Poly A mediated regulatory nanogold probe as well as preparation method and application thereof

A nano-gold probe and nano-gold particle technology, applied in the direction of nanotechnology for sensing, nanotechnology, nanomedicine, etc., to achieve the effect of solving non-specific adsorption

Active Publication Date: 2018-08-03
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, despite their individual efficiency, sensitivity, and convenience, none of the methods have tunable sensitivity and signal

Method used

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  • Poly A mediated regulatory nanogold probe as well as preparation method and application thereof
  • Poly A mediated regulatory nanogold probe as well as preparation method and application thereof
  • Poly A mediated regulatory nanogold probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] (1) Materials and equipment

[0067] The reagent that present embodiment adopts: trisodium citrate (C 6 h 5 Na 3 o 7 2H 2 O), phosphate, NaCl, MgCl 2 Reagents such as KCl and KCl were purchased from China National Pharmaceutical Group Corporation.

[0068] The above reagents were of analytical grade without further purification.

[0069] The water used in this embodiment is MilliQ water; MilliQ water: 18.2 MΩ.cm (Millipore).

[0070] The equipment used in this example includes a transmission electron microscope (TEM), an ultraviolet spectrophotometer (Hitachi U-3010), a fluorescence spectrophotometer (F-900, Edinburg), a pH meter, and a refrigerated high-speed centrifuge (Hitachi).

[0071] The diblock DNA, reporter molecule and target molecule used in the embodiment of the present invention were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. and purified by high performance liquid chromatography.

[0072] The embodiment of the present invention uses th...

Embodiment 2

[0088] In this example, the regulation of the number of assembled gold nanoparticles on the surface of the diblock DNA with different Poly A lengths is discussed.

[0089] The diblock DNA nano-gold probe solutions with different lengths of Poly A prepared in Example 1 were first quantified by an ultraviolet spectrophotometer. The calculation formula is CAuNPs-PolyA=A520nm*2.79*10-10M. The diblock nano-gold probe (0.15 nM final concentration) was added to mercaptoethanol (20 mM final concentration) and shaken overnight at room temperature. Then the supernatant fluorescently labeled DNA (fluorophore FAM, excitation wavelength 494nm, emission wavelength 520nm) was collected by centrifugation, and the fluorescence intensity was detected. Finally, the fluorescence value was substituted into the previous standard curve to calculate the concentration of fluorescently labeled DNA. Such as Figure 4 As shown, (A) As the length of PolyA increases, the number of diblock DNA assembled ...

Embodiment 3

[0091] In this example, the regulation of the recognition ability of the target sequence by diblock DNA with different numbers of Poly A nucleotides is discussed.

[0092] Nanogold probes with different lengths of Poly A provided in Example 1 were used. Add different concentrations of target molecules to different groups of nano-gold probes, respectively: 0, 0.01, 0.05, 0.1, 0.5, 1, 10, 50, 100, 150, 200nM, and detect the fluorescence intensity of each group after incubation. Each group of experiments was repeated three times. In this embodiment, the sequence of the target molecule is shown in SEQ ID NO.10, specifically 5' AGCCC CTGCC CACCG CACAC TG3'.

[0093] The result is as Figure 5 As shown, the intensity of the fluorescent signal increases with the addition of target molecules, and finally reaches a plateau. Different poly A numbers have a regulatory effect on the minimum detection limit of the probe. As the number of adenine nucleotides in Poly A increases, the det...

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Abstract

The invention relates to the field of biotechnology, and in particular to a nanogold probe. The nanogold probe comprises nanogold particles and a double-stranded probe which is connected to the nanogold particles, wherein the double-stranded probe comprises a first chain and a second chain; the first chain comprises a first segment and a second segment which are sequentially distributed from 5' to3'; the first segment and the second chain are complementary; the second segment, which serves as a cohesive end, is used for recognizing a target sequence; and the second chain is modified by a fluorescent gene. The nanogold probe provided by the invention, by adjusting nucleotide number of the second segment, can regulate and control hybridization efficiency of a recognition sequence and the target sequence, so that the recognition capacity of the nanogold probe on the target sequence is regulated and controlled.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Poly A-mediated adjustable nano-gold probe and its preparation and application. Background technique [0002] The detection of DNA / RNA has broad application space in biomedical research and clinical diagnosis. DNA detection is of great significance in molecular diagnosis of diseases, gene therapy, biomedical research, rapid detection of biological warfare agents and forensic applications. RNA plays an important role in biological processes, affecting the development of cells, tissues and individuals, and disease processes lead to specific RNAs being elevated in body fluids. For example, tumor-specific mRNA / miRNA can be used in the diagnosis and typing of cancer and has attracted extensive attention. [0003] Traditional nucleotide detection methods are divided into two categories, one is the detection method based on probe hybridization technology, including Northern blotting, mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6834B82Y5/00B82Y15/00
CPCB82Y5/00B82Y15/00C12Q1/6834C12Q2563/107
Inventor 宓现强王璐张欢樊春海王丽华左小磊
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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