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

Significance Digital assays have enormous untapped potential for diagnostics, environmental surveillance, and biosafety monitoring, but are currently confined to laboratory settings due to the instrumentation necessary to generate, control, and measure millions of droplets. We instead use a mobile phone-based imaging technique that is >100× faster than conventional microfluidic droplet detection, does not require expensive optics, is invariant to flow rate, and can simultaneously measure multiple fluorescent dyes in droplets. By using this time domain modulation with cloud computing, we overcome the low frame rate of digital imaging, and achieve throughputs as high as 1 million droplets per second. We integrate on-chip delay lines and a microbead processing unit, resulting in a robust device, suitable for low-cost implementation, with ultrasensitive measurement capabilities. 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 (R2=0.96).