Bacterial DNA Sample Preparation from Whole Blood Using Surface-Modified Si Pillar Arrays

Abstract

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.

Low-cost polymer microfluidic device for on-chip extraction of bacterial DNA

“…Experimental setup of microfluidic test module has been described in our previous report [24]. After washing with 1×PBS buffer (pH 7.4) twice, E. coli was diluted to concentration of 1 × 10 7 CFU/mL in sodium acetate buffer at pH 4, and introduced to the fabricated PDMS/Si chip.…”

“…Furthermore, the device functionality should be intact while performing the given bonding process. For example, the presence of ultra-thin organosilane (tridecafluoro-1,1,2,2-tetrahydrooctyl trimethoxysilane) film, which is an indispensable element for bacterial cell capture [24], restricts direct application of most conventional thermoplastic bonding techniques (i.e. adhesive, solvent, thermal bonding) because they can deteriorate integrity of organosilane and therefore lead to negative bioanalytical activities in terms of bacterial cell capture efficiency and PCR compatibility.…”

“…It was previously reported that bacterial DNA preparation can be done on microchips with surface-modified Si micropillars [24,25]. Bacterial cells can be selectively captured on them from buffer suspension or whole blood by optimizing surface tension of solid substrate and media pH based on bacterial adhesion thermodynamics [26].…”

“…66-69 erg/cm 2 ) [26]. Organosilane containing functional groups reactive to SiO 2 surface was chosen for controlling surface tension of the solid substrate in our previous report [24]. While performance of this approach was found to be equivalent to that of a commercial DNA extraction kit, the microchip was produced from expensive glass and silicon substrates using conventional photolithography processes including patterning, deep reactive-ion-etching and high temperature anodic bonding.…”

Enrichment of diluted cell populations from large sample volumes using 3D carbon-electrode dielectrophoresis

“…Hwang et al employed surface-modified micropillar arrays to extract Escherichia coli from up to 50% whole blood at concentrations above 10 3 cells/ml. 16 This approach required tailoring the pH of the sample to optimize trapping. They demonstrated high capture efficiency as long as the cell concentration was below 10 7 cells/ml due to saturation of the array.…”