Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices

Abstract

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection.

Manufacturing of poly(ethylene glycol diacrylate)-based hollow microvessels using microfluidics

“…The viability of utilizing a photopolymerization reaction with microuidics to solidify polymer-based materials and mixtures has been investigated extensively by other groups. [48][49][50][51][52][53] Moreover, unlike the other standard microvessel devices, [36][37][38][39][40][41][42] our multiaxial microvessel system has been optimized by testing a variety of chevron dimensions under various ow rates.…”

Comparative analysis of the microbial community and nutritional quality of sufu

“…In this study, 5.0 g of sufu was mixed with 25 ml of phosphate buffer (pH 7.2). The mixture was centrifuged at 5,000 rpm for 10 min, and then, 5 ml of the supernatant was subjected to genomic DNA extraction using a DNA kit (Sangon Biotech, Shanghai) (Almeida et al., 2018 ; Knob et al., 2018 ). The extracted genomic DNA was separated through 1% agarose gel electrophoresis.…”

Point of care technologies for sepsis diagnosis and treatment

“…Lab-on-a-chip systems with integrated bacterial separation, chemical or mechanical cell lysis, and DNA extraction for PCR or sequence specific capture have shown promise for rapid sepsis diagnosis. [78][79][80] Artificial spleen type devices for the direct removal of pathogens and endotoxins from whole blood were developed for therapeutic applications ( Figure 4A). Similar to dialysis, these devices process whole blood by capturing a broad range of infectious agents using ligand and antibody coated magnetic beads, [81][82][83][84][85] porous membranes, 86 biomimetic cell margination, 87,88 acoustophoresis, 89,90 and elasto-inertial-based migration.…”