Homogeneous Entropy-Driven Amplified Detection of Biomolecular Interactions

Abstract

While a range of artificial biochemical circuits is likely to play a significant role in biological engineering, one of the challenges in the field is the design of circuits that can transduce between biomolecule classes (e.g., moving beyond nucleic acid only circuits). Herein, we design a transduction mechanism whereby a protein signal is transduced into an amplified nucleic acid output using DNA nanotechnology. In this system, a protein is recognized by nucleic acid bound recognition elements to form a catalytic complex that drives a hybridization/displacement reaction on a multicomponent nucleic acid substrate, releasing multiple target single-stranded oligonucleotides in an amplified fashion. Amplification power and simple one-pot reaction conditions lead us to apply the scheme in an assay format, achieving homogeneous and rapid (∼10 min) analyte detection that is also robust (operable in whole blood and plasma). In addition, we demonstrate the assay in a microfluidic digital assay format leading to improved quantification and sensitivity approaching single-molecule levels. The present scheme we believe will have a significant impact on a range of applications from fundamental molecular interaction studies to design of artificial circuits in vivo to high-throughput, multiplexed assays for screening or point-of-care diagnostics.

Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis

“…For the analysis of clinical samples, a COVID-19 CRISPR-FDS assay result was considered positive if it was equal or greater than a cut-off threshold equal to the mean signal of the negative control samples plus three times its standard deviation (Kim et al, 2016). Using this criterion, 19 of 29 nasal swab samples were found to be SARS-CoV-2 positive (Fig.…”

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

“…TheD iCarlo group [54] developed am icrofluidic digital homogeneous entropy-driven biomolecular assay (dHEBA) for the detection of influenza A. Upon nucleoprotein binding,n ucleic acid labeled antibodies formed ac atalytically active complex that drove ah ybridization/displacement reaction on am ulticomponent nucleic acid substrate and generated many fluorescence-labeled oligonucleotides.T he dHEBAf ormat enabled the detection of influenza An ucleoprotein in ac oncentration of 4am in approximately 10 min without the need for ap urification step.…”

Fortschritte in der optischen Einzelmoleküldetektion: Auf dem Weg zu höchstempfindlichen Bioaffinitätsassays

“…Magnetische Mikrosphären wurden mit einem anti-CD63-Fängerantikçrper zum Immobilisieren von Exosomen beschichtet;nach der Inkubation der Probe wurden ein biotinylierter Nachweisantikçrper und ein Streptavidin-b-Galactosidase-Konjugat zu den Mikrosphären gegeben, die anschließend in Ebenfalls wurde ein kompetitiver Immunassay in Mikrotropfen zum Nachweis von a-fçtalem Protein (AFP) beschrieben. [53] Die Gruppe von Di Carlo [54] entwickelte einen digitalen, homogenen Entropie-getriebenen biomolekularen Assay (digital homogeneous entropy-driven biomolecular assay, dHEBA) zum Nachweis von Influenza A. Bei der Bindung von Nukleoproteinen bildeten Antikçrper,d ie mit Nukleinsäuren markiert waren, einen katalytisch aktiven Komplex, der eine Hybridisierungs-/Verdrängungsreaktion an einem Multikomponenten-Nukleinsäuresubstrat verursachte und viele fluoreszenzmarkierte Oligonukleotide freisetzte.D er dHEBA-Assay ermçglichte den Nachweis von Influenza-A-Nukleoprotein in einer Konzentration von 4am in ungefähr 10 min, ohne dass ein Waschschritt erforderlich war.…”