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Self-assembled super-quenched gold nanoparticle nano sensor and preparation method and application thereof

A gold nanoparticle and nanosensor technology, applied in the field of biological analysis, can solve problems such as affecting fluorescence quenching, affecting analysis accuracy and sensitivity, high background signal, etc., to avoid signal amplification steps, reduce sample consumption, and improve dissociation. The effect of efficiency

Pending Publication Date: 2021-12-21
SHANDONG NORMAL UNIV
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Problems solved by technology

However, the inventors found that the distance between the fluorophore and the AuNP surface can significantly affect the efficiency of fluorescence quenching, and in some cases, AuNPs can even enhance the fluorescence signal of adjacent fluorophores through metal-enhanced fluorescence (MEF), These factors tend to result in high background signal, which affects the accuracy and sensitivity of the assay

Method used

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  • Self-assembled super-quenched gold nanoparticle nano sensor and preparation method and application thereof
  • Self-assembled super-quenched gold nanoparticle nano sensor and preparation method and application thereof
  • Self-assembled super-quenched gold nanoparticle nano sensor and preparation method and application thereof

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Embodiment 1

[0069] Preparation of gold nanoparticle nanoprobes: AuNP nanoprobes were prepared by a typical salt aging method. The peptide probe (CSEVNLDAEFRK, see SEQ ID NO.1 for amino acid sequence) and auxiliary DNA (Cy5-SH-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-BHQ2, The nucleotide sequence is detailed in SEQ ID NO.2) Add 1 ml of AuNP solution (7.0×10 11 Particles per milliliter), placed in PBS buffer containing 0.1% Tween 20 (10 mmol per liter of phosphate solution (NaH 2 PO 4 / Na 2 HPO 4 ), pH 7.4). After incubating at room temperature for 20 minutes, gradually increase the NaCl concentration to 0.3 mol per liter within 24 hours, and then incubate at 4°C for 24 hours to complete the functionalization reaction. The solution was centrifuged 3 times at 12000 g for 15 minutes each time to remove unbound peptide probes and auxiliary DNA, and then resuspended in 60 microliters of PBS buffer (10 mmol per liter of phosphate solution, 0.3 liter NaCl, pH 7.4) and stored at 4°C.

[0070] Gold n...

Embodiment 2

[0077] Experimental verification of principle

[0078] The principle of the technical solution of the present invention: the C-terminus is designed as a sensing probe with a Cy5-labeled peptide segment (CSEVNLDAEFRK). In this sensing probe, the Swedish APP mutant fragment (SEVNLDAEFR) is selected instead of the wild-type APP fragment (SEVKMDAEFR) as the sensing probe. BACE-1 substrate, because it can be cleaved by BACE-1, and the cleavage efficiency is higher than wild type. In addition, cysteine ​​residues (C) containing sulfhydryl groups (SH) allow peptide probes to bind to the surface of AuNPs via Au-S bonds, while lysine (K) residues containing amino groups can bind peptide amines via Cy5 carboxyl groups. The condensation reaction of the Cy5 group facilitates the labeling of the Cy5 group. According to the distance between adjacent amino acids of 0.83 nm, the length of the polypeptide probe was determined to be 9.96 nm, so the spatial separation of AuNP and Cy5 would lead...

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Abstract

The invention discloses a self-assembled super-quenched gold nanoparticle nano sensor and a preparation method and application thereof. The self-assembled super-quenched gold nanoparticle nano sensor comprises: a peptide probe which is a peptide chain capable of being sheared by BACE-1 and is marked with fluorophore, an auxiliary DNA marked with a quenching agent, and gold nanoparticles. At least one peptide probe and at least one auxiliary DNA are assembled on the surfaces of the gold nanoparticles, the connecting ends of the auxiliary DNA and the gold nanoparticles are marked with fluorophores, and the fluorophores on the peptide probe are close to a quenching agent on the auxiliary DNA. The connecting ends of the auxiliary DNA and the gold nanoparticles are marked with fluorophores, so that the fluorophores are self-quenched due to an inner filtering effect caused by local high-concentration fluorophore molecules on the surface of the AuNP; the fluorophores and the quenching agent approach each other to induce fluorescence quenching; static quenching and fluorescence quenching of FRET (Fluorescence Resonance Energy Transfer) between the Cy5 and the AuNP are induced by the AuNP. The three quenching mechanisms can ensure that the fluorophores are completely quenched, so that the super-quenching is realized.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and belongs to a self-assembled super-quenching gold nanoparticle nanosensor used for BACE-1 imaging in living cells, in particular to a self-assembled superquenching gold nanoparticle nanosensor and its preparation method and application . Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Alzheimer's disease (Alzheimer's disease, AD) is a serious neurological disease, accounting for about 60-70% of human senile dementia, and its mediated progressive brain degeneration can lead to a series of cognitive dysfunction, such as Learning difficulties, memory loss, poor judgment, and disor...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6458G01N2021/6432
Inventor 张春阳马飞王琦
Owner SHANDONG NORMAL UNIV
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