Incorporation of Slow Off-Rate Modified Aptamers Reagents in Single Molecule Array Assays for Cytokine Detection with Ultrahigh Sensitivity.

Slow off-rate modified aptamers (SOMAmers) are attractive protein recognition reagents due to their high binding affinities, stable chemical structures, easy production, and established selection process. Here, biotinylated SOMAmer reagents were incorporated into single molecule array (Simoa)-based assays in place of traditional detection antibodies for six cytokine targets. Optimization and validation were conducted for TNF-α as a demonstration using a capture antibody/detection-SOMAmer detection scheme to highlight the performance of this approach. The optimized assay has a broad dynamic range (>4 log10 units) and an ultralow detection limit of 0.67 fM (0.012 pg/mL). These results show comparable sensitivity to our antibody pair-based Simoa assays, and tens to thousands-fold enhancement in sensitivity compared with conventional ELISAs. High recovery percentages were observed in a spike-recovery test using human sera, demonstrating the feasibility of this novel Simoa assay in detecting TNF-α in clinically relevant samples. Detection SOMAmers were also used to detect other cytokines, such as IFN-γ, IL-1ß, IL-2, IL-6, and IL-10, in human samples. Although not yet demonstrated, in principle it should be possible to eventually replace both the capture and detector antibodies with corresponding SOMAmer pairs in sandwich immunoassays. The combination of the ultrasensitive Simoa platform with the higher reliability of SOMAmer binding reagents will greatly benefit both biomarker discovery and disease diagnostic fields.

Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (ß-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.

Disease detection by ultrasensitive quantification of microdosed synthetic urinary biomarkers.

The delivery of exogenous agents can enable noninvasive disease monitoring, but existing low-dose approaches require complex infrastructure. In this paper, we describe a microdose-scale injectable formulation of nanoparticles that interrogate the activity of thrombin, a key regulator of clotting, and produce urinary reporters of disease state. We establish a customized single molecule detection assay that enables urinary discrimination of thromboembolic disease in mice using doses of the nanoparticulate diagnostic agents that fall under regulatory guidelines for "microdosing."