Multiplexed DNA and Protease Detection with Orthogonal Energy Transfer on a Single Quantum Dot Scaffolded Biosensor

Abstract

Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb → QD → AF647 → Cy5.5 (→ = ET step), which is detected in a time-gated modality, and QD → AF647 → Cy5.5, which is detected from direct excitation. The labeled DNAdisplaying QD construct is then further assembled with a Ru II -modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.

Enhancing enzymatic activity with nanoparticle display – an updated compendium and engineering outlook

“…210 Combining enzymes with QD's intrinsic photoluminescence and FRET or other energy transfer modalities can allow for sensor assemblies that are capable of extremely complex and concatenated Boolean logic functions. 211–213 This can allow such sensors to perform rudimentary computational analysis and provide a more complex and nuanced data output on the underlying processes being monitored.…”