Rapid and simple pressure-sensitive adhesive microdevice fabrication for sequence-specific capture and fluorescence dete
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RESEARCH PAPER
Rapid and simple pressure-sensitive adhesive microdevice fabrication for sequence-specific capture and fluorescence detection of sepsis-related bacterial plasmid gene sequences Yesman Akuoko 1 & Robert L. Hanson 1 & David H. Harris 1 & Jacob B. Nielsen 1 & Elaine Lazalde 1 & Adam T. Woolley 1 Received: 22 September 2020 / Revised: 6 November 2020 / Accepted: 10 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Microbial resistance to currently available antibiotics poses a great threat in the global fight against infections. An important step in determining bacterial antibiotic resistance can be selective DNA sequence capture and fluorescence labeling. In this paper, we demonstrate the fabrication of simple, robust, inexpensive microfluidic devices for DNA capture and fluorescence detection of a model antibiotic resistance gene sequence. We laser micromachined polymethyl methacrylate microchannels and enclosed them using pressure-sensitive adhesive tapes. We then formed porous polymer monoliths with DNA capture probes in these microchannels and used them for sequence-specific capture, fluorescent labeling, and laser-induced fluorescence detection of picomolar (pM) concentrations of synthetic and plasmid antibiotic resistance gene targets. The relative fluorescence for the elution peaks increased with loaded target DNA concentration. We observed higher fluorescence signal and percent recovery for synthetic target DNA compared to plasmid DNA at the same loaded target concentration. A non-target gene was used for control experiments and produced < 3% capture relative to the same concentration of target. The full analysis process including device fabrication was completed in less than 90 min with a limit of detection of 30 pM. The simplicity of device fabrication and good DNA capture selectivity demonstrated herein have potential for application with processes for bacterial plasmid DNA extraction and single-particle counting to facilitate determination of antibiotic susceptibility. Keywords Microfluidics . Laser micromachining . Rapid prototyping . Porous polymer monoliths . DNA analysis
Introduction Microbial resistance to antibiotics is a major threat to global healthcare [1, 2] with an estimated 10 million deaths and economic impact of over 1 trillion US dollars by the year 2050 [3]. Effective treatment of these infections requires knowledge of what antibiotics the bacteria are susceptible to, through antibiotic susceptibility tests (ASTs). Genotypic and phenotypic ASTs are currently used in detecting drug-resistant bacteria. Phenotypic ASTs provide specific resistance data but require hours to several days for bacterial growth, identification, and susceptibility testing, delaying effective antimicrobial therapy [4, 5]. Genotypic ASTs can be faster but require * Adam T. Woolley [email protected] 1
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA
advance knowledge of the gene, utilize expensive equipment, and often need bacterial
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