Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single‐Silver‐Nanoparticle Counting

Abstract

DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single-silver-nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon-burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single-nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag-NP-oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon-burst counts. The SSNPC method was used to measure the change in the photon-burst counts. The relationship between the change of the photon-burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence-specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2-5 orders of magnitude more sensitive than current homogeneous methods.

Detection of miRNA Expression in Intact Cells Using Activatable Sensor Oligonucleotides

“…The currently established methods for microRNA detection in situ rely on PCR and northern blotting, or high-affinity hybridization probes (Driskell, et al, 2009; Havelda, 2010; Husale, et al, 2009; Li, et al, 2009; Li, et al; Lu and Tsourkas, 2009; Mandir, et al, 2009; Nelson, et al, 2004; Nuovo, et al, 2009; Pohlmann and Sprinzl, 2010; Silahtaroglu, et al, 2007; Song, et al, 2010; Xu, et al, 2010). However, none of these methods are applicable in intact live cells.…”

Nanoparticle-based Optical Detection of MicroRNA

“…Exploiting the Brownian motion and the stronger resonance Rayleigh scattering of silver nanoparticles (AgNPs) [63], the single AgNPs counting system (SSNPC) by photon-burst counts with ultra-high sensitivity, high spatial resolution (0.5 fL), and good reproducibility was established to detect miRNA in homogeneous solution. The principle of the method is shown in Fig.…”

MicroRNAs and lung cancer: Biology and applications in diagnosis and prognosis

“…General techniques used for quantification of RNA, such as Northern blotting, reverse transcription-PCR (RT-PCR), RNA:RNA in situ hybridization, and microarray analysis, are also applicable for measurement of mature as well as precursor microRNAs. Cloning into an RNA library and sequencing,[33] single molecule sequencing,[34] surface-enhanced Raman spectroscopy,[35] surface plasmon resonance spectroscopy,[36] nanomechanical sensing,[37] and single-silver nanoparticle counting[38] are some other techniques being developed to detect and quantify microRNAs. The short length as well as sequence similarities of microRNAs does necessitate modification of such techniques, for example, through the use of stem-loop primers for reverse transcription,[39] and of locked nucleic acid-containing probes on microarrays.…”