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 100mL sample and elution of target DNA in 12min total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron Mediated Metallo b lactamase it was possible to extract and detect a 1pM 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.
Journal of chromatography. A