Back-Scattering Interferometry (BSI) Technology
BSI is an advanced, optical measurement technique for highly sensitive qualitative and quantitative molecular interaction assays. BSI uniquely offers homogeneous assay capability — no labeling or tethering of interactors, with sensitivity comparable to or exceeding the most advanced label-free methods currently available. BSI can also perform tethered, heterogeneous assays similar to Surface Plasmon Resonance (SPR) and other wave-guide technologies, but without the significant loss of sensitivity frequently experienced in these methods.
Benefits of BSI
- Homogeneous equilibrium binding Kd's from pM to mM; future developments in process for kinetics
- High sensitivity for small molecule — large protein target binding versus SPR or ITC
- Protein target — lead compound interaction detection is not limited by molecular weight of either binding partner
- Target classes currently investigated with BSI:
- Cell adhesion proteins
- Kinases
- Nuclear receptors
- Antibodies
- Heat shock proteins
- Chaperones
- Membrane bound enzymes
- Measurements in complex matrices
- 100% serum
- DMSO
- Cell lysates
- Surfactants
- Impure protein mixtures
- Cell membrane preparations
- Accurate measurement — deleterious matrix and labeling artifacts on binding measurements are removed by
BSI's label-free, tether-free capability
- Significant cost savings resulting from minimal sample preparation requirements
- Small sample volume (< 10 µL)
- 1000x less precious target protein than ITC methods
- Rapid methods development requiring:
- Little apriori knowledge of the molecular interactors
- Reduced target purification burden
- No labeling or surface attachment optimization
Components of the Technology
The Back-Scattering Interferometer employs a simple optical train comprised of a coherent light source (commonly a low-power He-Ne or red diode laser), a microfluidic channel (formed in glass, fused silica, or plastic), and a phototransducer (Figure 1). The interaction of the laser beam with the fluid-filled channel results in a high-contrast interference pattern. The profile of this fringe pattern changes in a predictable manner with molecular binding events within the optical channel. Analysis of fringe positional changes, performed by a phototransducer located in the direct backscatter direction, facilitates measurement of refractive index changes of less than 10-7 dn within a 1 nL detection volume.
Figure 1: Experimental setup for BSI. Molecular interaction assay samples are introduced within a temperature controlled microfluidic chip. Coherent light from a HeNe laser is directed towards the microchip fluidic channel, which functions to create the fringe pattern characteristic of BSI. The BSI fringe pattern is directed to a CCD array. An image is extracted and the positional shift of these fringes is monitored to provide binding signal.
Signal Analysis and Data Presentation
A proprietary algorithm is used to extract the exact movement of the fringes across the CCD camera and is represented below in Figure 2 as Gaussian traces which map the area of the fringe pattern. Movement of the fringes is shown on the computer screen as changes in the peak maxima in pixels and is plotted as time (seconds) vs pixels (millipixels). Each new sample injection causes an initial baseline disruption before coming to equilibrium at new pixel value for each new concentration. Analysis time is approximately 30 seconds per sample.
Figure 2: BSI signal processing. Interference fringes are imaged by the CCD camera. Custom software extracts wave from the fringe images. A proprietary algorithm is then applied to determine positional shifts of these fringes to a resolution of 50 nanometers. Change in fringe position as a function of time is plotted as the response curve for the binding event. Molecular binding is depicted as an increase in the binding curve's intensity. The plot demonstrates actual end-point data for a simple dose response. Each horizontal "step" corresponds to a different concentration of analyte following equilibration after the sample introduction spike.
Measurement of Equilibrium Binding Kd
Equilibrium binding Kd's of drug protein target — small molecule drug leads is determined by titrating the lead against a constant concentration of protein target and measuring the formation of the complex at equilbrium. Usually the concentration of target is 1/100th of the Kd and the drug lead is titrated from 1/10 to 5x the Kd. Because only 10 µL of sample is required per injection, only picomoles of precious protein target is required for measurements representing nM Kd binding.
Figure 3: BSI equilibrium binding Kd measurement. An equilibrium binding curve is generated from measuring the change in RI when titrating drug molecule B against a constant concentration of drug target A. The equilibrium signal created by AB at varying concentrations of B produce an equilibrium binding curve. The extraordinary signal increase from AB results from conformational changes in the target protein, associated changes in dipole moment and waters of hydration due to
binding by B.
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