DNA-Encoded Immunoassay in Picoliter Drops: A Minimal Cell-Free Approach.

Abstract

Immunoassays have emerged as indispensable bioanalytical tools but necessitate long preliminary steps for the selection, production, and purification of the antibody(ies) to be used. Here is explored the paradigm shift of creating a rapid and purification-free assay in picoliter drops where the antibody is expressed from coding DNA and its binding to antigens concomitantly characterized in situ. Efficient synthesis in bulk of various functional variable domains of heavy-chain only antibodies (VHH) using reconstituted cell-free expression media, including an anti-green fluorescent protein VHH, is shown first. A microfluidic device is then used to generate monodisperse drops (30pL) containing all the assay components, including a capture scaffold, onto which the accumulation of VHH:antigen produces a specific fluorescent signal. This allows to assess, in parallel or sequentially at high throughput (500Hz), the VHH-antigen binding and its specificity in less than 3 h,directly from a VHH-coding DNA, for multiple VHH sequences, various antigens anddown to DNA concentrations as low as 12 plasmids per drop. It is anticipated that the ultraminiaturized format, robustness, and programmability of this novel cell-free immunoassay concept will constitute valuable assets in fields as diverse as antibody discovery, point-of-care diagnostics, synthetic biology, and/or bioanalytical assays.