ABSTRACT
BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.
Subject(s)
Pathology, Molecular/methods , Plague/diagnosis , Polymerase Chain Reaction/methods , Yersinia pestis/genetics , Yersinia pestis/isolation & purification , DNA Primers , Equipment Design , Genes, Bacterial/genetics , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence Analysis , Pathology, Molecular/instrumentation , Polymerase Chain Reaction/instrumentation , Sensitivity and SpecificityABSTRACT
We report on the tuning of specific binding of DNA attached to gold nanoparticles at the individual particle pair (dimer) level in an optical trap by means of plasmonic heating. DNA hybridization events are detected optically by the change in the plasmon resonance frequency due to plasmonic coupling of the nanoparticles. We find that at larger trapping powers (i.e., larger temperatures and stiffer traps) the hybridization rates decrease by more than an order of magnitude. This result is explained by higher temperatures preventing the formation of dimers with lower binding energies. Our results demonstrate that plasmonic heating can be used to fine tune the kinetics of biomolecular binding events.
Subject(s)
Coated Materials, Biocompatible/chemical synthesis , DNA/chemistry , Gold/chemistry , Heating/methods , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Surface Plasmon Resonance/methods , Binding Sites , Coated Materials, Biocompatible/radiation effects , DNA/radiation effects , Energy Transfer , Gold/radiation effects , Kinetics , Light , Materials Testing , Metal Nanoparticles/radiation effectsABSTRACT
In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.
