Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

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• 作者：André M. Ferreira ; Daniela Cruz-Moreira ; Laura Cerqueira…
• 关键词：PNA ; Fish ; Microfluidics ; Modelling ; Fluid mechanics ; Oxygen plasma treatment
• 刊名：Biomedical Microdevices
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：19
• 期：1
• 全文大小：
• 刊物类别：Engineering
• 刊物主题：Biomedical Engineering; Biological and Medical Physics, Biophysics; Nanotechnology; Engineering Fluid Dynamics;
• 出版者：Springer US
• ISSN：1572-8781
• 卷排序：19

文摘

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.