SubscribeProteins, the building blocks of life, are diverse and complex molecules whose correct function is essential to all biological processes and whose malfunction lies at the heart of almost every disease.
Part of the complexity and subtlety of protein operation rests in the fact that it is not merely their amino acid composition but their 3-dimensional fold (conformation) that determines their functional characteristics. This conformation inevitably changes subtly in response to ligand binding, pH, ionic strength or temperature. The manner in which proteins perform their allocated function is entirely dependent on the driving forces provided by these structural changes.
For many years, biochemists and pharmacologists within the pharmaceutical industry have worked to determine precisely the consequences of changes in a protein's 3-dimensional shape, in order to gain a better understanding of how they operate or how drug molecules could be designed to assist in function or mediate in malfunction. Images of protein crystal structure are of intense interest to the pharmaceutical industry, but these are a static snapshot of what is actually an elegantly mobile mechanism and provide only one fragment of the information required to unravel the mysteries of proteins. Furthermore, imaging a protein using x-ray diffraction requires crystallisation of the protein, an extremely difficult process that has seriously restricted the number of useful images that have been obtained to date.
Researchers in the pharmaceutical industry have long demanded an analytical approach with which they can study a protein in a more native environment, measure protein structure in real time and at a resolution likely to yield information about its function, and directly correlate any structural changes with protein function. Dual Polarisation Interferometry (DPI) provides just such a method.
DPI quantitatively measures the structure of a protein in one dimension (i.e. its diameter or size) and the density (i.e. mass per unit volume or how tightly folded it is) by coupling the protein to a glass slide and probing its structure using non-diffractive optics. The method resolves protein structure to subatomic dimensions (well below 0.1 Å) in real time and has a growing acceptance among researchers in the field of protein structure-function characterization, an essential discipline in the pharmaceutical industry.
How DPI Works
In any waveguide structure, light is not wholly confined within the physical boundaries on the guiding medium but, rather, decays exponentially at the boundaries. Changes in the refractive index of materials within this decaying, or evanescent, field alter the speed of propagation of the field (the optical path length within the waveguide).DPI leverages this phenomenon to probe the optical properties of proteins that have been covalently, hydrophobically, or electrostatically attached to the sensing surface.
Interferometers detect the change in optical path length experienced by an optical field passing through the sensing path of the interferometer. Sensitivity is governed by, among other things, the interaction length and the signal-to-noise ratio of the detection scheme. Typically, integrated optical interferometers are configured in the Mach-Zehnder format by creating channel waveguiding regions in the top surface of an optical dielectric stack. (1)
DPI uses a much-simplified interferometer based on slab waveguides, with the reference arm buried beneath the sensing arm. Coherent light broadly illuminates the stack, traversing both waveguides, and upon exiting the structure diffracts into free space.
Because the waveguides are so close together, even within a few hundred micrometers of the end of the stack the diffracted wavefront generates the well-known pattern of Young's interference fringes in the far-field. Changes in the optical properties of proteins on the surface of the sensing waveguide translate into variations in the interference pattern, as captured by a high-resolution camera.
The optical tolerances are so forgiving that macroscopic movements of the input coupling beam on the order of hundreds of micrometers cause no change in the interference pattern. These loose tolerances allow the stack to be inserted and removed from the optical train without alignment, an essential characteristic for a disposable measurement platform.
For a given measurement of the interference pattern, one can determine an optogeometric parameter for a layer of protein. This parameter will not, however, unambiguously resolve the size or any other structural signature of the protein: A large protein in small number density will produce the same signal as a small protein in large number density.
Resolving this ambiguity requires two simultaneous measurements of the optical properties of the protein in two orthogonal polarizations (TE and TM) which, because the evanescent fields have two different profiles, can be mathematically converged on a single solution for the protein size and density. Knowing size and density, one can also trivially calculate the total mass, surface concentration, number of protein molecules, molecular footprint, and a host of other useful parameters.
Operating Principles
The phase changes of interest, ΔΦ, involve changes to the effective refractive index Ns of the mode in the upper (sensing) waveguide. Changes occurring at the surface do not alter the effective index of the lower (reference) mode Nr because the evanescent field of this mode decays rapidly in the region between the two guiding layers. The phase difference is given by:
ΔΦ = k0LΔNS (1)
Where k0 is the free space wave number, L is the interaction length, and ΔNs is the effective index change in the upper waveguide mode. Direct measurement of ΔΦ is obtained by continuously monitoring the relative phase position of the fringe pattern by performing a Fourier transformation relating intensity to position. The path length is fixed, so one can thus easily convert the experimental data to changes in effective refractive index.
A standard transfer matrix approach provides evaluation of the guided modes for the structure. This allows inclusion of an arbitrary number of layers in a model, each of which is represented by its own layer matrix. Within each layer, with propagation in the z-direction being parallel to the layers and the x-direction being perpendicular, the electromagnetic fields are proportional to:
(Ae-ikx + Beikx) ei(kzz-ωt) (2)
With ω fixed at the laser frequency, a scan through the propagation constant kz determines the transverse wave vector kx for each sub-layer via:
kx2 = ni2k02 - kz2 (3)
Where ni is the refractive index of the ith sublayer (ky = 0). Beginning with an evanescent solution in the region above the top layer of the waveguide structure, one can enforce matching of the tangential components of E and H for the TE and TM modes at each successive interface of the structure. This information yields the values of the coefficients A and B for each layer. A successive approximation method provides the optimum refractive-index value and the thickness (dimension) of the thin protein layer on the surface of the waveguide (see Figure 1).
Instrumentation and Results
The 26mm × 6mm slab waveguide is housed in a dual-zone, temperature-controlled enclosure that maintains thermal stability to within 1mK (see Figure 2). The clamping system also provides a fluidic interface to the sensing surface with a dead volume of approximately 2μl, allowing introduction of test materials as required. A helium neon laser (λ = 632.8nm) illuminates the end facet of the slab and a ferroelectric liquid crystal (FLC) halfwave plate switches the state of polarization of the input beam between TE and TM at typical frequencies of 50Hz. The high tolerance to relative movement of the incident beam comes into play here - the FLC plate produces small refractive displacements during switching but the output image remains stationary throughout.
A 1024 × 1024-element imaging device captures the diffraction fringes and passes the output to a digital signal processor. The image device output is synchronized with the FLC drive signal and sampled. The relative phase position is updated every 2ms using a spatial Fourier transform method. Further analysis by PC displays the data and processes it into thickness and refractive index values for the protein system. Millikelvin thermal control using an autonomous dual-zone control system and the opportunity for averaging yields an rms noise level of around ±0.1mrad, equivalent to about 0.1pg/mm2 mass loading or less than 0.1Å dimensional change.
DPI has been used to study protein structure and interactions, as well as surfactants, lipids, and many other interfacial chemistry systems. Researchers have verified the method against other physical measurements such as crystal structures or neutron methods.(2,3)
One brief example of DPI's powerful structural measurements involves the protein streptavidin (Mwt 55,000Da), which was immobilized to the sensing waveguide and challenged with biotin (Mwt 244 Da), a well-characterised small molecule binding partner with one of the strongest binding affinities known in biology. DPI analysis revealed that the protein (plus immobilizing linker chemistry) has a size of just over 66Å, which is in good agreement with what would be expected from streptavidin's known x-ray crystal structure (see Figure 3).
When challenged with biotin, the streptavidin undergoes a conformational change resulting in a reduction in size of 1.3Å. Simultaneously, as there is now a larger mass in a smaller volume, the density increases, providing an unambiguous marker of the protein function at a previously unimaginable fidelity. In more broad terms, the slab waveguide provides a convenient and disposable optical bench on which non-diffractive optics can probe a 100-nm region above the slab surface.
A multitude of optical methods compatible with this device could yield additional high-resolution measurements to further characterize the molecular system of interest. Already absorption studies (spectroscopy) have been observed, as have scattering signatures (morphology). The combination of wavelength and polarization (dichroism) promise to deliver both compositional and structural information highly relevant to the life scientist in years to come. We expect a portfolio of new analytical techniques to emerge, truly illuminating the molecular world...
References
(1). R. Rapola and T. Horbett J. Colloid Interface Sci. 136 480 (1990)
(2). G. Cross et al. Biosensors and Bioelectronics 19 383 (2003)
(3). G. Cross et al. J. Phys. D: Appl. Phys. 37 74 (2004).