Label-free molecular interaction determinations with nanoscale interferometry

J Am Chem Soc. 2004 Dec 22;126(50):16659-64. doi: 10.1021/ja047820m.


Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biotin / chemistry
  • Dimethylpolysiloxanes / chemistry
  • Immunoglobulin G / analysis
  • Immunoglobulin G / chemistry
  • Immunoglobulin G / metabolism
  • Microscopy, Atomic Force
  • Microscopy, Interference / methods*
  • Nanotechnology / methods*
  • Protein Array Analysis / methods*
  • Protein Binding
  • Proteins / analysis*
  • Proteins / chemistry
  • Proteins / metabolism
  • Sensitivity and Specificity
  • Silicones / chemistry
  • Staphylococcal Protein A / analysis
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / metabolism
  • Streptavidin / chemistry


  • Dimethylpolysiloxanes
  • Immunoglobulin G
  • Proteins
  • Silicones
  • Staphylococcal Protein A
  • baysilon
  • Biotin
  • Streptavidin