Journal Article

Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy

Kohli, Manish, Li, Nantao, Smith, Lucas D., Lu, Yi, Smith, Andrew M., Cunningham, Brian T., Canady, Taylor D.

2019

Overview

SignificanceHighly selective and sensitive detection of microRNA is a key challenge in the development of liquid-biopsy approaches. Technologies that can achieve high diagnostic performance without the requirement of complicated processing steps or expensive equipment are necessary for broad use. With these features in mind, we demonstrate a digital-readout microRNA diagnostic that fundamentally relies on microRNA-activated nanoparticle-photonic crystal hybrid coupling. The hybrid formation allows for clear detection of single-particle binding events due to enhanced nanoparticle absorption at the binding location. Whereas the applied photonics lend the assay concentration sensitivity, we additionally demonstrate broad placement single-base mismatch selectivity and complex media detection by applying free-energy tuned toehold probes. Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide “digital” resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe–nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (<1 pM) and high selectivity can be achieved on a direct readout diagnostic.

• Publisher: National Academy of Sciences
• Subject: Absorption / Biological Sciences / Biomarkers / Biomarkers, Tumor - analysis / Biomarkers, Tumor - chemistry / Biomarkers, Tumor - genetics / Biopsy / Biosensing Techniques - instrumentation / Biosensing Techniques - methods / Biosensors / Blood circulation / Circulating MicroRNA - analysis / Circulating MicroRNA - chemistry / Circulating MicroRNA - genetics / Crystal pulling / Diagnostic systems / Discrimination / Engineering / Gold / Gold - chemistry / Humans / Light intensity / Luminous intensity / Medical Sciences / Metal Nanoparticles - chemistry / MicroRNAs / Microscopy / Microscopy - instrumentation / miRNA / Nanoparticles / Nanostructures - analysis / Nanostructures - chemistry / Nucleic acids / Oligonucleotides / Oligonucleotides - chemistry / Photonic crystals / Photons / Physical Sciences / Point Mutation / Resonators / Ribonucleic acid / RNA / Selectivity / Sensitivity and Specificity / Signal to noise ratio / Surface chemistry / Surface matching / Wavelength