Nanoengineered metallic materials have been shown to have a number of exclusive physicochemical properties not available at neither larger (micro- and macroscopic) nor smaller (molecular) scales. Recently, these materials in particular have drawn significant attention due to their capability to enhance fluorescent signals of nearby fluorescent species through a phenomenon known as metal enhanced fluorescence (MEF). MEF originates from the localized surface plasmon resonance (LSPR), a collective oscillation of conduction-band electrons that can modify both the extinction coefficient and quantum yield of adjacent fluorescent molecules/species. [1] The extinction coefficient is a function of the electromagnetic field intensity experienced by the fluorescent molecules, and under an enhanced electromagnetic field, fluorescent molecules absorb photons and promote electrons into excited states at accelerated rates. LSPR of metallic nanostructures can also modify the quantum yield of fluorescent species by increasing their radioactive decay rate. The combined enhancement of extinction coefficient and quantum yield can result in significantly strengthened fluorescence from fluorescent species.

Adapter ligation is a critical first step in many microRNA analysis methods including microarray, qPCR, and sequencing. Previous studies have shown that ligation bias can have dramatic effects on both the fidelity of expression profiles and reproducibility across samples. We have developed a method for high efficiency and low bias microRNA capture by 3′ adapter ligation using T4 RNA ligase that does not require pooled adapters. Using a panel of 20 microRNA, we investigated the effects of ligase type, PEG concentration, ligase amount, adapter concentration, incubation time, incubation temperature, and adapter design on capture efficiency and bias. Of these factors, high PEG% was found to be critical in suppressing ligation bias. We obtained high average capture efficiency and low CV across the 20 microRNA panel, both in idealized buffer conditions (86%±10%) and total RNA spiking conditions (64%±17%). We demonstrate that this method is reliable across microRNA species that previous studies have had difficulty capturing and that our adapter design performs significantly better than the common adapter designs. Further, we demonstrate that the optimization methodology must be specifically designed for minimizing bias in order to obtain the ideal reaction parameters.

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification.