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Related Experiment Videos

Reversible sequential-binding probe receptor-ligand interactions in single cells.

Christoph Schreiter1, Marinela Gjoni, Ruud Hovius

  • 1Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratoire de Chimie Physique des Polymères et Membranes, 1015 Lausanne, Switzerland.

Chembiochem : a European Journal of Chemical Biology
|November 5, 2005
PubMed
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We developed a novel reversible sequential (ReSeq) binding assay for ultrasensitive single-cell receptor profiling. This method accurately measures receptor function, even at low expression levels, using fluorescent ligands and microfluidics.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Pharmacology

Background:

  • Accurate measurement of receptor function in single cells is crucial for understanding cellular processes and drug development.
  • Existing methods often lack the sensitivity or specificity required for analyzing low-expression receptors.

Purpose of the Study:

  • To introduce and validate a novel ultrasensitive assay, ReSeq, for profiling receptor function in single living cells.
  • To demonstrate the assay's utility using the nicotinic-acetylcholine receptor (nAChR) as a model system.

Main Methods:

  • Utilized reversible sequential (ReSeq) binding assay with fluorescent ligands exhibiting fast association-dissociation kinetics.
  • Performed equilibrium, kinetic, and competition-binding assays on single cells in microfluidic devices.

Related Experiment Videos

  • Employed fluorescent derivatives of alpha-conotoxin GI (alpha-CnTx) to target muscle-type nAChRs.

    • Main Results:

    • Achieved ultrasensitive profiling of nAChR function in single cells.
    • Determined binding constants for unlabeled alpha-CnTx and d-tubocurarine.
    • Demonstrated specific binding detection even with competing nAChR subtypes due to high alpha-CnTx selectivity.
    • Showcased assay accuracy and miniaturization through imaging of individual nAChRs and binding cycles.

    Conclusions:

    • The ReSeq assay offers a novel, highly sensitive, and accurate approach for single-cell receptor function analysis.
    • This method is effective even for cells with low receptor expression levels.
    • ReSeq has broad applicability for functional screening of compounds, membrane receptors, and characterization of rare primary cells.