Chapter One - Next-Generation AUC Adds a Spectral Dimension: Development of Multiwavelength Detectors for the Analytical Ultracentrifuge

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Abstract

We describe important advances in analytical ultracentrifugation (AUC) hardware, which add new information to the hydrodynamic information observed in traditional AUC instruments. In contrast to the Beckman-Coulter XLA UV/visible detector, multiwavelength (MWL) detection is able to collect sedimentation data not just for one wavelength, but for a large wavelength range in a single experiment. The additional dimension increases the data density by orders of magnitude, significantly improving the statistics of the measurement and adding important information to the experiment since an additional dimension of spectral characterization is now available to complement the hydrodynamic information. The new detector avoids tedious repeats of experiments at different wavelengths and opens up new avenues for the solution-based investigation of complex mixtures. In this chapter, we describe the capabilities, characteristics, and applications of the new detector design with biopolymers as the focus of study. We show data from two different MWL detectors and discuss strengths and weaknesses of differences in the hardware and different data acquisition modes. Also, difficulties with fiber optic applications in the UV are discussed. Data quality is compared across platforms.

Introduction

Analytical ultracentrifugation (AUC) is a powerful tool for the analysis of (bio)polymers and nanoparticles since the days of its invention in the 1920s by The Svedberg. A fundamental advantage is the fractionation of complex mixtures into their components. For nanoparticles, it has a size resolution in the Angström range (Cölfen & Pauck, 1997). Current detectors are based on the Beckman-Coulter Optima XL-A/I, which presently is the only commercially available AUC (UV/visible single wavelength and Rayleigh interference). A fluorescence detector is also available as a retrofit (MacGregor, Anderson, & Laue, 2004), and for nanoparticles, special turbidity detectors were developed on the basis of preparative ultracentrifuges, which allow for the detection of very broad particle size distributions by the application of gravitational sweep techniques (Mächtle, 1999, Müller, 1989). These detectors extend the range of applications for AUC and make it possible to exploit the presence of different chromophores, measure refractive index or turbidity for nonabsorbing molecules, or detect fluorescently tagged molecules with exquisite selectivity and high sensitivity. With the exception of the gravitational sweep technique (Mächtle, 1999), the observed signal is recorded as a function of radius and time and forms the basis for the extraction of hydrodynamic parameters. For the Beckman-Coulter XLA instrument, the wavelength of the light used to measure absorbance in the ultracentrifuge cell can be adjusted to match the chromophore of the analyte. However, this method of data acquisition has several important shortcomings: (1) In the XLA, a single scan requires about 2–5 min, depending on rotor speed and radial resolution, a time far too long to avoid temporal distortion for fast sedimenting analytes; (2) the total number of scans that can be collected, especially when multiple samples are measured, is limited by the slow scanning speed, reducing the number of scans available for analysis; and (3) the inability to collect data at multiple wavelengths during a single run in the XLA prevents the acquisition of spectral information. Many mixed systems display strong spectral diversity due to the extinction properties of their individual components. These properties could be exploited, but the XLA's design makes a multiwavelength (MWL) analysis prohibitive in terms of instrument time and sample requirements since each wavelength measurement requires an individual run. Though the Beckman-Coulter data acquisition software permits collection of three different wavelengths during a single experiment, the use of this feature is not practical because (a) the number of scans for each individual wavelength is reduced by two-thirds and (b) the monochromator is not guaranteed to reset to the same wavelength while cycling through the different wavelengths, causing changes in recorded absorbance due to changes in the extinction coefficients. Together, these limitations hinder scientific progress in the high-resolution analysis of UV/visible absorbance data from the analytical ultracentrifuge. To overcome these limitations, an MWL detector was developed for the AUC (Bhattacharyya, 2006, Bhattacharyya et al., 2006, Karabudak, 2009, Strauss et al., 2008) within the framework of the open AUC project (Colfen et al., 2010). The central part of this detector is a CCD array-based spectrometer, which is able to acquire a full UV/visible spectrum in 1 ms. Each of the 2048 pixels of the linear CCD array corresponds to a fixed wavelength reading. The basic design of the current detector is described below and by (Strauss et al., 2008). The data acquisition and control software of this detector was recently improved (Walter et al., 2014). This detector adds a spectral dimension to the hydrodynamic characterization by AUC and has enabled AUC experiments with so far unsurpassed information content (Backes et al., 2010, Karabudak, Backes, et al., 2010, Karabudak, Wohlleben and Colfen, 2010). Due to the attenuation of UV light intensity by fiber optic cables, the detector has so far primarily been used for wavelengths > 300 nm, which is sufficient for many colloidal samples, which absorb light in the visible range. However, limited measurements of important biopolymers like proteins have been made (Bhattacharyya, 2006, Walter et al., 2014, Walter et al., 2015), and no measurements of DNA have yet been published (Bhattacharyya, 2006, Walter et al., 2014, Walter et al., 2015). So far, the study of biopolymers with MWL AUC has not been the target of research. Here, we show that a fiber-based MWL detector system offers sufficient data quality to permit the investigation of biopolymer samples absorbing in the UV, despite the relatively low light intensity available in the UV. We predict this technology will prove particularly useful for the study of mixtures and heterointeracting biopolymer systems with components having distinct spectra, and build off of the work previously explored by AUC methods using several wavelengths for analysis (Cole, 2004, Lewis et al., 1994).

Section snippets

Development of MWL Absorbance Detectors

Note: In the literature, the terms MWL and MWA are used interchangeably for the same type of MWL detector. We reserve the term “MWL” for the early detector generations and refer to MWA as the third generation.

Second-Generation MWL Open AUC Design

The essential elements of the second-generation MWL optical detector (Strauss et al., 2008, Walter et al., 2014) are as follows, beginning with the light source: A 5-W Xenon flash lamp module (Hamamatsu 9455) is coupled to an OZ optics 200 μm UV/visible fiber with a vacuum feed-through adapter. The fiber end is positioned before a 10-mm biconvex quartz lens and the resulting collimated light is passed to a 45-degree flat mirror, directing it up through the optical path of the sample cell

Open AUC Data Format and Management

The MWL data are written by the acquisition software in a binary file format shown in SI 1 (http://dx.doi.org/10.1016/bs.mie.2015.06.033). Any version changes will be made available on the web (wiki.bcf2.uthscsa, n.d.-b). This step serves multiple purposes. First, it averages scans that were collected with adjacent wavelengths. The averaging of adjacent scans is reasonable since the resolution of the diffraction grating used in the optics is less than the pixel density of the CCD array, which

Data Visualization and Experimental Results

The data collected from a MWL experiment are first examined as the raw intensity counts from the spectrometer versus the radial dimension of a cell channel for one wavelength, compiling all of the scans from a run on to a single plot. An example is shown in Fig. 3, for an experiment with a mixture of nucleic acid and protein biopolymers, here DNA fragments and bovine serum albumin (BSA).

The right and left vertical indicators of Fig. 3 designate the region above the air–liquid meniscus where the

Qualitative Visualization of Data without Analysis

The absorbance data constituting a sedimentation profile provides the visual result familiar to the AUC researcher, as in Fig. 4. The 2D sedimentation profile is compiled to include wavelength spectra as a third visual dimension in a MWL data viewer such as UltraScan or the LabView-based viewer profiled above. Playing the scans through as the time domain of a 3D movie provides an instant and intuitive understanding of the time lapse sedimentation process in an experiment. The absorbance spectra

Summary and General Discussion of the Impacts of MWL AUC Data in the Field of Biopolymers

Two independent implementations of an MWL optics are detailed above. Both systems have an acceptable data quality at the wavelengths relevant for biopolymer analysis, despite the obstacle of fiber solarization, which significantly decreases the dynamic range for measurements at these wavelengths. Most importantly, the scope for multiplex analyses, by exploiting distinct absorbance spectra of various components in mixtures, is significantly increased and the number of addressable chromophores is

Acknowledgments

J.P. and H.C. thank the Center for Applied Photonics (CAP) at the University of Konstanz for financial support of this work. F.K. and K.S. gratefully acknowledge Tina Franke at Nanolytics for excellent technical assistance. We thank Aysha Demeler and Blanca Hernandez-Uribe for providing the DNA sample used in this study. B.D. wishes to acknowledge support by NSF Grant ACI-1339649.

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