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.

Electrochemical cell lysis device for DNA extraction.

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC) applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH(-)) at the cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device design, were two important parameters of the device performance. After optimizing the design and visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR) analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid processing. The device could potentially be applied as an on-chip DNA extraction component.

Bacterial DNA sample preparation from whole blood using surface-modified Si pillar arrays.

A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip.

Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance.

We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage deduced from QCM measurement and adopted a complementary fluorescence displacement method. Direct comparison of surface coverage deduced from frequency change in QCM measurement and determined by the fluorescence exchange reaction revealed that QCM results are highly overestimated and the amount of overestimation strongly depends on the type of buffer and the structure of the film. Discrimination capability of the surface attached 15-mer probe was also examined using a single-base mismatched target at various hybridization temperatures. Hybridization efficiency depending on the type of single base mismatch was investigated using surface plasmon resonance (SPR).