Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs.

We have developed a bead-packed microfluidic device with a built-in flexible wall to automate extraction of nucleic acids from methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs. The flexible polydimethylsiloxane (PDMS) membrane was designed to manipulate the surface-to-volume ratio (SVR) of bead-packed chambers in the range of 0.05 to 0.15 (µm(-1)) for a typical solid phase extraction protocol composed of binding, washing, and eluting. In particular, the pneumatically assisted close packing of beads led to an invariant SVR (0.15 µm(-1)) even with different bead amounts (10-16 mg), which allowed for consistent operation of the device and improved capture efficiency for bacteria cells. Furthermore, vigorous mixing by asynchronous membrane vibration enabled ca. 90% DNA recovery with ca. 10 µL of liquid solution from the captured cells on the bead surfaces. The full processes to detect MRSA in nasal swabs, i.e., nasal swab collection, prefiltration, on-chip DNA extraction, and real-time polymerase chain reaction (PCR) amplification, were successfully constructed and carried out to validate the capability to detect MRSA in nasal swab samples. This flexible microdevice provided an excellent analytical PCR detection sensitivity of ca. 61 CFU/swab with 95% confidence interval, which turned out to be higher than or similar to that of the commercial DNA-based MRSA detection techniques. This excellent performance would be attributed to the capability of the flexible bead-packed microdevice to enrich the analyte from a large initial sample (e.g., 1 mL) into a microscale volume of eluate (e.g., 10 µL). The proposed microdevice will find many applications as a solid phase extraction method toward various sample-to-answer systems.

Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria.

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 µL or 20 µL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system.