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Related Experiment Videos

A label-free continuous total-internal-reflection-fluorescence-based immunosensor.

Henrik A Engström1, Per Ola Andersson, Sten Ohlson

  • 1Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden.

Analytical Biochemistry
|August 29, 2006
PubMed
Summary
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This study developed a continuous optical immunosensor using intrinsic antibody fluorescence to monitor carbohydrate concentrations like maltose and panose in real-time. Weakly interacting antibodies enable continuous sensing without regeneration, offering fast and sensitive analyte detection.

Area of Science:

  • Biomedical Engineering
  • Analytical Chemistry
  • Immunosensing Technology

Background:

  • Monoclonal antibodies exhibit intrinsic fluorescence changes upon carbohydrate antigen binding.
  • Previous methods required antibody regeneration for continuous monitoring.
  • Extrinsic labeling of antibodies can be complex and introduce artifacts.

Purpose of the Study:

  • To develop a continuous optical immunosensor for real-time carbohydrate monitoring.
  • To utilize the intrinsic fluorescence of monoclonal antibodies for sensing.
  • To investigate the use of weakly interacting antibodies for continuous analyte detection.

Main Methods:

  • Continuous monitoring of maltose and panose concentration using total internal reflection fluorescence (TIRF).

Related Experiment Videos

  • Immobilization of four monoclonal antibodies with low affinity and fast dissociation kinetics.
  • Measurement of antibody-carbohydrate binding thermodynamics (DeltaH°, DeltaS°) via temperature dependence of K(d).

    • Main Results:

    • Achieved continuous monitoring of maltose and panose without sensor regeneration.
    • Demonstrated sensitivity in the low micromolar range (1-5 µM for panose, 10-15 µM for maltose).
    • Observed minimal intensity loss (3.5% per hour) during measurements, indicating sensor stability.
    • Antigen-antibody binding was enthalpically driven but diminished upon immobilization.

    Conclusions:

    • Weakly interacting antibodies are suitable for continuous immunosensing applications.
    • The developed immunosensor provides instantaneous analyte concentration information.
    • Intrinsic fluorescence sensing eliminates the need for extrinsic labeling, simplifying the system.