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Ang YS, Yung LY.
ACS Nano. 2012 Oct 23;6(10):8815-23.
Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Abstract
Single-nucleotide polymorphism (SNP) is an important biomarker for disease diagnosis, treatment monitoring, and development of personalized medicine. Recent works focused primarily on ultrasensitive detection, while the need for rapid and label-free single-nucleotide discrimination techniques, which are crucial criteria for translation into clinical applications, remains relatively unexplored. In this work, we developed a novel SNP detection assay that integrates two complementary nanotechnology systems, namely, a highly selective nanoparticle-DNA detection system and a single-particle sensitive nanopore readout platform, for rapid detection of single-site mutations. Discrete nanoparticle-DNA structures formed in the presence of perfectly matched (PM) or single-mismatched (SM) targets exhibited distinct size differences, which were resolved on a size-tunable nanopore platform to generate corresponding “yes/no” readout signals. Leveraging the in situ reaction monitoring capability of the nanopore platform, we demonstrated that real-time single-nucleotide discrimination of a model G487A mutation, responsible for glucose-6-phosphate dehydrogenase deficiency, can be achieved within 30 min with no false positives. Semiquantification of DNA samples down to picomolar concentration was carried out using a simple parameter of particle count without the need for sample labeling or signal amplification. The unique combination of nanoparticle-based detection and nanopore readout presented in this work brings forth a rapid, specific, yet simple biosensing strategy that can potentially be developed for point-of-care application.
Additional Information
Nanopore is a simple and elegant platform for label-free single molecule detection which however, suffers from the drawback of lack in selectivity. Also, it is difficult to pick up signals from small molecules above that of background noise without sophisticated device design. In this work, we addressed these challenges by marrying nanoparticle detection with nanopore readout using a commercially available qNano device (Izon Science). The basic idea is to use gold nanoparticle (AuNP) as an intermediate mediator to amplify the signal of a single nucleotide mismatch in DNA via distinct size differences of AuNP-DNA assemblies. This is complemented by the tunable resistive pulse sensing (TRPS) technique available on qNano for better signal-to-noise ratio. We wish to highlight that real-time measurement can be performed via our nanoparticle-nanopore approach, which can be further leveraged upon to develop an enabling tool for probing the dynamics of particle interaction in situ, be it for inorganic nanomaterials or complex biomolecules. Furthermore, the TRPS qNano platform is able to simultaneously detect nano- and micro-sized particle size, concentration counts and surface charge details. We believe that the ease of obtaining multi-dimensional information on a single-particle basis is a valuable tool to characterize and understand particle behavior for various applications.
