Ultrasensitive Microarray Detection of Short RNA Sequences with Enzymatically Modified Nanoparticles and Surface Plasmon Resonance Imaging Measurements

摘要

ABSTRACT: A novel multiplexed method for short RNAndetection that employs an enzymatic capture reaction ontonDNA-modified silica nanoparticles (SiNPs) followed by nano-nparticle-enhanced surface plasmon resonance imaging (SPRI) isndemonstrated. SiNPs functionalized with 50n-phosphorylatednsingle stranded DNA (ssDNA) are used with T4 RNA ligasento capture various short 20u000124 base single-stranded RNAn(ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specificallynadsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profilingnhas several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to anlarger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) thenSiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRInsignal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can benused to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 μL.