Surface-enhanced Raman detection of biomolecules based on hyperbranched nanostructures

A surface-enhanced Raman and nanostructure technology, which is applied in Raman scattering, measuring devices, and analytical materials, can solve the problem that the amplification factor of molecular signals is difficult to achieve consistency, specific quantitative detection cannot be achieved, and molecular concentration cannot be obtained from molecular signals, etc. problem, to achieve the effect of low cost, elimination of competition, and good reproducibility

Active Publication Date: 2019-04-30
EAST CHINA NORMAL UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing nanotechnology is difficult to obtain evenly SERS-enhanced coupled nano-sol or nano-substrate, which makes it difficult to achieve a consistent magnification of the molecular signal, so the detected molecular signal cannot be used to obtain the molecular concentration, that is, the specificity cannot be achieved. Quantitative detection

Method used

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  • Surface-enhanced Raman detection of biomolecules based on hyperbranched nanostructures
  • Surface-enhanced Raman detection of biomolecules based on hyperbranched nanostructures
  • Surface-enhanced Raman detection of biomolecules based on hyperbranched nanostructures

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation of hyperbranched nanoparticles:

[0072] figure 1 A schematic diagram of the experimental procedure for the preparation of hyperbranched nanoparticles is given.

[0073] Taking gold core and gold shell as an example, the specific preparation method is:

[0074] Scaffold DNA:

[0075] 5'-AAAAAAAAAAAAAAAAAAAAACAAGAGTTACTAGTCTCGTCGGAGTCGTATCGCTACAAGTCC-3'

[0076] (1) First take 100ul 10nM 13nm gold balls, add 4ul 100uM scaffold DNA, mix and add 2ul 500mm

[0077] Sodium citrate solution is aged, after reacting for 15-30min, centrifuge and wash three times at 12000rpm for 15min to remove unassembled

[0078] The DNA is dispersed with a buffer solution of 100ul 10mM PB (0.1M NaCl, pH=7.4);

[0079] (2) The mixture is diluted with 900ul 0.1M PBS (pH=7.4) buffer solution, and set aside;

[0080] (3) Take 100ul of the solution in (2), then add 50ul 1% PVP, 20ul 10mM NH 2 OH-HCl, repeat the above operation six times, and finally add 4ul, 8ul, 12ul, 16ul, 20ul...

Embodiment 2

[0082] Specific ultrasensitive quantitative detection of DNA

[0083] image 3 The flow chart of specific quantitative detection of DNA is given.

[0084] by figure 2 Middle No. 4 (sample 4) hyperbranched nanoparticles were used as SERS probes, 4-MBA was used as internal standard, and DTNB was used as external

[0085] Mark as an example:

[0086] Scaffold DNA:

[0087] 5'-AAAAAAAAAAAAAAAAAAAAACAAGAGTTACTAGTCTCGTCGGAGTCGTATCGCTACAAGTCC-3'

[0088] Probe 1: 5'-AAAAAAAAAA TTTTT ATgATgTTCg TTgTg-3'

[0089] Probe 2: 5'-gTgTT TAggATTTgC TTTTT AAAAAAAAAA-3'

[0090] DNA to be tested: 5'-gCAAA TCCTAAACAC CACAA CgAAC ATCAT-3'

[0091] 1. Preparation of hyperbranched nanostructured SERS probes

[0092] (1) First take 100ul 10nM 13nm gold balls, add 4ul 100uM scaffold DNA, mix and add 2ul 500mm sodium citrate solution for aging, after reacting for 15-30min, centrifuge and wash three times at 12000rpm for 15min, remove unassembled DNA, and use 100ul 10mM PB (0.1M NaCl, pH=7.4)...

Embodiment 3

[0103] Specifically recognizes RNAs of the let 7 family

[0104] image 3 A flowchart for the specific recognition of RNAs of the let 7 family is given.

[0105] by figure 2 The No. 4 hyperbranched nanoparticles are used as SERS as probe, 4-MBA as internal standard, and DTNB as external standard as an example:

[0106] Scaffold DNA:

[0107] 5'-AAAAAAAAAAAAAAAAAAAAACAAGAGTTACTAGTCTCGTCGGAGTCGTATCGCTACAAGTCC-3'

[0108] Probe 1: 5'-AAAAAAAAAAAAACTATGCAA-3'

[0109] Probe 2: 5'-CCTACTACCTCTAAAAAAAAAA-3'

[0110] let 7a:5'-UGAGGUAGUAGGUUGUAUAGUU-3'

[0111] let 7b:5'-UGAGGUAGUAGGUUGUGUGGUU-3'

[0112] let 7c:5'-UGAGGUAGUAGGUUGUAUGGUU-3'

[0113] let 7d:5'-AGAGGUAGUAGGUUGCAUAGUU-3'

[0114] let 7e:5'-UGAGGUAGGAGGUUGUAUAGUU-3'

[0115] let 7f:5'-UGAGGUAGUAGAUUGUAUAGUU-3'

[0116] let 7g:5'-UGAGGUAGUAGUUUGUACAGUU-3'

[0117] let 7i:5'-UGAGGUAGUAGUUUGUGCUGUU-3'

[0118] miR 98:5'-UGAGGUAGUAAGUUGUAUUGUU-3'

[0119] mirR 21:5'-UAGCUUAUCAGACUGAUGUUGA-3'

[0120] 1. Prepa...

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Abstract

The invention belongs to the technical field of biological molecule detection, and particularly relates to a hyperbranched nanostructure-based surface-enhanced raman biological molecule detection method. The method comprises the following steps: firstly, assembling a layer of DNA stent molecules and raman small molecules (I) on the surface of a small-particle-size nano metal ball; then generating a metal ball shell which has a certain thickness and is provided with a branch on the surface of a metal ball kernel by taking the DNA stent molecules as a template; finally, modifying the surface of the metal ball shell with raman small molecules (II) and corresponding probe molecules. Under the specificity binding action of an object to be detected and the probe molecules, nano particles of hyperbranched nanostructures are dragged to be gathered, and raman signals of two raman small molecules are detected; an internal standard method and a ratio method are cooperated to realize quantitative specificity detection of the object to be detected. The hyperbranched nanostructure-based surface-enhanced raman biological molecule detection method is easy to operate, quick and sensitive, can realize ultra-sensitive quantitative specificity detection of biological molecules, can be widely applied to the field of food safety detection, medical diagnosis, forensic examination and the like, and has important application prospect and development value.

Description

technical field [0001] The invention belongs to the technical field of biomolecular detection, and in particular relates to a quantitative surface-enhanced Raman biomolecular detection method based on a hyperbranched nanostructure. Background technique [0002] Sensitive and accurate detection, identification and / or quantification of low concentrations of biomolecules such as proteins, peptides, oligonucleotides, nucleic acids, esters, polysaccharides, hormones, neurotransmitters, metabolites, etc. is difficult tasks, but they have broad and potential applications in medical diagnostics, pathology, toxicology, epidemiology, biological warfare, environmental sampling, forensics, and a myriad of other fields. This requires that our detection devices must be gradually miniaturized, and at the same time ensure the high efficiency and sensitivity of detection, which is a great challenge faced by many traditional bioanalysis methods. [0003] Surface Enhanced Raman Scattering (SE...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 裴昊齐林瞿祥猛李丽
Owner EAST CHINA NORMAL UNIV
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