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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 116; no. 10; pp. 4489 - 4495
• Main Authors: Yelleswarapu, Venkata, Buser, Joshua R., Haber, Margalit, Baron, Jonathan, Inapuri, Eshwar, Issadore, David
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 05.03.2019
• Series: PNAS Plus
• Subjects: Assaying; Biological properties; Biological samples; Biological Sciences; Biomarkers; Coding; Complex media; Control equipment; Delay lines; Droplets; Enzyme-Linked Immunosorbent Assay; Fluorescence; Granulocyte-macrophage colony-stimulating factor; Granulocyte-Macrophage Colony-Stimulating Factor - blood; Humans; Inflow; Instrumentation; Integration; Interleukin 6; Interleukin-6 - blood; Laboratories; Microfluidic Analytical Techniques; Parallel operation; Physical Sciences; PNAS Plus; Point-of-Care Systems; Proteins; digital; portable; multiplex; cell phone; ELISA
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1814110116

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).