Diffusion NMR—be careful It seems like a straightforward idea: The diffusion coefficient of a molecule, such as a protein, changes when another molecule binds to it. As a result, diffusion NMR, which measures the diffusion coefficient noninvasively, has emerged as a powerful technique for identifying interacting systems and providing data to calculate binding constants. However, as Michael Shapiro and Aidi Chen of Novartis Pharmaceuticals report, an unintended nuclear Overhauser effect (NOE) can compromise the diffusion coefficient measurement. The problem arises during the diffusion time window (A) part of the NMR experiment (a stimulated echo-type of experiment)—when the diffusion is monitored. The magnetization is stored in the z direction during this window, and intermolecular NOE can take place, affecting the NMR signal decay and the subsequent results. Under conditions of slow-motion limit, strong NOE can build up rapidly during the A.
spectively. The probe transmission signal was measured with lock-in detection at the 50-kHz difference frequency. Unfortunately, the unaffected probe and back-reflected pump beams can affect the measurement by increasing the noise. The back-reflected pump beam caused greater problems, and two measures were taken to reduce its effects.
The Novartis scientists dem onstrated the problem by running a series of diffusion exper ments with human serum albumin and benzoic acid, a ligand known to bind to the protein. When the A is short, NOE is nc a problem, and an appropriate diffusion coefficient is determined. But, as A increases, the NOE affects the measurement, yielding diffusion coefficients that are less than 50% of the value determined at the shorte time. To further confirm the effect of the NOE the research the diffusion NMR Diffusion measurements with benzoic acid (H26), periment using the carboxyl showing the effect to NOE oo the proton signals as a carbon of benzoic acid which increases. The plotted data ara expected to be linear experiences little or no NOE with a slope yielding the diffusion coefficient; curved interactions In this case the lines indicatt the eresence of NOE. 13 C signals showed little variation with changing As nonbinding ligands by looking at how the Although it is a problem, this effect could decay rate is affected by varying A. .J. Am. be used to discriminate binding ligands from Chem. Soc. 1999,121, 5338-39)
The pump and probe beams were orthogonally polarized so that the pump could be filtered out while most of the probe was transmitted. In addition, a confocal arrangement consisting of a 50-pm pinhole filtered the llght totside oo f 1.25-um sample area. The instrument was used to measure undoped 80 A single quantum well Ga/As/
Al0 3Gao 7As samples. The only diiffeence between the samples was the ion implantation pattern—200-nm implanted stripes with 400-nm spaces for the first sample and 100-nm stripes with 2-um spaces for the second. A two-dimensional image of the amplitude of the pump-probe signal contains a pattern that matches the periodicity of the implantation pattern. A similar pattern did not appear in the topographic images, indicating that it not a topographic artifact. Measurements indicated that the carrier dynamics in the unimplanted regions of the sample were affected by diffusion, which has previously been shown to affect the decay of carriers in other semiconductor nanostructures. Because of the small scale of the effects, they could not have been seen in far-field measurements. Global pump/local probe configuration (left). Experimental setup (right). AOM is an acousto-optic (Rev. Sci. Instrum. modulator and GVD is group velocity dispersion. (Adapted with permission. Copyright 1999 American 1999 70 2758-64) Institute of Physics.)
Analytical Chemistry News & Features, August 1, 1999
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