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Catch-and-display immunoassay for digital biomarker detection
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DOI：10.1039/D6NH00038J.png)
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Abstract
En 中文
Digital immunoassays provide exceptional analytical sensitivity for detecting low-abundance biomarkers; but their broad adoption is limited by practical barriers. Commercial platforms are prohibitively expensive for routine use in individual laboratories; and laboratory-scale concepts typically describe specialized biosensors and sophisticated workflows. Here; we introduce a nanomembrane-based Catch-and-Display Immunoassay (CAD-IA); a cost-effective and laboratory-friendly digital immunoassay for common research settings. In CAD-IA; fluorescent nanoparticles are “captured” by the nanoscale pores of ultrathin silicon nitride membranes through a pipette powered filtration. The captured nanoparticles serve as optically isolated ‘hotspots’ for fluorescent immunocomplex formation when target antigen is present. Co-localization of the fluorescent particles and fluorescent immunocomplexes are then “displayed” and quantified by standard confocal microscopy to generate digital signals. CAD-IA is implemented using the µSiM-DX (microfluidic device featuring an ultrathin silicon membrane for diagnostics) platform; which is manually assembled from mass produced; cost-effective components. Using the traumatic brain injury (TBI) biomarker S100B as a model; we demonstrate that CAD-IA provides consistent digital outputs and linear quantification over a dynamic range of at least two orders of magnitude when digital and analog analyses are combined on the same image sets. We further demonstrate that the assay maintains linearity in serum matrices and achieves suitable sensitivity (LoD = 0.02 µg mL−1) for clinically relevant diagnostic with the addition of tyramide signal amplification (TSA). While further optimization of CAD-IA is possible; these results constitute a proof-of-concept demonstration of a novel digital immunoassay deployable in most research laboratories.
Keywords:
digital immunoassay
nanomembrane-based assay
fluorescent nanoparticles
biomarker detection
confocal microscopy
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