A digital microfluidic system for serological immunoassays in remote settings by Alphonsus H. C. Ng, Ryan Fobel, Christian Fobel, Julian Lamanna, Darius G. Rackus, Aimee Summers, Christopher Dixon, Michael D. M. Dryden, Charis Lam, Man Ho, Nooman S. Mufti, Victor Lee, Mohd Afiq Mohd Asri, Edward A. Sykes, M. Dean Chamberlain, Rachael Joseph, Maurice Ope, Heather M. Scobie, Alaine Knipes, Paul A. Rota, Nina Marano, Paul M. Chege, Mary Njuguna, Rosemary Nzunza, Ngina Kisangau, John Kiogora, Michael Karuingi, John Wagacha Burton, Peter Borus, Eugene Lam, and Aaron R. Wheeler Sci Transl Med Volume 10(438):eaar6076 April 25, 2018 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 1 Digital microfluidic cartridge and ELISA used for measles and rubella testing. Digital microfluidic cartridge and ELISA used for measles and rubella testing. (A) Isometric-view schematic of the DMF top and bottom plates assembled to form a cartridge. Inset: Cross-section image (not to scale) of a complete device bearing inkjet-printed electrodes (black) coated with dielectric (purple) and hydrophobic (yellow) layers. The flexible printing media (peach) substrate is affixed to a glass slide via double-sided adhesive tape (green). ITO, indium tin oxide. (B) Photograph of silver DMF electrodes printed on a flexible substrate using a commercial inkjet printer. (C) Top-view schematic of the DMF device with 92 driving electrodes and 10 reservoir electrodes. (D) Cartoon schematic of measles and rubella ELISAs. Paramagnetic particles coated with measles or rubella virus antigens are incubated with sample. Anti-measles or anti-rubella IgG (red) binds to the particles. Particles are washed and then incubated with anti-human IgG–horseradish peroxidase conjugate (purple). The particles are washed again and then exposed to a mixture of luminol and H2O2 (yellow-green). Enzymatic turnover of the product generates a chemiluminescent product (yellow). (E) Photographs of two assays performed in parallel (that is, “twoplex”) on a DMF cartridge (dyes are added to enhance droplet visibility; black arrows indicate the direction of droplet movement). (1) Droplets of particle suspension are dispensed; (2) blood samples are dispensed and merged with the particles; (3) particles are immobilized by activating the magnetic lens, and supernatant droplets are removed to an absorbent wick; (4) particles are washed in wash buffer (blue); (5) antibody conjugate (purple) is dispensed and incubated with the particles; (6) after further wash steps, droplets of luminol (yellow) and H2O2 (green) are dispensed, mixed, and (7) split; (8) particles are incubated with luminol and H2O2; and (9) particles are moved to the detection zone. Alphonsus H. C. Ng et al., Sci Transl Med 2018;10:eaar6076 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 2 Portable MR Box. Portable MR Box. (A) Photograph of the MR Box with transparent sides showing the photomultiplier tube (PMT), lens, webcam, and temperature and humidity sensors. The acrylic housing has a footprint of 25 cm × 20 cm. (B) Photograph showing detail of a DMF device being inserted into the MR Box interface. The DMF device sits atop a motorized magnetic lens and interfaces with the control system via pogo pin connectors. (C) Photograph (front view; transparent panels) of the MR Box showing the PMT, webcam, servo motor, switching boards, and high-voltage amplifier. With the lid closed, the MR Box measures 28 cm tall. (D) Table of innovations that are new to the MR Box hardware (see figs. S3 to S6) and software (see figs. S7 to S10), developed in preparation for the field tests. Alphonsus H. C. Ng et al., Sci Transl Med 2018;10:eaar6076 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 3 MR Box assay development and optimization in the laboratory. MR Box assay development and optimization in the laboratory. (A) Calibration curves generated from measurements of standards (filled circles) of measles IgG (left) and rubella IgG (right; inset: four lowest concentrations); error bars represent ±1 SD, n = 4 per condition. The coefficient of variance (CV) was <20% for each of the 10 conditions evaluated, except for the measles IgG (6.35 mIU/ml) measurement, which had CV of 27.5%. Curves were fitted (red lines) using a four-parameter logistic equation (measles, R2 = 0.9998; rubella, R2 = 0.9991) (B) Vertical scatterplots of serum panels tested in the laboratory for measles IgG, n = 8 (left) and rubella IgG, n = 20 (right). Green and red numbers in the scatterplots represent the number of samples correctly and incorrectly categorized by the MR Box, respectively. The inset plots show ROC curves for MR Box assays (red lines) and random guesses (dashed lines). The area under the curve (AUC) is reported in each plot. Alphonsus H. C. Ng et al., Sci Transl Med 2018;10:eaar6076 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 4 MR Box field trial in Kakuma refugee camp, Kenya. MR Box field trial in Kakuma refugee camp, Kenya. (A) Vertical scatterplot (left) of MR Box signals for samples determined to be positive (Pos) or negative (Neg) for anti-measles IgG by reference tests (n = 140). ROC curve (right) with AUC of 0.86 and threshold (X) selected for sensitivity of 86% (95% CI, 79 to 91%), specificity of 80% (95% CI, 44 to 97%), and overall agreement of 86% (95% CI, 79 to 91%) are shown. (B) Vertical scatterplot (left) of MR Box signals for samples determined to be positive or negative for anti-rubella IgG by reference tests (n = 135). ROC curve (right) with AUC of 0.90 and threshold (X) selected for sensitivity of 81% (95% CI, 72 to 88%), specificity of 91% (95% CI, 75 to 98%), and overall agreement of 84% (95% CI, 76 to 90%) are shown. Green and red numbers in the scatterplots represent the number of samples correctly and incorrectly categorized by the MR Box, respectively. Alphonsus H. C. Ng et al., Sci Transl Med 2018;10:eaar6076 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works