Remarkable Enhancement of Sensitivity with the Second Generation of Elliptically Polarization-detected Circular Dichroism Spectroscopy

Abstract

A new circular dichroism (CD) measurement system based on the detection of elliptically polarized light has been proposed. The approach has several unique and inherent advantages, such as sensitivities, reliability, and reproducibility, over conventional systems and Kliger's pioneering work. We have rederived the Jones calculus and introduced the angular term into the formulation revealing that this approach should improve the signal intensity by controlling the light ellipticity. Therefore, we have first demonstrated the hypothesis by the quantitative analysis of the signal intensity measuring of the split CD pattern of tris(bipyridine)-ruthenium(II) dichloride with the system. The CD signals increasing with decreasing the azimuth of retarder was clearly observed, enabling detection of CD signal with high sensitivity.Circular dichroism (CD) spectrometry is one of the most powerful and versatile methods for detection of interaction and binding of biomolecules, since these molecules consist of chiral unit modules.1 Thus, these conformational and structural changes and/or variety could be observed by CD spectra with high sensitivities. However, it is hard to measure CD spectra with good to excellent S/N, in general, as a result of small differences in extinction coefficients of asymmetric left and right circularly polarized light (¦¾). It is well known that the ¦¾ values are mostly three orders of magnitude lower than those of the UVvis (¾). Therefore, CD spectral studies have been predominantly confined to relatively high-concentration samples. In order to improve the sensitivity, a lock-in detection system with a photoelastic modulator (PEM) has been successfully employed in modern CD instruments. However, the modulation frequencies of asymmetric circular polarized lights of PEM (ca. 45 KHz) limit time-resolution of CD measurements. Therefore, CD spectral studies have been mostly focused on relatively slow events. Hence, novel CD detection methods for appropriate time resolution for observing early events such as protein folding are desired.These unique properties and advantages of CD measurements have driven our research interest and endeavor to creation of a novel methodology for detection and observation of CD spectra coping with both high sensitivity and high time resolution broadly in science and technology. To achieve this purpose, small differences in ¦¾ between large quantities of ¾ must be detected. For this purpose, several approaches for CD detection without PEM-based lock-in detection systems have been proposed recently. Hache et al. have reported that singular utilization for the polarization of light with a Babinet compensator could be applied for CD dynamics studies on 1,1¤-bi-2-naphthol. 2 Another approach had been proposed by Fiebig et al. 3 Their approach aimed to obtain CD signals more directly. The sophisticated way for transient absorption measurement with polarization-controlled white light could measure the differential absorption of circular polarized light generated by chiral sample...