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Gold nanorod detection probe, preparation method, detection method and application thereof

A technology of gold nanorods and detection probes, which is applied in the detection field of silicon nitride nanopore sensors, can solve the problems of high cost, time-consuming and limited resolution of nanopore preparation, and achieve improved connection efficiency, good dispersibility and uniformity effect

Pending Publication Date: 2021-06-15
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to factors such as processing technology and film thickness, the preparation of sub-2nm nanopores is costly and time-consuming, and does not use large-scale preparation applications
It is easy to prepare larger solid-state nanopores, but the resolution is limited by the fact that the size of the nanopore is similar to that of the detection molecule
At the same time, when short nucleotide sequences pass through solid-state nanopores, the speed is too fast, which also has higher requirements for signal acquisition.

Method used

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  • Gold nanorod detection probe, preparation method, detection method and application thereof
  • Gold nanorod detection probe, preparation method, detection method and application thereof
  • Gold nanorod detection probe, preparation method, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Synthesis of gold nanorods

[0051] Step 1: Seed synthesis: take 10mL of CTAB (0.1mol / L) solution and 0.01mol / L of HAuCl 4 Mix 10mL, add magneton, and then add 0.6mL of freshly prepared NaBH 4 (0.01mol / L) solution, stirring vigorously for 2 minutes while adding, and bathing in water at 30°C for 2 hours.

[0052] Step 2: Gold nanorod growth (64×16nm): take 100mL CTAB (0.1M) solution, 5mL of HauCl 4 (0.01mol / L) solution and 875uL AgNO 3 The solution (10mM) was mixed, and stirred by adding a magnet, adding 1.9mL of HCl (1mol / L) solution to adjust the pH to about 2, adding 0.8mL of 0.1mol / L ascorbic acid solution, stirring until the solution became colorless, adding 250uL of the gold seed solution prepared in step (1), stirred and mixed, placed in a water bath at 30°C for 8 hours.

[0053] Step 3: Centrifuge the gold nanorod solution grown in step (2) for 8 hours at 8000rpm for 30min in a 50mL centrifuge tube, absorb the supernatant to retain the precipitate...

Embodiment 2

[0055] Embodiment 2: Preparation of gold nanorod nucleic acid probe

[0056] Step (1): get the gold nanorods that above-mentioned step prepares, measure its ultraviolet absorption value, calculate the concentration of gold nanorods by the absorption value at its 400nm place, be concentrated to 5nM; Adopt gold nanorods and thiol DNA (such as SEQ ID NO: ID NO.1) the optimal concentration ratio is 1:50, take 80uL of gold nanorods (5nM) and add 2uL of CTAB (0.1M) solution, mix well, then add 2uL of aliquoted 10uM thiol DNA solution, shake Evenly, put it on the shaker at room temperature for 8h.

[0057] Step (2): Take out the sample after the reaction in step (1), centrifuge at 9000rpm, remove the supernatant, redissolve in 80uL ultrapure water, repeat twice, add 1.6uL 1% SDS solution, shake evenly , add 2uL aliquoted 10uM thiol DNA solution (as shown in SEQ ID NO.1), shake evenly, and put it on a shaker for 8h reaction.

[0058] Step (3): The sample prepared in step (2) was age...

Embodiment 3

[0066] Example 3: Detection of miRNA by nanopore sensor

[0067] Step 1: Take an equal amount of the above steps to prepare gold nanorod probes (first detection probe and second detection probe) with DNA strands complementary to half of the miRNA sequence at both ends, shake and mix evenly, take 5uL and add Add 100uL of different concentrations of miRNA solutions to be detected, and react for 3 hours to form a linear assembly structure.

[0068] Step 2: Use a 40nm nanopore sensor to detect the gold nanorods and assemblies modified with nucleic acid probes, and distinguish different assembly structures by the amplitude and residence time of the detected trajectory current signal, thereby realizing the detection of miRNA: Specifically: install the fluid, use a 40nm silicon nitride nanopore sensor, fill it with a 50mM NaCl solution, and add the solution sample prepared in step 1 into the trans chamber. Using patch clamp, apply a driving voltage to the chambers on both sides of t...

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Abstract

The invention discloses a gold nanorod detection probe, a preparation method, a detection method and application thereof. The detection probe comprises a gold nanorod and nucleic acid probes which are connected to the two ends of the gold nanorod and are complementary with target molecules. According to the gold nanorod detection probe, the preparation method, the detection method and the application thereof, the two ends of the gold nanorod are selectively modified through the sulfydryl nucleic acid probes, and the gold nanorod is specifically combined with target detection object miRNA molecules through base complementary pairing, so that the gold nanorod is linearly assembled; and when an assembly passes through a nanopore, a translocation signal with a high signal-to-noise ratio is caused, so that high-sensitivity detection of a silicon nitride nanopore sensor on miRNA micromolecules is realized. The gold nanorod detection probe, the preparation method, the detection method and the application thereof overcome the defects that biological micromolecules are high in speed and low in signal resolution in silicon nitride nanopore detection, meanwhile, the resolution and the detection flux of the solid nanopore are improved, and the application of the silicon nitride nanopore in the field of single molecule detection is expanded.

Description

technical field [0001] The invention belongs to the detection field of silicon nitride nanopore sensors, and in particular relates to a gold nanorod detection probe, a preparation method, a detection method and an application thereof. Background technique [0002] miRNA is a kind of endogenous non-coding RNA with regulatory function, which is involved in various metabolism and signal transduction pathways, such as growth and development, virus defense, cell proliferation and apoptosis, etc., and is associated with tumor occurrence, development, prediction, Diagnosis, treatment and postoperative are related. Therefore, miRNA, as a potential marker for cancer diagnosis, prediction and treatment, has broad clinical application prospects. miRNA plays multiple roles in non-small cell lung cancer, including playing the role of tumor-promoting gene or tumor suppressor gene in the process of lung cancer, regulating the proliferation and differentiation of lung cancer cells, particip...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/6886C12N15/11
CPCC12Q1/6825C12Q1/6886C12Q2600/178C12Q2563/137C12Q2563/157C12Q2565/607C12Q2565/631
Inventor 翁丽星武灵芝严馨曾祥杰
Owner NANJING UNIV OF POSTS & TELECOMM
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