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ForteBio Interactions Newsletter Biosensor photo

SPRING 2012    VOLUME 5    ISSUE 1

WWW.FORTEBIO.COM

BioLayer Interferometry (BLI) — How Does it Work?

Darick Dayne, Ph.D., Product Manager, Marketing, DDayne@fortebio.com

ForteBio introduced its first BLI breakthrough in 2005 with the Octet instrument, and one question we’ve been asked quite often since then is “how does it work”? So we thought it was high-time to answer that question for both new and experienced users alike.

The technology employed by the Octet platform is based on the principles of optical interferometry, namely the interaction of light waves. The basic phenomenon is quite simple. When two propagating waves are perfectly in phase (e.g. the peaks and troughs of the waves match up exactly), the resulting wave has an amplitude equal to the sum of the two waves due to constructive interference (Figure 1A). When the two propagating waves are completely out of phase (e.g. the peaks of one wave matches up exactly with the trough of the other), the resulting wave will have zero amplitude due to destructive interference (Figure 1B).

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FIGURE 1

 

 

Figure 2 shows a magnified view of a ForteBio glass fiber-based biosensor, where the surface chemistry occurs at the very tip of the glass fiber. When the biosensor is in the Octet or BLItz platforms, white light (inclusive of all wavelengths in the visible spectrum) is sent down the glass fiber (Figure 3) and is reflected back up to the instrument from two interfaces: 1) the interface between the glass fiber and the proprietary bio-compatible layer, and 2) the interface between the surface chemistry and solution (Figure 4).

Since the two reflections come from the same white light source in the instrument, they both contain the same wavelengths in the visible spectrum (Figure 5). When the two reflections are taken apart and the same color channel (e.g. wavelength) from each reflection is monitored and analyzed together, an interference pattern emerges. In the example shown in Figure 6, the two red light waves might undergo complete constructive interference, resulting in a doubling of amplitude. The two green light waves might undergo partially constructive interference, resulting in a certain amplitude smaller than that of the red channel. The two violet light waves might undergo complete destructive interference, resulting in zero amplitude. If the relative amplitudes of the red, green, violet and all other wavelengths were plotted, an interferometric profile would be derived (Figure 7). This forms the basis of BLI measurements on the Octet and BLItz instruments.

When molecules bind to the surface of the biosensor, the path length of the reflection (the one reflecting from the interface between surface and solution) increases while that of the other reflection remains the same (Figure 8). This changes the interference patterns for all wavelengths. In this example, the two waves of the red wavelength from the two reflections are no longer perfectly in phase, which causes the slight drop in the resulting amplitude. The two waves of the green wavelength are further out of phase, causing a further drop in the resulting amplitude. However, the two waves of the violet wavelength are no longer completely out of phase and thus the resulting amplitude is no longer zero (Figure 9). Plotting this for all the wavelengths results in a new interferometric profile that is shifted to the right from the original profile (Figure 10).

Biolayer

 

Biolayer

 

 

Biolayer

 

Biolayer

 

 

This optical construct allows the real-time monitoring of molecular binding events occurring on the surface of the biosensor. As more molecules bind to the surface, the interferometric profile shifts further to the right (Figure 11). Conversely, as molecules dissociate from the surface, the interferometric profile shifts back left to its initial position. When this shift is measured over a period of time and its magnitude plotted as a function of time, a classic association/dissociation curve is obtained (Figure 12).

The Octet and BLItz platform’s superior technology harnesses the real-time label-free detection power of BLI in a manner that makes them simple to use and easy to maintain for researchers in a variety of laboratory settings. Almost all surface-based technologies on the market today require samples to flow across a sensor in finicky and hard-to-maintain microfluidic systems that are also prone to frequent breakdowns. However, Octet and BLItz platforms create flow without microfluidics and place biosensors into samples directly for true Dip and Read simplicity, enabling scientists to accelerate their research by maximizing productivity.