Nanoencapsulated Microcrystalline Particles for Superamplified Biochemical Assays

Abstract

We report on the preparation and utilization of a novel class of particulate labels based on nanoencapsulated organic microcrystals with the potential to create highly amplified biochemical assays. Labels were constructed by encapsulating microcrystalline fluorescein diacetate (FDA; average size of 500 nm) within ultrathin polyelectrolyte layers of poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) via the layer-by-layer technique. Subsequently, the polyelectrolyte coating was used as an "interface" for the attachment of anti-mouse antibodies through adsorption. A high molar ratio of fluorescent molecules present in the microcrystal core to biomolecules on the particle surface was achieved. The applicability of the microcrystal-based label system was demonstrated in a model sandwich immunoassay for mouse immunoglobulin G detection. Following the immunoreaction, the FDA core was dissolved by exposure to organic solvent, leading to the release of the FDA molecules into the surrounding medium. Amplification rates of 70-2000-fold (expressed as an increase in assay sensitivity) of the microcrystal label-based assay compared with the corresponding immunoassay performed with direct fluorescently labeled antibodies are reported. Our approach provides a general and facile means to prepare a novel class of biochemical assay labeling systems. The technology has the potential to compete with enzyme-based labels as it does not require long incubation times, thus speeding up bioaffinity tests.

Integrated Nanoparticle–Biomolecule Hybrid Systems: Synthesis, Properties, and Applications

“…Particularly attractive are the recently developed nanoencapsulated microcrystalline particles prepared by the layer-bylayer technique which offer large marker/biomolecule ratios and superamplified bioassays. [240] Related analytical procedures that combined multiple amplification pathways based on enzyme-functionalized liposomes and the accumulation of the biocatalytic reaction product were reported as ultrasensitive DNA assays. [241] Such bioassays relied on the large surface area of the liposomes that carry a large number of enzyme molecules; the accumulated product was detected by means of chronopotentiometry.…”

Biofunctional organic nanocrystals for quantitative detection of pathogen deoxyribonucleic acid

“…The amplicons were hybridized with the immobilized HPV 16,18 or 45 probes and detected using FDA/FITC/HRP-labeled streptavidin. Figure 1 shows the calibration curves of the assays performed with the streptavidin-FDA nanocrystal labels.…”

“…In a previous study, we applied a novel signal amplification technology based on a new class of biofunctional fluorogenic nanocrystals to improve the sensitivity and the limits of detection in immunoassays [18][19][20][21]. A simple two-step approach was used to encapsulate nanocrystalline fluorescein diacetate (FDA) with an average size of 107 nm with ultrathin polyelectrolytic distearoylphosphatidylethanolamine (DSPE) lipids coupled with amino(poly(ethylene glycol)) (PEG2000-Amine) [19].…”

An Organic Nanoparticle based High Signal Amplification Immunoassay with Improvement of Nonspecific Binding

“…Recently, C.P Chan and Dieter Trau et al reported a DSPE-PEG (2000) amine based polyelectrolyte multilayers fluorescein diacetate (FDA) nanocrystals [18][19][20][21]; Yvonne Bruemmel et al [18] studied the influence of different surface modifications on the performance of fluorescent based immunoassays. These methods increase the F/P ratio by releasing fluorescein molecules through the hydrolysis of the FDA nanoparticles after dissolution in DMSO, which undoubtedly can achieve very high sensitivity and very low limit of detection in principle.…”