DNA-encoded immunoassay in picoliter drops: a minimal cell-free approach

bioRxiv (Cold Spring Harbor Laboratory)(2022)

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摘要
AbstractBased on the remarkably specific antibody-antigen interaction, immunoassays have emerged as indispensable bioanalytical tools for both fundamental research and biomedical applications but necessitate long preliminary steps for the selection, production and purification of the antibody(ies) to be used. Here, we adopt a paradigm shift exploring the concept of creating a rapid and purification-free assay where the antibody is replaced by its coding DNA as a starting material, while exploiting a drop microfluidic format to dramatically decrease sample volume and accelerate throughput and sorting capability. The methodology consists in the co-encapsulation of a DNA coding for the variable domain of the heavy chain of heavy-chain only antibodies (VHH), a reconstituted cell-free expression medium, the target antigen and a capture scaffold where VHH:antigen accumulate to create a detectable signal, inside picoliter drop compartments. We first demonstrate successful synthesis of a functional hemagglutinin (HA)-tagged anti-GFP VHH, referred to as NanoGFP, at a high yield (15.3 ± 2.0 µg·mL-1) in bulk and in less than 3 h using PURExpress cell-free expression medium. We then use a microfluidic device to generate stable water-in-oil drops (30 pL) encapsulating NanoGFP-coding DNA, PURExpress medium, EGFP antigen and HA tag-specific magnetic nanoparticles prior to incubating at 37 °C the resulting emulsion under a magnetic field, inducing both in situ synthesis of NanoGFP and accumulation of NanoGFP:EGFP complexes on magnetically assembled particles. This allows us to assess, for the first time and in less than 3 hours, the binding of an antigen to a cell-free synthesized antibody, in a large number of picoliter drops down to a DNA concentration as low as 12 plasmids per drop. We also show that the drops of this immunoassay can be further sequentially analyzed at high throughput (500 Hz), thus offering capability for library screening, sorting and/or rare event detection. We finally demonstrate the versatility of this method by using DNA coding for different VHH (e.g., anti-mCherry protein), by characterizing VHH specificity in the presence of antigen mixtures, and by showing that antigens can be either inherently fluorescent or not. We thus anticipate that the ultraminiaturized format (pL), rapidity (3 h), programmability (DNA-encoded approach) and versatility of this novel immunoassay concept will constitute valuable assets for faster discovery, better understanding and/or expanded applications of antibodies.
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关键词
picoliter drops,immunoassay,dna-encoded,cell-free
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