We recently developed capillaric circuits (CCs) – advanced capillary microfluidic devices assembled from capillary fluidic elements in a modular manner similar to the design of electric circuits (Safavieh & Juncker, Lab Chip, 2013, 13, 4180–4189).
Quantitative models of Förster resonance energy transfer (FRET)-pioneered by Förster-define our understanding of FRET and underpin its widespread use. However, multicolour FRET (mFRET), which arises between multiple, stochastically distributed fluorophores, lacks a mechanistic model and remains intractable. mFRET notably arises in fluorescently barcoded microparticles, resulting in a complex, non-orthogonal fluorescence response that impedes their encoding and decoding. Here, we introduce an ensemble mFRET (emFRET) model, and apply it to guide barcoding into regimes with extreme FRET. We further introduce a facile, proportional multicolour labelling method using oligonucleotides as homogeneous linkers. A total of 580 barcodes were rapidly designed and validated using four dyes-with FRET efficiencies reaching 76%-and used for multiplexed immunoassays with cytometric readout and fully automated decoding. The emFRET model helps to expand the barcoding capacity of barcoded microparticles using common organic dyes and will benefit other applications subject to stochastic mFRET.
We present the nELISA, a miniaturised, high-throughput, and high-fidelity protein profiling platform. DNA oligonucleotides are used to pre-colocalize antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection while ensuring spatial separation between non-cognate antibody pairs. Read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact to performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements across the platform spanning 8 orders of magnitude. We then performed a large-scale PBMC secretome screen, with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel cytokine responses, that were conserved across donors and stimulation conditions. We also validated its use in phenotypic screening, and proposed applications for the nELISA in drug discovery.
Capillaric circuits (CCs) are advanced capillary microfluidic devices that move liquids in complex pre-programmed sequences without external pumps and valves -relying instead on microfluidic control elements powered by capillary forces. CCs were thought to require high-precision micro-scale features manufactured by photolithography in a cleanroom, which is slow and expensive. Here we present rapidly and inexpensively 3D-printed autonomous CCs. Molds for CCs were fabricated with a benchtop 3D-printer, Poly(dimethylsiloxane) replicas were made, and fluidic functionality was verified with aqueous solutions. We established design rules for 3D-printed CCs by a combination of modelling and experimentation. The functionality and reliability of 3D-printed trigger valves -an essential fluidic element that stops one liquid until flow is triggered by a second liquid -was tested for different geometries and different solutions. Trigger valves with geometries up to 80-fold larger than cleanroom-fabricated ones were found to function reliably. We designed 3D-printed retention burst valves that encode sequential liquid drainage and delivery using capillary pressure differences encoded by varying valve height and width. Using an electrical circuit analogue of the CC, we established circuit design rules for ensuring strictly sequential liquid delivery. We realized a 3D-printed CC with reservoir volumes 60 times larger than cleanroom-fabricated circuits and autonomously delivered eight liquids in a pre-determined sequence in < 7 min, exceeding the number of sequentially-encoded, self-regulated fluidic delivery events previously reported. Taken together, our results demonstrate that 3D-printing enables rapid prototyping of reliable CCs with improved functionality and potential applications in diagnostics, research and education.
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