Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection†

doi:10.7503/cjcu20150016

Abstract

Abstract:

A simple, rapid and low-cost polydopamine nanoparticle(PDA NP)-based fluorescence resonance energy transfer(FRET) assay with deoxyribonucleaseⅠ(DNaseⅠ)-assisted target recycling signal amplification was developed for microRNA(miRNA) detection. Under the optimized experimental conditions, the recovery ratio of Cy5 fluorescence intensity increased linearly with logarithm of miR-21 concentration from 10 pmol/L to 100 nmol/L, with a detection limit of 7 pmol/L. Moreover, satisfactory results were obtained while the proposed method was applied to detect miRNA in 10% human serum.

Key words: Polydopamine nanoparticle, microRNA, DNaseⅠ, Fluorescence resonance energy transfer

CLC Number:

O657

TrendMD:

MA Lina, LIU Fuyao, GAO Jiaxue, WANG Zhenxin. Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection^†[J]. Chem. J. Chinese Universities, 2015, 36(6): 1061.

Figures/Tables 7

Table 1 Oligonucleotide sequences used in experiments*

Name	Sepuence	Number of bases
P-miR-21	5'-Cy5-TCAACATCAGTCTGATAAGCTA-3'	22
miR-21	5'-UAGCUUAUCAGACUGAUGUUGA-3'	22
P-7f	5'-Cy5-AACTATACAATCTACTACCTCA-3'	22
let-7f	5'-UGAGGUAGUAGAUUGUAUAGUU-3'	22
let-7a	5'-UGAGGUAGUAGGUUGUAUAGUU-3'	22

Fig.1 TEM micrographs of as-prepared PDA NPs with different average diameters(A—C) and their UV-Vis spectra(D) Average diameter/nm: (A), a. 94±9; (B), b. 206±15; (C), c. 306±17.

Scheme 1 Schematic representation of PDA NP-based FRET assay with DNase I-assisted target recycling signal amplification for the detection of target miRNA

Fig.2 Effects of quenching time of 94(A), 206(B) and 306 nm(C) PDA NPs on quenching efficiency in the concentration of 20 μg/mL

Fig.3 Effects of concentration of PDA NPs(A), cleavage/reaction time(B), NaCl concentration(C) and MgCl2 concentration(D) on the recovery ratio of Cy5 fluorescence intensity(FR) in the presence of 66 nmol/L Cy5-ssDNA

Fig.4 Fluorescence spectra of Cy5 in the presence of various concentrations of miR-21 in buffer(A) and 10% human serum(B), respectively and calibration curves of recovery ratio of Cy5 fluorescence intensity(FR) vs. logarithmic concentration of miR-21(C) in buffer(a) and 10% human serum(b), respectivelyThe error bars mean standard deviations(n=6).

Fig.5 Specificity evaluations for miRNA detection in buffer and 10% human serum, respectivelyThe concentration of the each miRNA targets is 100 nmol/L.

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