluid pipeline with the direction of hydraulic pressure ; Each microsphere is defined as a unique coding address because it is coated with different proportions of special fluorescent dyes, so that many coding microspheres are arranged into a map similar to an "array". The solid surface of the microspheres is modified with different group molecules, such as carboxyl, hydroxyl, biotin, streptavidin, etc., and forms microsphere probes with specific biomolecules through chemical reactions. These biomolecules can be Capture antibodies, nucleot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533G01N21/64
CPCG01N21/6486G01N33/533G01N33/54326
Inventor 刘楠
Owner 河南大学深圳研究院
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