Nucleic Acid Extraction Method and Microfluidic Device for Molecular Diagnostics

Abstract

A novel bacterial DNA extraction method with microfluidic device has been developed for molecular diagnostics. In order to incorporate the conventional complex DNA sample preparation processes into microdevice, a solid phase DNA extraction composed of bacterial cell capture, washing, in-situ lysis, DNA elution was attempted, and it demonstrated that the full process of pathogen capture to DNA isolation from various human specimens could be automated in a single microchip. At first, to facilitate the bacterial cell capture onto the solid surface of flow-through microdevice, the thermodynamically-favorable conditions for bacterial adhesion such as surface tension of solid surface and medium pH were optimized. The 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 E. coli, S. epidermis and S. mutans in buffer solution was over 75% with a flow rate of 400 μL/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 polymerase chain reaction (PCR). Next, in order to manufacture a low-cost, disposable microchip, micropillar arrays of high surface-to-volume ratio (SVR, 0.152 μm-1) were constructed on polymethyl methacrylate (PMMA) by hot embossing with an electroformed Ni mold, and their surface was modified with SiO2 and an organosilane compound in subsequent steps. To seal open microchannels, the organosilane layer on top plane of the micropillars was selectively removed through photocatalytic oxidation via TiO2/UV treatment at room temperature. As a result, the underlying SiO2 surface was exposed without deteriorating the organosilane layer coated on lateral surface of the micropillars that could serve as bacterial cell adhesion moiety. Afterwards, a plasma-treated polydimethylsiloxane (PDMS) substrate was bonded to the exposed SiO2 surface, completing the device fabrication. To optimize manufacturing throughput and process integration, the whole fabrication process was performed at 6 inch wafer-level including polymer imprinting, organosilane coating, and bonding. Preparation of bacterial DNA was carried out from urine samples with the fabricated PDMS/PMMA chip according to the suggested procedure: bacterial cell capture, washing, in-situ lysis, and DNA elution. The polymer-based microchip presented here demonstrated similar performance to Glass/Si chip in terms of bacterial cell capture efficiency and PCR compatibility. And then, a miniaturized bead-beating device has been developed to automate nucleic acids extraction from gram-positive bacteria. The microfluidic device was fabricated by sandwiching a monolithic flexible 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 (MRSA), were captured on surface-modified glass beads from 1 mL of initial sample solution and in-situ lyzed by bead-beating operation. Then,10 L or 20 L of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume faction 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 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. Finally, such a bead-packed microfluidic device with a built-in flexible wall was further applied to automate extraction of nucleic acids from MRSA in nasal swab. The flexible PDMS membrane was designed to manipulate the SVR of bead-packed chamber 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 swab, i.e., nasal swab collection, pre-filtration, on-chip DNA extraction, and real-time 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.