Sequence-specific DNA solid-phase extraction in an on-chip monolith: Towards detection of antibiotic resistance genes

Abstract

Antibiotic resistance of bacteria is a growing problem and presents a challenge for prompt treatment in patients with sepsis. Currently used methods rely on culturing or amplification; however, these steps are either time consuming or suffer from interference issues. A microfluidic device was made from black polypropylene, with a monolithic column modified with a capture oligonucleotide for sequence selective solid-phase extraction of a complementary target from a lysate sample. Porous properties of the monolith allow flow and hybridization of a target complementary to the probe immobilized on the column surface. Good flow through properties enable extraction of a 100 μL sample and elution of target DNA in 12 minutes total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron-Mediated Metallo-β-lactamase it was possible to extract and detect a 1 pM sample with 83% recovery. Temperature-mediated elution by heating above the duplex melting point provides a clean extract without any agents that interfere with base pairing, allowing various labeling methods or further downstream processing of the eluent. Further integration of this extraction module with a system for isolation and lysis of bacteria from blood, as well as combining with single-molecule detection should allow rapid determination of antibiotic resistance.

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

“…For Schiff-base immobilization of DNA, the monolith was fabricated as described in [18]. In brief, the device was filled with a polymerization mixture containing 24% GMA, 16% PEGDA, 40% 1-propanol and 20% 1,4-butanediol with 3% BME photoinitiator (all w/w).…”

“…We used the same setup as in previous work [18]. Briefly, monolith-modified devices were connected to a Fluigent (Lowell, MA) pressure-driven pumping and valving system.…”

“…Heating of the monolith section of the device was performed with a thermoelectric module (TE-23-1.0-1.3P, TE Technology, Traverse City, MI) operated by a temperature controller (TC-48-20, TE Technology) and equipped with control thermistors (MP-2444, TE Technology). Device temperature was approximated from the previously observed difference between the heater thermistor temperature and a thermistor on the opposite side of the device [18]. The confocal laser-induced fluorescence setup probed a point 2 mm beyond the monolith as described in prior publications [18,22] and shown in Figure 1A.…”

“…Device temperature was approximated from the previously observed difference between the heater thermistor temperature and a thermistor on the opposite side of the device [18]. The confocal laser-induced fluorescence setup probed a point 2 mm beyond the monolith as described in prior publications [18,22] and shown in Figure 1A. Hybridization buffer was 20 mM Tris-HCl pH 8 with 500 mM NaCl and 50 mM MgCl 2 .…”

“…In previous work [18] we demonstrated the applicability of porous polymer monoliths prepared in microfluidic channels and modified with oligonucleotides using the Schiff-base method, for sequence-specific capture of complementary oligonucleotides. Here, we demonstrate single-step fabrication of monoliths functionalized with a capture oligonucleotide in a microfluidic device.…”