Optokinetically Encoded Nanoprobe-Based Multiplexing Strategy for MicroRNA Profiling

Abstract

Multiplexed real-time analysis on multiple interacting molecules and particles is needed to obtain information on binding patterns between multiple ligands and receptors, specificity of bond formations, and interacting pairs in a complex medium, often found in chemical and biological systems, and difference in binding affinity and kinetics for different binding pairs in one solution. In particular, multiplexed profiling of microRNA (miRNA) in a reliable, quantitative manner is of great demand for the use of miRNA in cell biology, biosensing, and clinical diagnostic applications, and accurate diagnosis of cancers with miRNA is not possible without detecting multiple miRNA sequences in a highly specific manner. Here, we report a multiplexed molecular detection strategy with optokinetically (OK) coded nanoprobes (NPs) that show high photostability, distinct optical signals, and dynamic behaviors on a supported lipid bilayer (SLB) (OK-NLB assay). Metal NPs with three distinct dark-field light scattering signals [red (R), green (G), and blue (B)] and three different target miRNA half-complements were tethered to a two dimensionally fluidic SLB with mobile (M) or immobile (I) state. In situ single-particle monitoring and normalized RGB analysis of the optokinetically combinatorial assemblies among three M-NPs and three I-NPs with dark-field microscopy (DFM) allow for differentiating and quantifying 9 different miRNA targets in one sample. The OK-NP-based assay enables simultaneous detection of multiple miRNA targets in a highly quantitative, specific manner within 1 h and can be potentially used for diagnosis of different cancer types. We validated the OK-NLB assay with single-base mismatched experiments and HeLa cell-extracted total RNA samples by comparing the assay results to the quantitative reverse transcription polymerase chain reaction (qRT-PCR) results.

Biocomputing with Nanostructures on Lipid Bilayers

“…This dimensionality reduction is of paramount importance, as the tethered structures could be characterized by various optical microscopy methods that enable parallel analysis on them with high spatiotemporal resolution. The most notable example is a nanoparticle‐modified supported lipid bilayer platform that allows for multiplexed, ultrasensitive analysis of nucleic acids (DNA and microRNA) via massively parallel dark‐field analysis on single plasmonic nanoparticles . The tethered nanoparticles were modified with surface DNA ligands, which were designed in a way that the dynamic particle‐to‐particle interactions are driven by ligand‐mediated molecular recognition events.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…It is worth noting that this review mainly focuses on fluorescent and bioluminsescent RNA imaging probes, but there are several promising RNA nanoprobes with other optical methods such as surface-enhanced Raman scattering (SERS) 12–15 and dark field 16–18 methods. SERS-based RNA imaging probes have two distinct types: label-free SERS probes 13–15 and nanotag-containing SERS probes 17, 18 .…”

“…The nanotags in the latter type of Raman probe are generally Raman-active compounds and recognition nucleotide sequences, which image target RNAs through base-pairing and are structurally similar to the Nano-MBs in this study. Dark-field based RNA imaging probes 16 are generally GNPs modified with recognition nucleotide sequences, which have plasmon coupling and change scattering signals in the presence of target RNAs. Both SERS-based and dark-field based RNA imaging probes are highly specific, highly sensitive and multiplexed and their applications mainly focus on the detection of miRNA tumour markers.…”

Super‐resolution Geometric Barcoding for Multiplexed miRNA Profiling

“…Unlike methods based on spectrally separable fluorophores or plasmonic interactions [10] our method is not limited by the number of spectrally separable probe species and could potentially combine the advantages of a highly sensitive and specific single-molecule assay [7] with a high degree of multiplexing capability (> 100 ×). [11] …”

“…These measurements were consistent with previous reports and further confirmed the specific capture and multiplexed, accurate quantitation ability of our method (Figure S9). [10,14] …”