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Hairpin DNA-templated silver nanoclusters as novel beacons in strand displacement amplification for microRNAs detection Jingpu Zhang, Chao Li, Xiao Zhi, Gabriel Alfranca Ramón, Yanlei Liu, Chunlei Zhang, Fei Pan, and Daxiang Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03729 • Publication Date (Web): 17 Dec 2015 Downloaded from http://pubs.acs.org on December 19, 2015
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Analytical Chemistry
Hairpin DNA-templated silver nanoclusters as novel beacons in strand displacement amplification for microRNAs detection Jingpu Zhanga,b, Chao Lia, Xiao Zhib, Gabriel Alfranca Ramóna, Yanlei Liua,b, Chunlei Zhanga, Fei Pana,c, Daxiang Cuia,c* a
Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and Microfabrication
of the Ministry of Education, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering; bSchool of Biomedical Engineering, cNational Center for Translational Medicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China * To whom correspondence should be addressed. Tel: 0086-21-34206886; Fax: 0086-21-34206886; Email:
[email protected] 1
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Analytical Chemistry
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ABSTRACT MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated
silver
nanoclusters
(AgNCs/HpDNA)
were
prepared
and
integrated
into
strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. Based on the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way. INTRODUCTION
MicroRNAs (miRNA) are endogenous noncoding RNAs with a length of ~22 nt, regulating the expression level of mRNA 1. MiRNAs dysregulation has been reported to contribute to various diseases including cancer 2. Especially, a growing number of miRNAs with abnormal expression are being identified as potential biomarkers for early diagnosis, prognosis of cancer or monitoring tumor development 2. Among those biomarkers, miR-16-5p and miR-19b-3p in plasma were identified by our group as novel circulating biomarkers to indicate the progression of gastric cancer using miRNA profiling microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) test 3. As golden standard, qRT-PCR is widely used to screen miRNA biomarkers with high sensitivity and specificity. However, qRT-PCR is time and labor-consuming due to a prior reverse transcription and the consequent amplification and detection in thermo-cycler via the deliberately designed primers or even matched TaqMan probes 4. Therefore, 2
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Analytical Chemistry
the development of novel convenient and rapid method for miRNA detection is needed. Isothermal amplification is gaining increasing interest as it avoids a thermal cycle process during the synthesis of DNA 5. Strand-displacement amplification (SDA) reaction was developed as the first generation of isothermal amplification inspired by the base excision mechanism for DNA repair, and has developed into many subtypes, such as multiply primed SDA, nicking-initiated
SDA,
loop-mediated
isothermal
amplification
(LAMP),
and
structure-switching-triggered SDA 6. Without the need of particular restriction site and nicking enzymes, structure-switching-triggered SDA is highly adaptable to oligos of short length, in which primers anneal to a probe instead of the target with a hairpin structure following after its unfolding upon the hybridization of the target 6. The modified molecular beacon (MB) with a fluorophore at one end of the hairpin structure and a quencher at the other end was generally used as the signal probe in structure-switching-triggered SDA
7, 8
. Despite the sensitive and multiplex detection
capability of MB, the extra conjugations of the dyes make it costly and limited. With the development of DNA-templated silver nanoclusters (AgNCs/DNA), novel fluorescence probes have become available beyond those modified by conventional fluorophores and quantum dots (QDs). Fluorescent metal nanoclusters (
