Validation of a centrifugal microfluidic sample lysis and homogenization platform for nucleic acidextraction with clinical samples

Abstract

The applications of microfluidic technologies in medical diagnostics continue to increase, particularly in the field of nucleic acid diagnostics. While much attention has been focused on the development of nucleic acid amplification and detection platforms, sample preparation is often taken for granted or ignored all together. Specifically, little or no consideration is paid to the development of microfluidic systems that efficiently extract nucleic acids from biological samples. Here, a centrifugal microfluidic platform for mechanical sample lysis and homogenization is presented. The system performs sample lysis through a magnetically actuated bead-beating system followed by a centrifugal clarification step. The supernatant is then transferred for extraction using a unique siphon. Several other new microfluidic functions are implemented on this centrifugal platform as well, including sample distribution, a unique hydraulic capillary valve, and self-venting. Additionally, the improved system has features with a small footprint designed specifically for integration with further downstream processing steps. Biological validation of the platform is performed using Bacillus subtilis spores and clinical samples (nasopharyngeal aspirates) for respiratory virus detection. The platform was found to be as efficient as in-tube bead-beating lysis and homogenization for nucleic acid extraction, and capable of processing 4 samples in batch to near PCR-ready products in under 6 min.

Whole Blood Immunoassay Based on Centrifugal Bead Sedimentation

“…Extensive development efforts have yielded multiple types of immunoassay applications, including simple modifications of data compact discs, sophisticated assay platforms, and printed protein arrays (4 -11 ). In addition, there has been substantial progress toward centrifugal systems with self-contained sample processing capabilities for detection of both DNA/RNA and protein (12)(13)(14)(15)(16)(17). In a notable recent example, researchers at the Samsung Advanced Institute of Technology developed a fully integrated, portable immunoassay platform capable of directly processing whole blood (11 )…”

Contactless microfluidic pumping using microchannel-integrated carbon black composite membranes

“…2,3 For example, microfluidic large scale integration (LSI) uses microchannel networks with integrated pressure-actuated elastomeric pumps and valves to control, route, and mix fluid in channels for on-chip genetic analysis of single cells. 4 Others have used centrifugal forces, 5 surface acoustic waves (SAW), 6 and electrical forces to perform fluidic metering mixing and aliquoting for applications including DNA extraction, plasma separation, 7 cell lysis, 8 protein crystallization, 9 immunoassays, 10 and single cell analysis. 11 The majority of these microfluidic platforms, however, require large and often expensive external actuation equipment for operation, which can confine their usefulness to the laboratory a) Author to whom correspondence should be addressed.…”

“…LSI, for example, utilizes thousands of elastomeric pump and valve components for precise and local fluid manipulation, but requires bulky external computer controlled vacuum or pressure sources for component actuation. Centrifugal-based microfluidics 5 can drive fluidic routing, mixing, and metering on rotating substrates with on-chip valves and channels, but the motor required for actuation is expensive, and this platform lacks flexibility; the spinning chip cannot be interfaced with external electrical readouts or sensors. Platforms based on SAW 12 and electrical forces 13 offer an effective means to transport liquid on-chip, but require expensive and often large electrical equipment.…”

Theory, fabrication and applications of microfluidic and nanofluidic biosensors

“…Often high temperatures and/or large electric fields may be needed and therefore fabrication methods for biomaterials have to be adapted to meet the device-bonding requirements. Polymers are attractive materials for substrates because of their low cost, disposability and ability to adapt to several biomaterial and diagnostic applications [113]. For example, a polymer-based disposable device capable of detecting 0.25-10 ppm of anaesthetic propofol within 60 s has been already demonstrated (figure 5) [114].…”