Construction method and application of single-particle biological probe

A technology of biological probes and construction methods, applied in the field of biological detection, can solve the problems of low content of microRNAs, and achieve the effects of improving material mass transfer rate, strong spatial positioning ability, and strong rigidity

Inactive Publication Date: 2018-05-29
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, due to the low content of microRNAs, the detection limit of miR-21 by the plasmonic nanobiosensor constructed based on simple ssDNA-modified single metal nanoparticle-formed probe molecules can only reach 1 fM.

Method used

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  • Construction method and application of single-particle biological probe
  • Construction method and application of single-particle biological probe
  • Construction method and application of single-particle biological probe

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Embodiment 1

[0057] This embodiment relates to a method for preparing tsDNA, including the following steps:

[0058] The design of tsDNA is based on complementary base pairing, with as little secondary structure as possible and GC content above 50% as the principle. Three different sizes of tsDNA are designed with side lengths of 7bp, 17bp and 26bp. For the synthesis of tsDNA, please refer to "A DNA nanostructure-based biomolecular probe carrier platform for electrochemical biosensing, Pei H, Lu N, Wen Y, et al. Advanced Materials, 2010,22(42):4754-4758", the specific method as follows:

[0059] 1. Dissolve each ssDNA, measure the absorbance at 260nm under an ultraviolet spectrophotometer, obtain the molar extinction coefficient of each ssDNA from Dalian Bao Biological Company, and determine the actual concentration of each ssDNA;

[0060] 2. Mix the four pieces of ssDNA forming tsDNA in the TM buffer in equal proportions, add tris(2-carboxyethyl)phosphine with a final concentration of 1-10 mM t...

Embodiment 2

[0068] This embodiment relates to a method for preparing an indium tin oxide conductive film glass substrate with Au@AgNCs fixed on the surface, including the following steps:

[0069] 1. Synthesis of nano-gold seeds: We use a common seed growth method to prepare gold seeds with a diameter of 20-40nm. In order to prepare gold nanoparticles with a diameter of 1~5nm, first add 0.2~0.8mL of ice water to a fresh 5~15mM sodium borohydride solution under rapid stirring and add 5~15mL of 50~150mM CTAB and 0.2~0.5mL of 5~ In the mixed solution of 10 mM chloroauric acid, the brown solution is obtained and kept in a water bath at 20 to 30°C for 2 to 5 hours to ensure that the excess sodium borohydride is completely decomposed before use. Then, the gold seed solution was prepared by the following method, adding 2-5 mL of 50-200 mM ascorbic acid solution to 5-15 mL of 100-200 mM CTAC solution and 5-10 mL of 0.2-0.5 mM chloroauric acid mixed solution. Color and transparent solution, after st...

Embodiment 3

[0075] This embodiment relates to a method for modifying Au@Ag NCs by tsDNA, which includes the following steps:

[0076] Take 100-200 μL of the 1pM tsDNA solution synthesized in Example 1 and drop it onto the surface of the indium tin oxide conductive film glass fixed with Au@Ag NCs in Example 2, and incubate at room temperature for 2-6 hours. Then use ultrapure water to remove the excess tsDNA solution, and blow dry with nitrogen to prepare a single Au@Ag NC LSPR probe based on tsDNA.

[0077] Named according to the size of the side length of the tetrahedron, three different tetrahedrons with side lengths of 7bp, 17bp and 26bp are tsDNA respectively 7 , TsDNA 17 And tsDNA 26 . Because of the strong covalent interaction between silver and sulfur, tsDNA with a side length of 17bp 17 For example, such as Figure 7 Shown in tsDNA 17 Modified sulfhydryl groups on the three vertices of, one of the edges (l 1 ) Vacate a section of ssDNA molecules for capturing miR-21 molecules, on the o...

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Abstract

The invention provides a construction method and application of a single-particle biological probe. The construction method comprises the following steps: separately preparing gold and silver core-shell nano cubic (Au@AgNCs) collosol and a tsDNA solution, and fixing Au@AgNCs on the surface of indium tin oxide conductive film glass; and dropwise adding the tsDNA solution on the surface of the indium tin oxide conductive film glass fixed with Au@AgNCs, after incubating at a room temperature, washing with an ultra-pure water, and blow-drying with nitrogen to obtain a single-particle LSPR probe based on tsDNA. The particle size of the prepared single-particle probe is between 50 nm and 60 nm, the detection sensitivity reaches 1 aM, and LSPR scattered spectrum peak is positioned at 540-560 nm.Compared with the prior art, the construction method has the beneficial effects that compared with a silver nano cube, Au@AgNCs has similar plasma characteristics and more stable structure; and compared with single-chain DNA molecules with one-dimensional structures and hairpin type DNA molecules with two-dimensional structures, tsDNA with three-dimensional structures has higher rigidity and structural stability, and facilitates multi-functionalization and the like.

Description

Technical field [0001] The invention relates to a construction method and application of a single particle LSPR (local surface plasmon resonance) probe based on tsDNA (tetrahedral structure DNA), and belongs to the technical field of biological detection. Background technique [0002] MicroRNAs are a type of endogenous non-protein coding RNAs with a length of 20-24 nucleotides. They usually play a very important role in the biological processes of early development, cell proliferation, differentiation, and apoptosis. Abnormal expression of MicroRNAs occurs in malignant tumor cells at the pre-cancerous stage, and is related to many cancers (such as: lung, liver cells, large intestine, breast cancer, etc.). MicroRNA-21 (miR-21) is present in many of the aforementioned human cancer tissues, and the expression level in lung squamous cell carcinoma tissue is twice as much as that in normal tissues. Therefore, microRNAs can be used as biomarkers for the diagnosis and prognostic treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6834C12Q1/6811C12Q1/44G01N21/552
CPCC12Q1/44C12Q1/6811C12Q1/6834G01N21/554G01N2333/922C12Q2525/207C12Q2565/628
Inventor 汪联辉张颖张磊翁丽星周浩
Owner NANJING UNIV OF POSTS & TELECOMM
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