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

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 mu L sample and elution of target DNA in 12 min total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron-Mediated Metallo-beta-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. (C) 2017 Elsevier B.V. All rights reserved.