title: Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).
abstract: Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.
authors.affiliation: LEPABE- Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.|CEFT-Transport Phenomena Research Center, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.
authors.forename: André M
authors.initials: AM
authors.lastname: Ferreira
authors.affiliation: LEPABE- Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.|CEFT-Transport Phenomena Research Center, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cruz-Moreira
authors.affiliation: LEPABE- Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.|Biomode 2, S.A.-Edifício GNRation, Praça Conde de Agrolongo, n°, 123 4700-312, Braga, Portugal.
authors.forename: Laura
authors.initials: L
authors.lastname: Cerqueira
authors.affiliation: CEFT-Transport Phenomena Research Center, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal.
authors.forename: João M
authors.initials: JM
authors.lastname: Miranda
authors.affiliation: LEPABE- Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering of University of Porto, Rua Dr. Roberto Frias, s, /n 4200-465, Porto, Portugal. nazevedo@fe.up.pt.
authors.forename: Nuno F
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D004867:Equipment Design; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D010100:Oxygen / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D058626:Plasma Gases / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D020135:Peptide Nucleic Acids; D058626:Plasma Gases; D010100:Oxygen
keywords: Fluid mechanics; Microfluidics; Modelling; Oxygen plasma treatment; PNA-Fish