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Microfluidic biosensor for cholera toxin detection in fecal samples

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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 GM1-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 processing steps, including filtration and centrifugation, were optimized 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 electrochemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrated excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices.

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Acknowledgments

The authors thank Wijit Wonglumsom of Mahidol University for help with the bacterial biochemical tests. We thank Sutas Boonyong of Nakhon Pathom Hospital, Thailand, for help in obtaining the stool samples. We also thank John Connelly for IDUA electrode fabrication. This work was performed in part at the Cornell Nanofabrication Facility, a member of the National Nanofabrication Users Network, which is supported by the National Science Foundation (grant ECS-0335765). This research was also supported in part by the Thailand Research Fund through the RGJ-PhD program in Thailand and the Commission on Higher Education, Ministry of Education, Thailand.

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Author notes

  1. Antje J. Baeumner

    Present address: Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93053, Regensburg, Germany

Authors and Affiliations

  1. Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Nakhon Pathom, 73170, Thailand

    Natinan Bunyakul & Chamras Promptmas

  2. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, 14853, USA

    Antje J. Baeumner

Authors
  1. Natinan Bunyakul
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  2. Chamras Promptmas
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  3. Antje J. Baeumner
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Correspondence to Antje J. Baeumner.

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Published in the topical collection celebrating ABCs 13th Anniversary.

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Bunyakul, N., Promptmas, C. & Baeumner, A.J. Microfluidic biosensor for cholera toxin detection in fecal samples. Anal Bioanal Chem 407, 727–736 (2015). https://doi.org/10.1007/s00216-014-7947-9

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  • DOI: https://doi.org/10.1007/s00216-014-7947-9

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