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Gold nanopore array based SERS (surface-enhanced Raman spectroscopy) detection method for DNA methylation and application of method

A surface-enhanced Raman and detection method technology, which is applied in Raman scattering, measuring devices, and material analysis through optical means, can solve problems such as complex amplification, labeling procedures, large amount of DNA, and unstable measurement. Simple operation, high reproducibility, good Raman enhancement effect

Active Publication Date: 2018-11-16
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods require complex amplification and labeling procedures, or have problems such as complex operation, unstable measurement, and large amount of DNA, which limit their wide application and development.

Method used

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  • Gold nanopore array based SERS (surface-enhanced Raman spectroscopy) detection method for DNA methylation and application of method
  • Gold nanopore array based SERS (surface-enhanced Raman spectroscopy) detection method for DNA methylation and application of method
  • Gold nanopore array based SERS (surface-enhanced Raman spectroscopy) detection method for DNA methylation and application of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of basic solution

[0045] 1. Preparation of gold nanopore arrays

[0046] Gold nanohole arrays were prepared using an electron beam exposure system and thermal evaporation coating. A 200-nm thick PMMA electron-beam etchant layer was spin-coated on a Si wafer, and then the PMMA-coated substrate was produced under an electron beam with 200-nm apertures and 385-nm edges. 50 μm × 50 μm nanowell array with a distance between them. After developing for 70 seconds in a PMMA developer with a mass ratio of 1:3 methyl isobutyl ketone / isopropanol (MIBK / IPA), a porous structure was produced, followed by IPA rinsing and post-baking at 95°C for 30 minutes, and then in A 50nm-thick gold film was evaporated on the substrate to complete the preparation of the nanopore array. Before use, the substrate was cleaned in UV ozone for 20 minutes with a 18.2MΩ·cm -1 Rinse with deionized water and rinse with N 2 dry. A clean gold nanopore array substrate is obtained. Characteri...

Embodiment 2

[0050] Raman Performance Evaluation of Gold Nanohole Arrays

[0051] The enhancement effect of the gold nanohole array was studied by Raman spectroscopy; a laser of 785nm was selected.

[0052] With the Rh6G solution prepared in Example 1, detect the surface-enhanced Raman spectrum of the solution;

[0053] A piece of gold nanopore array prepared in Example 1 is immersed in the prepared Rh6G solution, and the Raman spectrum of the Rh6G solution is detected by a Raman spectrometer, as shown in figure 2 As shown, the direct detection of 10 at the nanopore -6 Raman signal of mol / L rhodamine 6G. It can be found that there is an obvious Raman enhancement effect in the nanohole area, and the Raman enhancement factor is close to 10 6 . Randomly select different regions in the nanopore array for detection, such as image 3 As shown, it can be found that any region of the nanohole array has a very uniform and stable Raman response, and a Raman signal with good reproducibility can...

Embodiment 3

[0055] Gold Nanopore Arrays for Detecting DNA Methylation

[0056] Take the DNA solution prepared in Example 1 with a concentration of 100 μmol / L and drop-coat it on the gold nanopore array, take it out after 0.5 hours, dry it with nitrogen, and use a Raman instrument with a 785nm laser to detect and study the properties of S1 and S2. Raman spectroscopy. Compare the characteristic differences of their Raman spectra, such as Figure 4 Shown, the characteristic peak of methyl (at 1365cm -1 ) intensity is significantly enhanced, while the characteristic peak of the base cytosine (located at 785cm -1 ) intensity is relatively weakened, and DNA methylation detection can be realized. Take S2 ​​to prepare a set of solutions with different concentrations of DNA, the concentration range is 0.1 μmol / L to 100 μmol / L; the Raman signal of this series of DNA solutions with different concentrations is detected by Raman spectrometer, as shown in Figure 5 As shown, the DNA concentration o...

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Abstract

The invention discloses a DNA methylation detection method based on SERS (surface-enhanced Raman spectroscopy) of a gold nanopore array and an application of the method. The method comprises the following steps: (1) the periodic gold nanopore array is prepared by an electron beam lithography system through electron beam evaporation coating to serve as an SERS enhancing substrate; (2) surface Ramanenhancing performance of the SERS enhancing substrate is inspected by a Raman beacon molecule Rhodamine 6G, and the enhancing substrate is applied to DNA methylation detection; (3) DNA methylation detection based on a surface Raman enhancing effect is established. According to the detection method, the surface Raman enhancing performance of the prepared gold nanopore array is evaluated by the Raman beacon molecule Rhodamine 6G, and it is discovered that the gold nanopore array has good Raman enhancing effect on Rhodamine 6G and has efficient repeatability; the gold nanopore array is applied to DNA methylation detection. The method has the advantages of being high in sensitivity, simple to operate, high in repeatability and capable of being used for detecting DNA methylation.

Description

technical field [0001] The invention belongs to the technical field of biological Raman detection, and relates to a DNA methylation detection method, in particular to a DNA methylation detection method based on a gold nanopore array surface-enhanced Raman effect and an application thereof. Background technique [0002] DNA methylation is an important epigenetic modification that plays an important regulatory role in maintaining cell proliferation and differentiation, genetic imprinting, and gene expression. Abnormal expression of methylation is closely related to many diseases (such as neurodegenerative diseases, Prader-Willi syndrome, metabolic disorders, etc.), and even closely related to tumors. High-level and low-level expression of methylation can be used as an important indicator for early diagnosis and prognosis of tumor detection. It also helps to provide important information for studying the expression mechanism of normal and pathological genes at the molecular le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 吴萍罗晓俊蔡称心
Owner NANJING NORMAL UNIVERSITY
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