Abstract
In this work, a simple and label-free fluorescence “off” to “on” platform was designed for the sensitive and selective detection of microRNA (miRNA) in cancer cells. This method utilized a padlock DNA-based rolling circle amplification (P-RCA) to synthesize fluorescent poly(thymine) (PolyT) which acted as a template for the synthesis of copper nanoparticles (CuNPs) within 10 minutes under mild conditions. While the repeated PolyT sequence was used as the template for CuNP synthesis, other non-PolyT parts (single strand-DNAs without the capacity to act as the template for CuNP formation) served as “smart glues” or rigid linkers to build complex nanostructures. Under the excitation wavelength of 340 nm, the synthesized CuNPs emitted strong red fluorescence effectively at 620 nm. To demonstrate the use of this method as a universal biosensor platform, lethal-7a (let-7a) miRNA was chosen as the standard target. This sensor could achieve highly sensitive and selective detection of miRNA in the presence of other homologous analogues for the combination of P-RCA with the fluorescent copper nanoparticle. Overall, this novel label-free method holds great potential in the sensitive detection of miRNA with high specificity in real samples.
Highlights
MicroRNAs are non-coding short RNAs present in animals, plants, and some viruses [1].Previous evidence has shown that miRNAs are vital regulators for post-transcription, leading to the silencing of genes through target degradation or translational repression [2,3]
The in vitro polymerization of nucleotides due to their signal amplification ability has been widely used in the analysis of miRNAs, including polymerase chain reaction (PCR) [5], strand displacement amplification (SDA) [6] and rolling circle amplification (RCA) [7]
The oligonucleotides was purchased from AIT Biotech (Singapore) and Bacillus phage phi29 (Phi29) DNA polymerase were high-performance liquid chromatography (HPLC)-purified
Summary
MicroRNAs (miRNAs) are non-coding short RNAs present in animals, plants, and some viruses [1]. The degree of nonspecific recognition caused by these signal reporters raises their environmental susceptibility and reduces sensitivity Owing to their distinctive linear, geometric nanosized structures, unique molecular recognition characteristics, and strong affinities for metal ions, certain types of nucleic acids have been utilized as templates for synthesizing metallic nanostructures [9,10,11,12,13]. The nanoparticles exhibited comparable fluorescence intensities to the widely used silver nanoparticles, quickening the detection process Both single and double-stranded poly(thymine) (PolyT)-templated copper nanoparticles (CuNPs) present great potential for applications in biochemical analysis, owing to their easy synthesis, admirable photo-physical properties, and excellent biocompatibility [26,27,28,29,30]. Represent miRNA and the light blue lines represent the corresponding single-stranded DNA (ssDNA)
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