A light wave has a wavelength, amplitude and direction. The wavelength is determining the colour of the light wave. The amplitude is the intensity of the light wave. Visible light ranges from 400 nm (violet) to 700 nm (red) (1), (4).
When two light waves interact the result of the interaction depends on the phase and amplitude of the waves. If the waves have the same amplitude but opposite phase (red and green) they will cancel out. This is called destructive interference. If the waves have the same phase and amplitude the resulting wave has the same phase but the amplitude is the sum of the two amplitudes. This is called constructive interference. The two examples are at the extreme of the possibilities because most of the interferences are a combination of phase shift and amplitude change.
The sensor is attached to an optical fibre which guides the light beams from the light source to the sensor tip and back to the spectrometer. Between the sensor tip and the optical fibre is an air gap to suppress internal reflection. The sensor tip is made of high grade optical glass (SiO2) with an index of refraction of about 1.4 - 1.5 or a high grade polymer like polystyrene or polyethylene with an index of refraction between 1.3 – 1.8.
Between the optical glass layers is the first refraction layer made of Ta2O5 with an index of refraction 2.1. The layer is made by conventional vapour deposition and is typically between 5 and 30 nm. The distal optical glass layer is between 400 and 1000 nm. The tip of the optical layer is modified with for instance bifunctional reagents containing a siloxane group for chemical attachment to SiO2 and a hydroxyl, amine, carboxyl or other reactive group to make a surface suitable for ligand immobilization.
In Bio-Layer Interferometry a white light source with all wavelengths from the visible light is used. The white light (I1) is send down an optical glass fibre and reflected back (I2) at the glass – optical surface and the interface between the surface chemistry and the solution (I3). All the light that is not reflected travels through the sensor into the liquid (I4). Although the reflected light (I2) from the first reflective layer is constant, the reflected light from the surface – liquid boundary is not. If molecules attach or detach the (optical) thickness of this layer is changed and consequently the path length the reflected light. Hence, the path length of the reflected light changes and therefore the interference at the detector changes.
Because only molecules which are part of the layer thickness contribute to the path length change, the composition of the liquid below the tip has no influence on the signal. This makes it possible to do measurements in opaque and high optical active solutions, like DMSO and glycerol.
The total wavelength-dependent intensity of the reflected wavelength is:
Where I is the intensity of the reflected light, I2 and I3 the intensities of the two reflected light beams, Δ is the optical path difference and λ is the wavelength. Assuming that the intensities of both of the reflected light are equal the formula becomes:
This formula makes it possible to determine the optical thickness of the extra layer based on the intensity of the reflected light and the wavelength.
The reflected light from both interfaces is compared per wavelength for interferences. For each wavelength the amplitude (intensity) is determined and plotted in an intensity plot. When the initial values are set, any binding of dissociation to the sensor tip will change the interference pattern and therefore the intensity plot. When molecules bind to the sensor the interferometric pattern shifts to the right and when molecules dissociate the interferometric pattern shifts to the left. The measurement of the interferometric pattern can be done in real-time thus enabling the monitoring the interaction of molecules. By plotting the changes as a function of time an association/dissociation curve is obtained.
Like SPR, BLI requires the immobilization of a ligand to the sensor. In principle the same chemistries and modifications are possible as with SPR. BLI does not use a flow, but the sensor is dipped in the analyte solution and vibrated to minimize analyte depletion around the sensor tip. With this set-up the analyte consumption is low and association and dissociation times can be long compared to most SPR instruments.
|(1)||Chapter 37: Interference of light.|
|(2)||ForteBio website. Goto reference|
|(3)||Chen, D.Fiber optic direct-sensing bioprobe using phase-tracking approache. United States Patent(1996).|
|(4)||Pedrotti, L. S.Fundamentals of photonics / Basic physical optics.|
|(5)||Tan, H., D. Chen, Y. Tan, et al.Fiber-optic assay apparatus based on phase-shift interferometry. United States Patent(2004).|