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Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry

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Science  21 Sep 2007:
Vol. 317, Issue 5845, pp. 1732-1736
DOI: 10.1126/science.1146559
  • Fig. 1.

    (A) Experimental setup for BSI. (B) Microfluidic chip with serpentine mixer and restriction. (C) Photograph of representative fringe patterns showing a RI-induced position shift of the fringes, a representation of a binding event, and observed signal for a control and reactive pair binding event.

  • Fig. 2.

    (A) Real-time association plots are shown for PA binding IgG at various nanomolar concentrations. In the stop-flow interaction experiments, a fixed concentration of 2.5 nM PA was used, and sequential experiments with increasingly larger concentrations of the FC region from IgG (i.e., from 10 to 40 nM) were performed. PA was buffered at a pH = 7.2 with 15 mM Na2HPO4, 50 mM NaCl, 0.1 mM EGTA, and 0.02% sodium azide. All IgG solutions were made using the same buffer as PA. The temperature of the solutions and microfluidic chip were held constant at 25°C ± 0.01°C throughout the experiment. (B) Extracted rates are plotted versus IgG concentration. (C) End-point values are plotted as a function of IgG concentration and analyzed by Prism software.

  • Fig. 3.

    Association curves of CaM with (A) Ca2+ using a constant buffered concentration of 5 μM CaM and Ca2+ concentrations from 12.5 to 100 μM. All CaM solutions used contained a small amount of EGTA to chelate any free Ca2+. A5 μM CaM solution and a 100 μM Ca2+ solution, both containing excess EGTA (i.e., 400 μM), served as the control. (B) TFP using a constant concentration of 2 μMactivated CaM. TFP solutions were made in the same buffer and held at the same pH as CaM. A 2 μM solution of CaM and a 25 μM solution of TFP, both in the absence of Ca2+,were mixed to serve as a control. (C) Calcineurin using a constant concentration of 10 nM activated CaM and a control of 10 nM solution of CaM and a 100 nM solution of Calcineurin, both in the absence of Ca2+.(D) M13 using a concentration of activated CaM kept constant at 5 nM. A 5 nM solution of CaM and a 50 nM solution of M13, both devoid of CaM activating Ca2+ served as the control.

  • Fig. 4.

    IL-2-Ab binding curves with interaction assay performed in cell-free media. The IL-Ab concentration was held constant at 2 nM. Both the IL-2 and IL-Ab solutions were made using RPMI 1640 cell media with 1% fetal bovine serum and 10 μg/mL Cipro. A blank [0 M of both IL-2 and IL-Ab,(black)], as well as two controls [0 pM IL-2 reacted with 2 nM IL-Ab (blue); 100 pM IL-2 mixed on chip with 0 nM IL-Ab (red)] were evaluated.

  • Table 1.

    A comparison of free-solution, label-free molecular interaction techniques.

    TechniqueCompatibility with μ-fluidicsMinimum sample volumeDetection limits
    Microcal ITC (3) No 1.3 mL 1.0 × 10-6 M (1.3 nmol)
    Enthalpy arrays (4) No 500 nL 5.0 × 10-5 M (25 pmol)
    BSI Yes 350 pL 8.57 × 10-11 M (30 zmol)
  • Table 2.

    The binding affinities determined by BSI compared with reported values.

    K d
    Binding partnersBSIReportedDetection method of published data
    KineticEndpoint
    CaM-Ca2+ 3.40 μM 18.23 μM 1-10 μM Equilibrium and flow dialysis (43)
    CaM-TFP 4.73 μM 7.82 μM 4.5-5.8 μM Affinity chromatography (44)
    CaM-CaN 15.64 nM 11.39 nM 4-16 nM Radioisotope (46, 47) and affinity chromatography (45)
    CaM-M13 2.89 nM 9.87 nM 1.9-5.5 nM SPR (48)
    PA-IgG 7.92 nM 6.05 nM 5-34.5 nM Acoustic waveguide (41) and others (40)
    IL-2-Ab 25.91 pM 10-60 pM Radioisotope (52)

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  • Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry
    Darryl J. Bornhop, J. C. Latham, Amanda Kussrow, D. A. Markov, Richard D. Jones, Henrik S. Sørensen

    Supporting Online Material

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