Abstract
In this paper, we present a method for the ultrasensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes, using a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22 oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 attomoles). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis, as well as a range of other RNA based detection applications.
EXPERIMENTAL
Materials
The silane, 3-N-((6-(N’-Isopropylidene-hydrazino))nicotinamide)propyltriethyoxysilane (HyNic Silane), and succinimdyl 4-formyl benzoate (S-4FB) were purchased from SoluLink. PBS was reconstituted with deionized water from Dulbecco’s Phosphate Buffered Saline packets purchased from Sigma-Aldrich (St. Louis, MO), and the buffer pH adjusted to pH 7.4 (PBS-7.4) or pH 6.0 (PBS-6) with sodium hydroxide or hydrochloric acid. A 20× saline-sodium phosphate-EDTA buffer (SSPE) was purchased from USB Corp. for use in a high stringency hybridization buffer. All other reagents were purchased from Fisher, unless otherwise noted, and used as received.
Fabrication of Silicon Photonic Microring Resonators and Measurement Instrumentation
The fabrication of sensor chips and operational principles of the measurement instrumentation have been previously reported.34, 49 Briefly, sensor substrates, each containing 32 uniquely-addressable microring resonators within a 6×6 mm footprint, were fabricated at a commercial-scale silicon foundry on 8” silicon-on-insulator wafers using conventional deep-UV photolithography and dry etching methods, before being diced into individual chips. After immobilization of DNA capture probes (described below), the sensor chips are loaded into a biosensor scanner (Genalyte, Inc.), and the wavelengths of optical resonance of the entire array of microring elements are monitored in near real-time using an external cavity laser, integrated control hardware, and data acquisition software.
Read More: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146633/