3D-Printed Modular Microfluidic Device Enabling Preconcentrating Bacteria and Purifying Bacterial DNA in Blood for Improving the Sensitivity of Molecular Diagnostics.

Abdurhaman Teyib Abafogi,Jaewon Kim,Danny Van Noort,Jinyeop Lee,Sungsu Park,Merem Omer Mohammed

doi:10.3390/s20041202

Abstract

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3DpmμFD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3DpmμFD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

Highlights

Sepsis is a life-threatening immune response caused by a bacterial infection in blood [1,2,3], causing approximately 6 million deaths worldwide each year [4]
At 4 s, bacterium-antibody conjugated magnetic nanoparticles (Ab-magnetic nanoparticles (MNPs)) complexes in the W-shaped microchannel were attached at higher concentrations to the left side of the second curve than in other regions due to the rate reduction occurred at 2 s (Figure 3a, iii), whereas those in the helical microchannel were attached along its inner wall of the channel, evenly spaced at a relatively low concentration, unlike in the case of W-shaped microchannel (Figure 3b, iii)
At 6 s, most of the bacterium-Ab-MNP complexes in the W-type microchannel were attached to the channel wall and the remaining small amount of bacterial -Ab-MNP complexes were moved to the outlet (Figure 3a, iv)

Summary

Introduction

Sepsis is a life-threatening immune response caused by a bacterial infection in blood [1,2,3], causing approximately 6 million deaths worldwide each year [4]. Molecular diagnostics based on gene amplification is rapid and accurate for detection of microorganisms [6,7]. GDNA purification methods can improve the limit of detection (LOD) of molecular diagnostics for sepsis by providing purified gDNA at high concentrations [11] for use with polymerase chain reaction (PCR) and quantitative PCR (qPCR). Existing purification methods include alkaline extraction [12], gradient centrifugation [13], and magnetic silica bead (MSB)-based gDNA extraction [14]. Among these methods, MSB-based gDNA extraction has been frequently used because of its simplicity [15]

Methods

Results

Conclusion