For CD measurement in a cell with a path length of 1 mm, a sample volume of about 400 μL is required. However, for very valuable samples, a microsampling disk can be used to measure even smaller quantities. CD spectra can be measured simply by placing a drop of the sample into the water-repellent coating area in the center of the assembled disc (Fig. 13). The volume that can be measures is from 2 μL (optical path length of 0.2 mm) to 10 μL (optical path length of 1 mm). Fig. 14 shows the results for a 10 μL drop of a bovine thymus DNA solution and for a sample in a normal 400 μL cylindrical cell. It can be seen that the CD spectra are almost identical despite the 40-times difference in sample size.
Fig. 13 Sample loading
Fig. 14 CD spectrum of bovine thymus DNA
Rapid kinetics using stopped-flow method
The reaction process for optically active substances can be traced using the stopped-flow method. This method can be used to evaluate reaction rates, short-lived intermediates, and reaction inhibitors and accelerators. In particular, it has been applied to folding analysis of proteins. Using this method to track CD signal changes over periods of milliseconds, it is possible to visualize protein denaturation and structural changes in chiral molecules. Fig. 15 shows the change in the CD signal during hydrolysis of D-glucuronolactone with an aqueous KOH solution.
Fig. 15 Hydrolysis of aqueous solution of D-glucuronic acid lactone
Multi-sample screening (HTCD)
The effectiveness of CD for monitoring protein structural changes has already been described. In recent years, in addition to CD, simultaneous measurements of UV absorption and fluorescence, and multiparameter measurements involving both temperature and pH changes, have become common. At the same time, the need for fully automated measurements of many samples is rapidly increasing. In order to realize tailor-made medical treatments such as nucleic acid medicines and antibody medicines, screening of the required chiral molecules is indispensable, and CD instruments must be capable of high-throughput measurements.
Fig. 16 High-throughput CD system
Fig. 17 Spectrum QC test program
The pharmaceutical application that introduced at Biophysical Society 2019 is below.
Please click following link and check it.
New Approach to Evaluating the Stability of Antibodies Using Circular Dichroism Spectroscopy Screening