Analytical and Bioanalytical Chemistry

, Volume 393, Issue 1, pp 177–186 | Cite as

Cholera toxin subunit B detection in microfluidic devices

  • Natinan Bunyakul
  • Katie A. Edwards
  • Chamras Promptmas
  • Antje J. Baeumner
Original Paper

Abstract

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM1 was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL−1 for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.

Keywords

Biosensor Microfluidic Cholera toxin Liposomes Electrochemical detection 

Abbreviations

AP

alkaline phosphatase

BSA

bovine serum albumin

DPPC

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

DPPE

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine

DPPG

1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt

HEPES

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

HSS

HEPES-saline-sucrose

IMS

immunomagnetic separation

OG

n-octyl-β-D-glucopyranoside

PBS

phosphate-buffered saline

SRB

sulforhodamine B

TBS

Tris-buffered saline

Notes

Acknowledgements

The authors thank Dr. John March of Cornell University for their use of the inverted fluorescence microscope. We thank Barbara Leonard for help with the preparation of the liposomes. We thank Dr. Natalya Zaytseva for helpful advice on microfluidic analysis. We also thank John Connelly and Dr. Sam Nugen for microfabrication training. This research was supported in part by the Cornell University Agricultural Experiment Station federal formula funds, Project No. 123-464, received from Cooperative State Research, Education and Extension Service, US Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture. 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 (TRF) through the RGJ-PhD program in Thailand, and the Commission on Higher Education, Ministry of Education, Thailand.

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Natinan Bunyakul
    • 1
    • 2
  • Katie A. Edwards
    • 1
  • Chamras Promptmas
    • 2
  • Antje J. Baeumner
    • 1
  1. 1.Department of Biological and Environmental EngineeringCornell UniversityIthacaUSA
  2. 2.Department of Clinical Chemistry, Faculty of Medical TechnologyMahidol UniversityBangkokThailand

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