This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL-1 level from a 5 μL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

The Democratic Republic of the Congo (DRC) has a high measles incidence despite elimination efforts and has yet to introduce rubella vaccine. We evaluated the performance of a prototype rapid digital microfluidics powered (DMF) enzyme-linked immunoassay (ELISA) assessing measles and rubella infection, by testing for immunoglobulin M (IgM), and immunity from natural infection or vaccine, by testing immunoglobulin G (IgG), in outbreak settings. Field evaluations were conducted during September 2017, in Kinshasa province, DRC. Blood specimens were collected during an outbreak investigation of suspected measles cases and tested for measles and rubella IgM and IgG using the DMF-ELISA in the field. Simultaneously, a household serosurvey for measles and rubella IgG was conducted in a recently confirmed measles outbreak area. DMF-ELISA results were compared with reference ELISA results tested at DRC’s National Public Health Laboratory and the US Centers for Disease Control and Prevention. Of 157 suspected measles cases, rubella IgM was detected in 54% while measles IgM was detected in 13%. Measles IgG-positive cases were higher among vaccinated persons (87%) than unvaccinated persons (72%). In the recent measles outbreak area, measles IgG seroprevalence was 93% overall, while rubella seroprevalence was lower for children (77%) than women (98%). Compared with reference ELISA, DMF-ELISA sensitivity and specificity were 82% and 78% for measles IgG; 88% and 89% for measles IgM; 85% and 85% for rubella IgG; and 81% and 83% for rubella IgM, respectively. Rubella infection was detected in more than half of persons meeting the suspected measles case definition during a presumed measles outbreak, suggesting substantial unrecognized rubella incidence, and highlighting the need for rubella vaccine introduction into the national schedule. The performance of the DMF-ELISA suggested that this technology can be used to develop rapid diagnostic tests for measles and rubella.

Microfluidic sample manipulation is a key enabler in modern chemical biology research. Both discrete droplet‐based digital microfluidic (DMF) assays and continuous flow in‐channel assays are well established, each featuring unique advantages from the viewpoint of automation and parallelization. However, there are marked differences in the applicable microfabrication materials and methods, which limit the interfacing of DMF sample preparation with in‐channel separation systems, such as the gold standard microchip electrophoresis. Simultaneously, there is an increasing demand for low‐cost and user‐friendly manufacturing techniques to foster the adaptation of microfluidic technology in routine laboratory analyses. This work demonstrates integration of DMF with in‐channel separation systems using only low‐cost and accessible (non‐cleanroom) manufacturing techniques, i.e., inkjet printing of silver for patterning of the driving electrodes and UV curing of off‐stoichiometric thiol–ene (OSTE) polymers both for dielectric coating of the electrode arrays and replica molding of the microchannel network. As a dielectric, OSTE performs similar to Parylene C (a gold standard dielectric in electrowetting), whereas its tunable surface and bulk properties facilitate straightforward bonding of the microchannel with the dielectric layer. In addition, a new chip design that facilitates efficient droplet transfer from the DMF part to the microchannel inlet solely by electrowetting is showcased.

Finger-stick blood sampling is convenient for point of care diagnostics, but whole blood samples are problematic for many assays because of severe matrix effects associated with blood cells and cell debris. We introduce a new digital microfluidic (DMF) diagnostic platform with integrated porous membranes for blood-plasma separation from finger-stick blood volumes, capable of performing complex, multi-step, diagnostic assays. Importantly, the samples can be directly loaded onto the device by a finger "dab" for user-friendly operation. We characterize the platform by comparison to plasma generated via the "gold standard" centrifugation technique, and demonstrate a 21-step rubella virus (RV) IgG immunoassay yielding a detection limit of 1.9 IU mL(-1), below the diagnostic cut-off. We propose that this work represents a critical next step in DMF based portable diagnostic assays-allowing the analysis of whole blood samples without pre-processing.

Serosurveys are useful for assessing population susceptibility to vaccine-preventable disease outbreaks. Although at-risk populations in remote areas could benefit from this type of information, they face several logistical barriers to implementation, such as lack of access to centralized laboratories, cold storage, and transport of samples. We describe a potential solution: a compact and portable, field-deployable, point-of-care system relying on digital microfluidics that can rapidly test a small volume of capillary blood for disease-specific antibodies. This system uses inexpensive, inkjet-printed digital microfluidic cartridges together with an integrated instrument to perform enzyme-linked immunosorbent assays (ELISAs). We performed a field validation of the system’s analytical performance at Kakuma refugee camp, a remote setting in northwestern Kenya, where we tested children aged 9 to 59 months and caregivers for measles and rubella immunoglobulin G (IgG). The IgG assays were determined to have sensitivities of 86% [95% confidence interval (CI), 79 to 91% (measles)] and 81% [95% CI, 73 to 88% (rubella)] and specificities of 80% [95% CI, 49 to 94% (measles)] and 91% [95% CI, 76 to 97% (rubella)] (measles, n = 140; rubella, n = 135) compared with reference tests (measles IgG and rubella IgG ELISAs from Siemens Enzygnost) conducted in a centralized laboratory. These results demonstrate a potential role for this point-of-care system in global serological surveillance, particularly in remote areas with limited access to centralized laboratories.

Microfluidics has become an important tool that is useful for a wide range of applications. A drawback for microfluidics is that many of the techniques that are commonly used to fabricate devices are not widely accessible, not scalable to high‐volume manufacturing processes, or both. Recently, a number of printing strategies that were originally developed for other applications have been applied to microfluidic device fabrication. These techniques, which include inkjet printing (IJP), screen printing (SP), and solid wax printing (SWP), are proposed to have a transformative effect on the field. Here microfluidics and printing, are introduced and a list of favorite examples is provided that highlights the accessibility and scalability that the combination is bringing to the field.

Microfluidic platforms are an attractive option for incorporating complex fluid handling into low-cost and rapid diagnostic tests. A persistent challenge for microfluidics, however, is the mismatch in the "world-to-chip" interface - it is challenging to detect analytes present at low concentrations in systems that can only handle small volumes of sample. Here we describe a new technique termed pre-concentration by liquid intake by paper (P-CLIP) that addresses this mismatch, allowing digital microfluidics to interface with volumes on the order of hundreds of microliters. In P-CLIP, a virtual microchannel is generated to pass a large volume through the device; analytes captured on magnetic particles can be isolated and then resuspended into smaller volumes for further processing and analysis. We characterize this method and demonstrate its utility with an immunoassay for Plasmodium falciparum lactate dehydrogenase, a malaria biomarker, and propose that the P-CLIP strategy may be useful for a wide range of applications that are currently limited by low-abundance analytes.

The diagnosis of infectious disease is typically carried out at the point-of-care (POC) using the lateral flow assay (LFA). While cost-effective and portable, LFAs often lack the clinical sensitivity and specificity required for accurate diagnoses. In response to this challenge, we introduce a new digital microfluidic (DMF) platform fabricated using a custom inkjet printing and roll-coating process that is scalable to mass production. The performance of the new devices is on par with that of traditional DMF devices fabricated in a cleanroom, with a materials cost for the new devices of only US $0.63 per device. To evaluate the usefulness of the new platform, we performed a 13-step rubella virus (RV) IgG immunoassay on the inkjet printed, roll-coated devices, which yielded a limit of detection of 0.02 IU mL-1, well below the diagnostic cut-off of 10 IU mL-1 for RV infection and immunity. We propose that this represents a breakthrough for DMF, lowering the costs to a level such that the new platforms will be an attractive alternative to LFAs for the diagnosis of infectious disease at the POC.

We present the fabrication and use of the first microfluidic device with integrated fluid handling capabilities coupled to ambient ionization for mass spectrometry. Devices are fabricated using an inkjet printing process to deposit silver nanoparticle ink on a paper substrate. Paper spray ionization emitters are formed using an adhesive contact mask to remove the substrate’s hydrophobic coating and expose the hydrophilic surface needed for paper spray mass spectrometry. A benzoylecgonine standard was analyzed on device. Product ion peaks at m/z 168 and m/z 272 were observed.