A microfluidic system integrated with buried optical fibers for detection of Phalaenopsis orchid pathogens $ Chih-Lin Lin a , Wen-Hsin Chang a , Chih-Hung Wang a , Chia-Hwa Lee d , Tzong-Yueh Chen e , Fuh-Jyh Jan d,n , Gwo-Bin Lee a,b,c,nn a Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan b Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan c Institute of NanoEngineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan d Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan e Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan article info Article history: Received 19 May 2014 Received in revised form 25 July 2014 Accepted 11 August 2014 Available online 17 August 2014 Keywords: Microfluidics Optical fibers Phalaenopsis orchid Pathogen detection Reverse transcription loop-mediated isothermal amplification abstract Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time- consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25 fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 min. & 2014 Elsevier B.V. All rights reserved. 1. Introduction Economically valuable orchids are threatened on a global scale by a variety of pathogenic organisms. Therefore, there is an urgent need to rapidly and accurately detect orchid pathogens prior to their infecting a larger proportion of the population. Viruses are among the most significant pathogens of orchids of the genus Phalaenopsis, which are an important agricultural species in Taiwan, in particular. Traditionally, immune-interactions (Eun and Wong, 1999), polymerase chain reaction (PCR) (Seoh et al., 1998), and enzyme-linked immunosorbent assays (ELISA) (Arimoto et al., 1992) have been the most common detection methods for viruses in Phalaenopsis spp. samples. However, these methods typically require time-consuming processing steps, spe- cialized equipment, and well-trained personnel; therefore, they are poorly suited for rapid, accurate, and sensitive on-site detec- tion. Furthermore, experienced pathologists may not be available Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics http://dx.doi.org/10.1016/j.bios.2014.08.013 0956-5663/& 2014 Elsevier B.V. All rights reserved. Abbreviations: CaCV, capsicum chlorosis tospo virus; DNA, deoxyribonucleic acid; EMVs, electromagnetic valves; ELISA, enzyme-linked immunosorbent assays; LAMP, loop-mediated isothermal amplification; LOD, limit of detection; PCR, polymerase chain reaction; PDMS, polydimethylsiloxane; RT-LAMP, reverse-tran- scription loop-mediated isothermal amplification; RNA, ribonucleic acid; ag, attograms; bp, base-pair; ddH2O, double-distilled water; fg, femtograms; ng, nanograms; pg, picograms ☆ The preliminary results of this study have been presented in the 17th Interna- tional Conference on Solid-State Sensors, Actuators and Microsystems (Transducers 2013 & Eurosensors XXVII). n Corresponding author. Tel.: þ886 4 22840780x360; fax: þ886 4 22854145. nn Corresponding author at: Department of Power Mechanical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan. Tel.: þ886 3 5715131x33765; fax: þ886 3 5722840. E-mail addresses: fjjan@nchu.edu.tw (F.-J. Jan), gwobin@pme.nthu.edu.tw (G.-B. Lee). Biosensors and Bioelectronics 63 (2015) 572–579