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 from n = 14 human subjects using our mobile duplex assay, and showed excellent agreement with the gold standard system ([Formula: see text]).

Keywords: ELISA; cell phone; digital; multiplex; portable.

Fig. 1.

Miniaturization and parallelization of droplet dELISA. (A) A schematic of the conventional workflow for dELISA, which requires multiple hands-on steps and is rate-limited by the serial partitioning of the sample into droplets and the serial detection of the fluorescence of each individual droplet. (B) The $\mathrm{\mu }$MD parallelizes droplet generation, incubation, and detection to miniaturize dELISA fully onto a mobile platform and increase its throughput by 100×. (C) Antibody-functionalized, color-coded beads are used in a duplex dELISA assay, wherein individual beads are encapsulated into droplets and read out if they have captured a single target protein.

Fig. 2.

Integrated $\mathrm{\mu }$MD workflow. (A) A schematic of the $\mathrm{\mu }$MD chip, showing both a top view and a bottom view. Each cartoon shows a schematic of the modules that are incorporated onto the $\mathrm{\mu }$MD. (B) A photograph of the disposable $\mathrm{\mu }$MD chip, with the channels filled with dye to make them visible. (C) A micrograph showing the droplet generator encapsulate microbeads into d = 40 $\mathrm{\mu }$m droplets. The arrows highlight the microbeads. (Scale bar = 50 $\mathrm{\mu }$m.) (D) A fluorescence micrograph of the droplets after the delay line. (Scale bar = 50 $\mathrm{\mu }$m.) (E) A schematic of the $\mathrm{\mu }$MD platform, consisting of a mobile phone, three light sources, and the disposable $\mathrm{\mu }$MD chip.

Fig. 3.

Software workflow for phase and velocity invariant optofluidic fluorescence droplet detection. (A) The algorithm for detecting droplets. (B) Truth table for interpreting the readout of the $\mathrm{\mu }$MD’s three-color (r, red ELISA signal; g, green beads; b, blue beads) fluorescence measurement. (C) Schematic showing the $\mathrm{\mu }$MD platform collecting data, which are sent to the cloud to be processed, and then returned to the mobile phone to report the results of the assay to the user. (D–F) A sample workflow for a droplet that contains a green bead and is positive for its target. (D) The video’s image frames are segmented into 1D vectors. (E) A 3D correlation results in a data matrix where the phase is first identified. (F) From this 2D “slice” of the data matrix, the velocity of the droplet is found. (G) The position is recorded for each peak in the correlation space.

Fig. 4.

Flow rate-invariant droplet generation and detection allow inexpensive, compact implementation of dELISA. (A) By using the Millipede geometry, droplet size is invariant to dispersed phase flow rate. (B) For a range of continuous flow rates (45 mL/h to 65 mL/h) and dispersed flow rates (2 mL/h to 14 mL/h), the generated droplets remained monodispersed with syringe pumps (CV = 5.3$\%$) and with inexpensive peristaltic pumps (CV = 6.0$\%$). (C) To evaluate the enzymatic amplification of captured protein in the droplets, we inspected the droplets after the delay line with fluorescence microscopy. (D) After a 3.2-min delay, the distribution of droplets positive and negative for enzyme were measured. (Scale bar for A and C = 50 $\mathrm{\mu }$m.)

Fig. 5.

Benchmarking and characterization of ultrasensitive, duplex protein detection in complex media. (A) Single-plex detection of GM-CSF spiked into PBS. The limit of detection LOD = 0.0045 pg/mL (320 aM). (B) Single-plex detection of IL6 spiked into PBS. LOD = 0.0070 pg/mL. (C) The same samples of FBS spiked with varying concentrations of GM-CSF were measured using the $\mathrm{\mu }$MD and Quanterix’s Simoa. Good agreement was found between the two measurements, $R^2$ = 0.95. (D) The LOD, LOQ, dynamic range, and CV are reported for the $\mathrm{\mu }$MD’s and Simoa’s measurement of GM-CSF in FBS. (E) The duplex assay is tested by measuring various concentrations of GM-CSF and IL6 spiked into FBS. (F) Varying concentrations of GM-CSF into FBS resulted in insignificant cross-talk with the measurement of IL6 and did not significantly change the LOD for GM-CSF. (G) Conversely, varying concentrations of IL6 into FBS resulted in insignificant cross-talk with the measurement of GM-CSF and did not significantly change the LOD for IL6. Insets for F and G show these measurements on a linear scale. (H) Twenty-two various concentrations of GM-CSF and IL6 were spiked into FBS and measured. Good agreement was found between the spiked and measured results, for both GM-CSF ($R^2=0.99$) and IL6 ($R^2=0.99$).

Fig. 6.

The measurement of endogenous protein in human serum. (A) Human serum was collected from n = 14 healthy controls, and an aliquot was measured using our $\mathrm{\mu }$MD’s duplex IL6, GM-CSF assay and was measured on Quanterix’s commercial assay. (B) Good agreement between Simoa and the $\mathrm{\mu }$MD was found for measurements of both IL6 and GM-CSF ($R^2=0.96$).