PAPER IN FOREFRONT Microfluidic biosensor for cholera toxin detection in fecal samples Natinan Bunyakul & Chamras Promptmas & Antje J. Baeumner Received: 30 March 2014 /Revised: 28 May 2014 /Accepted: 4 June 2014 /Published online: 24 June 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Sample preparation and processing steps are the most critical assay aspects that require our attention in the development of diagnostic devices for analytes present in complex matrices. In the best scenarios, diagnostic devices should use only simple sample processing. We have therefore investigated minimal preparation of stool samples and their effect on our sensitive microfluidic immunosensor for the detection of cholera toxin. This biosensor was previously developed and tested in buffer solutions only, using either fluorescence or electrochemical detection strategies. The microfluidic devices were made from polydimethylsiloxane using soft lithography and silicon templates. Cholera toxin subunit B (CTB)-specific antibodies immobilized onto superparamagnetic beads and ganglioside GM 1 -containing liposomes were used for CTB recognition in the detection system. Quantification of CTB was tested by spiking it in human stool samples. Here, optimal minimal sample process- ing steps, including filtration and centrifugation, were opti- mized using a microtiter plate assay owing to its high- throughput capabilities. Subsequently, it was transferred to the microfluidic systems, enhancing the diagnostic characteristic of the biosensor. It was found that the debris removal obtained through simple centrifugation resulted in an acceptable removal of matrix effects for the fluorescence format, reaching a limit of detection of only 9.0 ng/mL. However, the electron transfer in the electrochemical format was slightly negatively affected (limit of detection of 31.7 ng/ mL). Subsequently, cross-reactivity using the heat-labile Escherichia coli toxin was investigated using the electro- chemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrat- ed excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices. Keywords Biosensor . Microfluidic . Liposomes . Cholera toxin . Fecal sample Introduction From the introduction of the first microfluidic device devel- oped in the 1970s [1], microfluidic device technology has entered various fields, including chemical, biological, and diagnostic analyses, owing to its advantages in terms of the small amounts of reagent and sample required, high sensitiv- ity, rapid response, portability, and its simple adaptation to multifunctionality and high-throughput analyses [2]. Diagnostic assay development is moving toward the use of micro total analysis systems in which detection and sample preparation are integrated. In the case of microfluidic immunosensors, antibodies are used as biorecognition ele- ments, similarly to traditional immune biosensors, which find ample application in point-of-care diagnostics [3]. Both opti- cal and electrochemical detection approaches have been real- ized with microfluidic immunosensors. Their application has been successfully demonstrated for a variety of analytes. For Published in the topical collection celebrating ABCs 13th Anniversary. N. Bunyakul : C. Promptmas Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand A. J. Baeumner (*) Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA e-mail: baeumner@cornell.edu A. J. Baeumner e-mail: antje.baeumner@ur.de Present Address: A. J. Baeumner Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93053 Regensburg, Germany Anal Bioanal Chem (2015) 407:727–736 DOI 10.1007/s00216-014-7947-9