Droplet Manipulation and Droplet Microfluidics for Rapid Amplification and Real-Time Detection of Nucleic Acids

摘要

Molecular diagnostics offer quick access to information for healthcare decision-making towards personalized therapeutics, but complicated procedures requiring extensive labor and infrastructure restrict their use. Droplet-based technologies can expand the accessibility of molecular diagnostics by miniaturizing devices, shortening sample-to-answer times, decreasing costs and increasing throughput. Methods for droplet manipulation are central to the automation of molecular diagnostics protocols. The innovative method, wire-guided droplet manipulation (WDM), is the actuation of liquid droplets in a hydrophobic milieu with a wire, or needle, guide. In this work, WDM is demonstrated for the automation of the polymerase chain reaction (PCR) on reprogrammable platforms for the diagnosis of cardiovascular infections. WDM is used to minimize thermal resistance by convective heat transfer for PCR amplification at a maximum speed of 8.67 s/cycle. The oil-water interfacial boundary is shown to passively partition molecular contaminants from sample matrices, including blood and heart valve tissue. Molecular self-assembly at the oil-water interface is used to increase PCR efficiency with blood in situ and is used as an innovative sensing modality for real-time monitoring of PCR amplification. Temperature feedback controlled droplet actuation is achieved by using a thermocouple loop as a functionalized wire-guide. Our novel methodology for real-time PCR, droplet-on-thermocouple silhouette real-time PCR (DOTS qPCR), utilizes interfacial effects to achieve droplet actuation, relief from PCR inhibitors and amplification sensing, for a sample-to-answer time as short as 3 min 30 s. DOTS qPCR addresses three major issues for rapid PCR—sample preparation, rapid thermocycling and sensitive real-time detection—on an inexpensive, disposable device with smartphone-based detection. In contrast, commercially available real-time PCR systems rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection we anticipate extending this technology towards trending biological research applications such as single cell, single nucleus, and single DNA molecule analyses, especially in droplet microfluidic platforms.