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Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes

Small, ISSN: 1613-6829, Vol: 12, Issue: 8, Page: 1035-1043
2016
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Article Description

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes. A versatile cyclic draining-replenishing (CDR) methodology effectively "erases" size-associated diffusion constraints characteristic to bulky nanoparticle probes, dramatically enhancing the speed of on-surface binding. Application of CDR to enzyme-linked immunosorbent assays yields reaction-limited assay kinetics with large polymeric enzyme probes. Similarly, a reliable immuno-labeling of cellular targets with fluorescent nanoparticles could be achieved in just 10 min.

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