Controlling the Bioreceptor Spatial Distribution at the Nanoscale for Single Molecule Counting in Microwell Arrays

Abstract

The ability to detect low concentrations of protein biomarkers is crucial for the early-stage detection of many diseases, and therefore indispensable for improving diagnostic devices for healthcare. Here, we demonstrate that by integrating DNA nanotechnologies like DNA origami and aptamers we can design innovative biosensing concepts for reproducible and sensitive detection of specific targets. DNA origami structures decorated with aptamers were studied as a novel tool to structure the biosensor surface with nanoscale precision in a digital detection bioassay, enabling control of the density, orientation and accessibility of the bioreceptor to optimize the interaction between target and aptamer. DNA origami was used to control the spatial distribution of an in-house generated aptamer on superparamagnetic microparticles, resulting in an origami-linked digital aptamer bioassay to detect the main peanut antigen Ara h1 with 2-fold improved signal to noise ratio and 15-fold improved limit of detection compared to a digital bioassay without DNA origami. Moreover, the sensitivity achieved was 4 orders of magnitude higher than commercially available and literature-reported ELISA techniques. In conclusion, this novel and innovative approach to engineer biosensing interfaces will be of major interest to scientists and clinicians looking for new molecular insights and ultrasensitive detection of a broad range of targets, and, for the next generation of diagnostics.

DNA Origami as Emerging Technology for the Engineering of Fluorescent and Plasmonic-Based Biosensors

“…Compared to directly aptamer coated microparticles (232 fM, 45 pg/mL) a LOD of one order of magnitude lower was measured (18 fM, 3.5 pg/mL) after a reaction time of 3h 30 min. Reported LODs for ELISA and lateral flow assays were 12 ng/mL and 450 ng/mL, respectively [54]. These results showed a clear improvement in sensitivity when using DNA origami structures.…”

“…These interactions can be translated into optical signals by plasmonic or by fluorescence excitation [50,51]. Optical DNA origami sensors are developing into sensitive, rapid, accurate, flexible, and multi-target sensing tools with a low limit of detection (LOD) [18,19,21,[52][53][54][55]. They are used in biology, environment monitoring, and the food safety industry, and have a great potential for applications in biomedicine to detect and monitor diseases and pathogens [56].…”

“…In 2019, Deams et al [54] presented a cost-effective and sensitive enzyme-linked-hybrid aptamer immunosorbent assay to detect the peanut antigen Ara h1. Magnetic microparticles were coated with 2D DNA origami structures, each functionalized with aptamers to specifically bind at least one Ara h1 protein molecule.…”

“…As the endosome function is to transport materials from the outside to the inside of the cell, the DNA nanoribbons could also be used for delivering siRNA to the cytoplasm for gene silencing purposes, for example in cancer cells (load capacity of 45.7 wt %). In 2019, Deams et al [54] presented a cost-effective and sensitive enzyme-linked-hybrid aptamer immunosorbent assay to detect the peanut antigen Ara h1. Magnetic microparticles were coated with 2D DNA origami structures, each functionalized with aptamers to specifically bind at least one Ara h1 protein molecule.…”

“…(B) Schematic representation of a metallic microparticle (grey sphere) coated with DNA origami structures (light blue). The inset shows the signaling principle when the target is detected[54]. The blue helices are aptamers to which the target can bind.…”