PCR-Free, Multiplexed Expression Profiling of microRNAs Using Silicon Photonic Microring Resonators

Abstract

We describe an approach for multiplexed microRNA analysis using silicon photonic microring resonators to detect cDNA reverse transcription products via a subsequent enzymatic signal enhancement strategy. Key to this method is a modified stem loop primer that facilitates downstream signal amplification via enzymatic turnover and improves the sensor signal 20-fold when compared to traditional stem loop primers. This approach facilitates targeted microRNA quantification in only 2.5 hours and without requiring target amplification via the polymerase chain reaction (PCR) . Primers for 7 miRNA targets were orthogonally designed to avoid cross-hybridization between capture probes. This approach was applied to the detection of total RNA from human tissues and found to display differential expression profiles consistent with literature precedent. This development holds promise as an alternative to single-plex RT-qPCR methods and more expensive RNA-seq by offering a cost-effective method to analyze targeted miRNA panels in emerging diagnostic applications.

Whispering-Gallery Sensors

“… 90 , 91 , 92 Techniques have also been employed to further increase the analytical sensitivity for nucleic acids, either through the additional secondary labels and/or enzymatic reactions. 93 , 94 …”

Whispering-Gallery Mode Resonators for Detecting Cancer

“…Gene-related detection often uses polymerase chain reaction (PCR) to replicate analytes to detectable levels, as most of the detection in this application is known for extremely low target concentrations compared to background molecules. The challenge of optical resonator detection also focuses on how the process can be speeded-up by reducing the gene amplification time by eliminating PCR from the system or using other on-chip methods [ 38 , 130 , 131 ].…”

“…Such processes are time- and resource-consuming. Here, there is a trend for minimizing the PCR step or replacing it entirely [ 38 , 130 ]. The domain of interest is mainly the method for detecting the nucleic acid–based analyte in small volumes [ 17 , 20 ] extracted from both tumor cells and other body fluids.…”

Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators

“…On gold-coated sensor surfaces, this is typically done with thiol-ended self-assembled monolayers (SAMs), while silanization is often the method of choice for glass-or silicon-based sensors [19]. These SAMs and silane films typically introduces amino-, thiol-carboxyl-or epoxy-groups to the substrate that can subsequently be used to covalently immobilize capture probes either through a direct covalent bond induced through carbodiimide chemistry [20] or via the use of a linker such as bis(sulfosuccinimidyl)suberate (BS3) [21,22] or glutaraldehyde [23,24].…”

“…Sabaté del Rio et al report on DNA functionalization of ring resonators through copper-catalyzed chemistry on rings silanized with a 2% mixture of 11-aziduondecyltriethoxysilane in toluene and an overnight incubation [41]. These sensors where subsequently used for real-time and label-free monitoring of solid-phase recombinase polymerase amplification, with a limit of detection of 7.8 × 10 −13 M. Both Valera et al [21] and Graybill et al [22] report on silanization with a 5% APTES solution in acetone for 5 min before using the BS3 linker to immobilize antibodies and aminated ssDNA capture probes respectively. Graybill reports on multiplexed detection of expressed microRNAs, while Valera reported on biosensing of monocyte chemotactic protein 1.…”