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Method for preparing core-shell SERS structure based on amplification of nucleic acid strand by terminal deoxynucleotidyl transferase

A technology for terminal transferase and nucleic acid amplification, which is applied in the field of preparation of nucleocapsid SERS structures, can solve the problems of few and rare preparations of nucleocapsid SERS structures based on DNA bases, and achieve high-efficiency and uniform SERS effects, simple methods, and The effect of high sensitivity

Inactive Publication Date: 2019-02-05
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technical solution involves creating an enzyme called rastersmall (RSP) particles attached onto gold surfaces instead of just being able to attach them themselves into this type of material. These tiny objects have unique properties like their ability to absorb light energy from specific wavelength ranges while still maintaining its original shape when suspended at different locations within these materials. They may even help identify harmful organism's gene products without actually killing oneself. By attaching these minute metallic structures together they become larger sores than traditional probes made of silver nitrate gelatin layers alone. Additionally, there was no loss of any Raman signals during processing due to weak interactions between the atoms inside the particle itself. Overall, this technique allows us to make better ways to detect diseases caused by various types of substances including cancer cells, blood clotting proteins, tissue stains, viruses, germs, and others.

Problems solved by technology

Technologies aim towards improving the performance of SRS probes by introducing specific types of metallic structures called "spikes," such as AuNPs, Ag NPs, Pb Nanotubules, Silver Colon Grounds, and silica particles. These structural modifications help create more effective SERS agents while reducing their size. Additionally, they provide potential applications in biology due to improved ability to control the amount of detected substances.

Method used

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  • Method for preparing core-shell SERS structure based on amplification of nucleic acid strand by terminal deoxynucleotidyl transferase
  • Method for preparing core-shell SERS structure based on amplification of nucleic acid strand by terminal deoxynucleotidyl transferase
  • Method for preparing core-shell SERS structure based on amplification of nucleic acid strand by terminal deoxynucleotidyl transferase

Examples

Experimental program
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Effect test

Embodiment 1

[0036] (1) Add 10 μL thiol-modified initial primer chain (SH-polyA 10 , 100 μM), diluted with water to the final concentration of the initial chain is 5 μM. After shaking at room temperature overnight, add 100mM PB buffer (phosphate buffer) until the final concentration of phosphate buffer is 10mM; after overnight at room temperature, add aging solution (10mM PB, 2M NaCl, pH7.4) so ​​that the final concentration of NaCl in the solution is 0.15 M.

[0037] After the mixture solution was left overnight at room temperature, it was centrifuged at 4°C and 12,000 rpm for 20 min, and washed by centrifugation for 4 times. The precipitate, namely the nanogold-starting chain complex, was resuspended in 200 μL of PB (10 mM, 0.15 M NaCl, pH 7.4) solution, Store at 4°C.

[0038] (2) 4 μL nano gold-starting chain complex (ie Au-PolyA 10 , the 5'-end is connected to the surface of gold nanoparticles), add 1 μL terminal transferase (concentration 10U / μL), 1 μL dATP (concentration 10mM), 2 ...

Embodiment 2

[0045] (1) 10 μL of thiol-modified initial chain (SH-polyT 15 , 100 μM), the starting chain final concentration was 5 μM. After shaking at room temperature overnight, add 100mM PB buffer (phosphate buffer) until the final concentration of phosphate buffer is 10mM. After overnight at room temperature, add aging solution (10mM PB, 2M NaCl, pH 7.4) to make the final concentration of salt in the solution 0.15M . After overnight at room temperature, the mixture solution was centrifuged and washed 4 times at 4°C at 12000rpm / min for 20min, and the precipitate was resuspended in 200μL of PB (10mM, 0.15M NaCl, pH7.4) solution and stored at 4°C.

[0046] (2) 4 μL complex gold-starting chain (Au-PolyT 15 ), add 1 μL terminal transferase (concentration 10U / μL), 1 μL dTTP (10 mM), 2 μL Reaction Buffer to make up the volume to 10 μL, mix the solution well and incubate at room temperature for 1 h; bathe in 70 ° C for 15 min to inactivate the enzyme .

[0047] Take 4 μL of the sample, and...

Embodiment 3

[0050] (1) 10 μL of thiol-modified initial chain (SH-polyA 10 , 100 μM), the starting chain final concentration was 5 μM. After shaking at room temperature overnight, add 100mM PB buffer (phosphate buffer) until the final concentration of phosphate buffer is 10mM. After overnight at room temperature, add aging solution (10mM PB, 2M NaCl, pH 7.4) to make the final concentration of salt in the solution 0.15M . After overnight at room temperature, the mixture solution was centrifuged and washed 4 times at 4°C at 12000rpm / min for 20min, and the precipitate was resuspended in 200μL of PB (10mM, 0.15M NaCl, pH7.4) solution and stored at 4°C.

[0051] Follow the same system and method to assemble the initial chain SH-polyT on the surface of gold nanoparticles 15 , operate with embodiment 2.

[0052] (2) 4 μL complex Au-polyA 10 Add 1 μL of terminal transferase, 1 μL of ddATP (10 mM), 2 μL of Reaction Buffer, mix the solution with a total volume of 10 μL and incubate overnight at ...

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Abstract

The invention relates to the field of biological detection and the field of nanomaterial functional application, and discloses a method for preparing a core-shell SERS structure based on the amplification of a nucleic acid strand by terminal deoxynucleotidyl transferases. The method comprises the following steps of: assembling a starting strand on the surface of a nanomaterial, carrying out the DNA amplification reaction on the surface of the terminal deoxynucleotidyl transferase, transferring deoxynucleotide to the starting primer strand assembled on the surface of the nanomaterial so as to form long single-stranded DNA on the surface of the nanomaterial and then obtain a nanomaterial-long-single-stranded-DNA complex; and covering a metal shell layer outside the nanomaterial-long-single-stranded-DNA complex by the chemical reduction method so as to form the core-shell structure. The core-shell SERS structure has long strand DNA which can be used as a Raman reporter small molecule so as to output efficient and uniform SERS signals to enhance the intensity of Raman signals. Bases are located between the core and the shell, so that the number of ''hot spots'' in the core-shell structure is increased, the adsorption of target molecules or antibodies on the surface of the core-shell structure is affected, the sensitivity of the detection can be greatly improved, and the method canbe applied to the field of biological analysis and detection.

Description

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Claims

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Application Information

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Owner SHANGHAI OCEAN UNIV
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