Next-Generation AUC Adds a Spectral Dimension

Development of Multiwavelength Detectors for the Analytical Ultracentrifuge

Joseph Z. Pearson*; Frank Krause†; Dirk Haffke*; Borries Demeler‡; Kristian Schilling†; Helmut Cölfen*,1    * Physical Chemistry, Department of Chemistry, University of Konstanz, Konstanz, Germany
† Nanolytics GmbH, Potsdam, Germany
‡ Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
1 Corresponding authors: email address: Helmut.Coelfen@uni-konstanz.de

1 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, & 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, 2015), and no measurements of DNA have yet been published (Bhattacharyya, 2006; Walter et al., 2014, 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, Shrager, & Kim, 1994).

2 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.

2.1 First-Generation MWL

In order to address the shortcomings of the current XLA design, the user community has focused on new detector development and new data management paradigms. The design of the MWL system for the Beckman-Coulter Optima L, XL, and XL-A platform has been iterated through several generations. The first-generation design, described in Bhattacharyya et al. (2006), featured the flash lamp and spectrometer outside of the vacuum chamber, each connected to the optical system by fiber optic cables passing through the vacuum chamber wall. This resulted in low light intensities.

2.2 Second-Generation MWL Developments

A second-generation design, described in Strauss et al. (2008), removed the fiber optic component on the detector side and installed the spectrometer directly above the objective lens of the optical path. The detection systems profiled in this chapter have evolved from the second-generation design, highlighting advancements of two independent implementations. One, a continuation of the open source device developed in the Cölfen lab (Bhattacharyya, 2006; Karabudak, 2009), and a second redesign developed at Nanolytics’ in 2013. Both implementations are based on the Beckman-Coulter ultracentrifuge platforms (Nanolytics: Optima L and Open AUC: Optima XL/XL-A), and share similar hardware architecture. They differ mainly in the data acquisition software. In these designs, at incremental radial positions, a complete UV/visible spectrum is captured on a CCD chip, resulting in radial scans of a sample sector. The Open AUC MWL developments have proceeded with incremental advances to the second-generation design of (Strauss et al., 2008), outlined below, and have been...