No abstract

There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67 sec/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1 ng/µL or 105 genomes/µL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity.

A novel polymerase chain reaction (PCR) device was developed that uses wire-guided droplet manipulation (WDM) to guide a droplet over three different heating chambers. After PCR amplification, end-point detection is achieved using a smartphone-based fluorescence microscope. The device was tested for identification of the 16S rRNA gene V3 hypervariable region from Escherichia coli genomic DNA. The lower limit of detection was 103 genome copies per sample. The device is portable with smartphone-based end-point detection and provides the assay results quickly (15 min for a 30-cycle amplification) and accurately. The system is also shock and vibration resistant, due to the multiple points of contact between the droplet and the thermocouple and the Teflon film on the heater surfaces. The thermocouple also provides realtime droplet temperature feedback to ensure it reaches the set temperature before moving to the next chamber/step in PCR. The device is equipped to use either silicone oil or coconut oil. Coconut oil provides additional portability and ease of transportation by eliminating spilling because its high melting temperature means it is solid at room temperature.

PCR device amplifies DNA directly from tissue samples and achieves smartphone-based quantification at early thermal cycles.

Wire-guided droplet manipulation (WDM) is a simple method of manipulating liquid droplets in a hydrophobic environment to conduct experiments, reactions, and assays. In WDM, a wire (or needle tip) manipulates microlitersized liquid droplets within an immiscible liquid or on a hydrophobic surface. The attributes of this format for liquid handling address some of the challenges facing the use of conventional techniques. Specifically, WDM provides solutions for development of automated, sample-to-answer, point-of-care systems with potential applications in medicine, life science research, forensics, veterinary diagnostics, and disease control.The widespread applicability of this technique is due to its inherent simplicity stemming from the attractive force between the droplet and the wire. The physics of this interaction will be explained in this chapter. WDM can be applied to standard protocols and is easily reprogrammable for different liquid handling applications. Dilution, mixing, centrifugation, and thermocycling have all been automated by WDM (You and Yoon, J Biol Eng 6:15, 2012). If desired, the principles of droplet manipulation can be easily integrated into the common scientific automation strategy, using commercially available robotic pipetting systems. WDM is automatable, reprogrammable, easy to use, and robust. These are essential features of rapid, all-in-one, sample-to-answer systems to be used at the point-of-care.The applications of WDM within molecular biology that have been demonstrated include DNA extraction (lysing, precipitation, washing, and rehydration), nanoparticle surface deposition for fabrication of a protein nanoarray, immunoassay, PCR thermocycling, and real-time PCR.