Making the undetectable detectable: Pushing limits of biomolecular recognition by ultra-sensitive multiplexed single-particle liposome readouts.

Abstract

Biomolecular recognition is fundamental for all biological and chemical processes. However, current screening methods are often reliant on large robotic devices, require long running times and much manpower as well as excessive materials cost. Here I will present our recent work implementing multiple miniaturized screening assays for ultra-sensitive and high-throughput readout at the single-particle level of DNA-DNA recognition, sub-attoliter cargo delivery, transporter function and label-free DNA biosensing. I will focus on the single-particle combinatorial multiplexed liposome fusion mediated by DNA for parallelized multistep and non-deterministic fusion of individual sub-attoliter nanocontainers (SPARCLD). Our new toolbox allowed us to attain efficient (>93%) and leakage free stochastic fusion sequences for arrays of surface-tethered target liposomes with six freely diffusing populations of cargo liposomes, each functionalized with individual lipidated single-stranded DNA and fluorescently barcoded by a distinct ratio of chromophores. Because each lipid nanoparticle is an autonomous nanocontainer the stochastic fusion sequence resulted in massive number of distinct permutations of cargo delivery loadings and sequences. Real-time recordings and machine learning analysis allowed the rapid classification of >16,000 fusions and 566 distinct fusion sequences within hours. The high-density arrays of surface-tethered target nanocontainers (∼42,000 containers per mm2) offers entire combinatorial multiplex screens using only picograms of material and is implanted for screening transporter function and label free DNA biosensing.