Mobile platform for rapid sub–picogram-per-milliliter, multiplexed, digital droplet detection of proteins

Abstract

Digital droplet assays—in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually—have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are currently mainly confined to laboratory settings, due to the instrumentation necessary to serially generate, control, and measure tens of millions of droplets/compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology is based on three key innovations: (i) the integration and parallel operation of a hundred droplet generators onto a single chip that operates >100× faster than a single droplet generator, (ii) the fluorescence detection of droplets at >100× faster than conventional in-flow detection using time domain-encoded mobile phone imaging, and (iii) the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. To demonstrate the power of this approach, we performed a duplex digital ELISA. We characterized the performance of this assay by first using spiked recombinant proteins in a complex media (FBS) and measured a limit of detection, 0.004 pg/mL (300 aM), a 1,000× improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. We additionally measured endogenous GM-CSF and IL6 in human serum fromn= 14 human subjects using our mobile duplex assay, and showed excellent agreement with the gold standard system (R2=0.96).

Formation of uniform reaction volumes using concentric amphiphilic microparticles

“…This inevitably escalates the imaging acquisition burden and prolongs readout time with the need to isolate potential combinations of barcodes for data analysis. [46] A potential solution is to further enhance the throughput of the droplet generator and reader [4] to shorten the time needed to accumulate a sufficient number of droplets containing only one particle of each single type. Our dropicle system provides an alternative approach to overcome this obstacle, as the single solid phase is designed into the amphiphilic particles during manufacturing, and therefore not subject to Poisson loading statistics.…”

“…Uniformity in the compartment volumes ensures the reaction conditions are relatively similar and reactions across volumes can be compared. Microfluidic wells [1][2][3] or droplet generators [4][5][6] enable uniform compartmentalization of a sample fluid volume into many smaller reactions, however skilled users or specialized and costly commercial instruments have been required for reproducible implementation. In addition, for many affinity assays, a solid phase, such as microbeads, is desired to bind a target and allow washing to remove excess reagents or minimize non-specific binding.…”

“…a microbead and a target cell or molecule inside a droplet approaches ~1% of the entire population of droplets, whereas the remaining droplets would have undesired combinations of the components. [4,14,15] Moreover, for multiplexing, multiple types of microbeads with distinct barcoding signatures should be encapsulated in separate droplets with the targets of interest, which is further limited by multiplicative probabilities to triple or larger Poisson distributions for duplex or greater multiplexing. Therefore, an instrument-free compartmentalization system that forms uniform droplets embedded with a single solid phase per droplet can address many of the challenges with current approaches.…”

Mobile imaging platform for digital influenza virus counting

“…33 Issadore et al described an integrated fluidic system with a smartphone-based imaging unit for solution handling and detection using the digital ELISA. 43 However, a swift and simple digital bioassay that would meet urgent needs for early stage disease detection has not yet been developed. Previously, we reported a digital influenza virus counting (DIViC) method based on a fluorogenic assay of neuraminidase activity of the influenza virus.…”

Microfluidic SlipChip device for multistep multiplexed biochemistry on a nanoliter scale

“…), especially when there are multiple steps. 6,[8][9][10][11][12][13][14] Although droplet-encapsulation methods 15 provide virtually unlimited scalability to the number of compartments, and have progressed greatly in high-throughput detection, [16][17][18] washing, 19 and sorting, 20 they have limitations with respect to reagent additions to the originally encapsulated volumes, imaging, and reaction parallelization.…”