Protein - protein and ligand - substrate interactions are central to understanding basic cellular function and for evaluating therapeutics. However, the tools available to quantify these interactions without modifying the proteins or ligands have important limitations. Surface immobilization of the substrate, receptor, or protein or the attachment of a signaling fluorophore influences molecular interactions. It is desirable to eliminate the need for the surface chemistry normally used in binding assays, since it presents persistent problems including: complicated kinetics due to surface bound targets, lack of durability and longevity, high cost, non-specific binding, and difficulty in quantification of the immobilized targets. The ability to perform pure liquid - phase molecular binding analysis in a mu-TAS format would eliminate many of the problems that arise from the surface chemistry, while facilitating small volume samples to be studied. Preliminary observations presented here indicate that molecular interactions, such as protein - protein or ligand - substrate binding, can be studied in the absence of these perturbations, at high sensitivity, in picoliter volumes and in free-solution using on-chip interferometric backscatter detection (OCIBD). The proposed technology uses a simple and inexpensive optical train to facilitate these homogeneous, label-free measurements. OCIBD monitors minute refractive index changes within a microfluidics channel formed in silica or PDMS. OCIBD performed in a non-modified PDMS channel has recently facilitated quantification of reversible and irreversible protein - protein binding without a fluorescent label, the direct measurement of DNA interactions and fluorescent tag perturbations on delta/G. These determinations were performed in picoliter volumes and at attomolar levels and confirmed by Isothermal Titration Calorimetry (ITC). It has been possible to quantify reaction kinetics and binding affinities (Ko) for protein-IgG label-free in free-solution, with detection limits of 4 attomoles for IgG. Recently it has been possible to detect 10,800 molecules of IL-2 with backscattering interferometry. This proposal expands on these results, culminating a multiplexed OCIBD for homogeneous (free-solution) molecular interaction assays.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB003537-02
Application #
7086118
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Kelley, Christine A
Project Start
2005-07-01
Project End
2009-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
2
Fiscal Year
2006
Total Cost
$271,108
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Kussrow, Amanda; Enders, Carolyn S; Bornhop, Darryl J (2012) Interferometric methods for label-free molecular interaction studies. Anal Chem 84:779-92
Lau, Jolene L; Baksh, Michael M; Fiedler, Jason D et al. (2011) Evolution and protein packaging of small-molecule RNA aptamers. ACS Nano 5:7722-9
Baksh, Michael M; Kussrow, Amanda K; Mileni, Mauro et al. (2011) Label-free quantification of membrane-ligand interactions using backscattering interferometry. Nat Biotechnol 29:357-60
Kussrow, Amanda; Baksh, Michael M; Bornhop, Darryl J et al. (2011) Universal sensing by transduction of antibody binding with backscattering interferometry. Chembiochem 12:367-70
Kussrow, Amanda; Enders, Carolyn S; Castro, Arnold R et al. (2010) The potential of backscattering interferometry as an in vitro clinical diagnostic tool for the serological diagnosis of infectious disease. Analyst 135:1535-7
Morcos, Ereny F; Kussrow, Amanda; Enders, Carolyn et al. (2010) Free-solution interaction assay of carbonic anhydrase to its inhibitors using back-scattering interferometry. Electrophoresis 31:3691-5
Kussrow, Amanda; Kaltgrad, Eiton; Wolfenden, Mark L et al. (2009) Measurement of monovalent and polyvalent carbohydrate-lectin binding by back-scattering interferometry. Anal Chem 81:4889-97
Latham, Joey C; Stein, Richard A; Bornhop, Darryl J et al. (2009) Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry. Anal Chem 81:1865-71