Minimizing Stray Light Advanced plate readers facilitate high-throughput dynamic light scattering. by Sigrid C. Kuebler, Senior Application Scientist, Wyatt Technology Corp.
For the past 30 years, laboratory scientists have performed conventional dynamic light scattering (DLS) measurements in the same labor-intensive way. DLS typically required someone to measure one sample after another in a single cuvette—manually: a process that was both time consuming and fraught with the potential for contamination and error.
With the emergence of microarray technology for drug research, the need for equally rapid screening methods has become increasingly important. Using disposable standard 96-, 384- and 1536-well plates, a high-throughput plate reader has been developed to address this growing need for rapid, automated and reliable DLS measurements.
While this concept of using standard well plates has previously been applied to other detection methods, notably UV and fluorescence assays, it has never before been used with a DLS instrument.
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In a DLS measurement, time-dependent fluctuations in the scattered light signal are measured using a fast photon counter. DLS measurements can determine the hydrodynamic radius of macromolecules or particles.
Light scattering is a technique that can be applied in either a batch or a chromatographic mode. In either instance the sample may be recovered at the end of the measurement. Since light scattering provides the weight-averaged molar mass for all molecules in solution, it is generally more useful to utilize the chromatography mode; each technique has unique advantages.
While absolute molecular weights can also be determined using mass spectrometry, membrane osmometry and sedimentation equilibrium (analytical centrifugation), only light scattering covers so broad a range of macromolecules including their oligomeric states. Most importantly, light scattering permits measurement of the solution properties of macromolecules. Sedimentation equilibrium runs require up to 72 hours, and a size exclusion chromatography/light scattering study may be completed in well under an hour, a batch mode analysis is finished within a few minutes. These comparatively short run times, coupled with the absolute determination of molar mass, size and the second virial coefficient (A2) make light scattering the method of choice for accurate and fast macromolecular characterization.
DLS measures the translational diffusion of molecules in a solution using Brownian motion. For example, as the molecules diffuse, their relative positions change with time. This causes fluctuations in the intensity of the scattered light because of interference. Small molecules diffuse quickly and generate signals that fluctuate slowly. The diffusion coefficient from these fluctuations is determined by autocorrelation analysis. Assuming the molecule is a uniform sphere, the Stokes-Einstein relationship enables the determination of the molecule’s hydrodynamic radius from the diffusion coefficient
Figure 1. Workflow from Interference of Light to Autocorrelation analysis to Stokes-Einstein Relationship.
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When performing light-scattering measurements, care must be taken to ensure high quality of the incident and scattered light (i.e., the optical paths need to be tightly controlled, and stray light must be minimized). Dust should be excluded from the sample, and the temperature needs to be controlled for the highest quality measurements and temperature studies. A high-throughput plate reader can provide a stable, temperature-controlled enclosure that only needs to be accessed when changing well plates. Additionally, the inclusion of a built-in attenuator can automatically ensure the optimum instrument sensitivity for each sample.
Automating DLS
Flow-through DLS instrumentation requires invasive measurements, where the sample is transferred from a microwell plate by a robot into a measurement cell for analysis. Methods use a robotic tip (either disposable or not) for sample transfer, and as such introduce the likelihood of contamination during the transfer processes. Also well plates are challenging to sample with a robotic tip and typically only smaller plates (96- and 384-well) can be used. Because significant time is spent transferring the sample, this process can account for as much as 90% of the analysis time. Moreover, if the study of aggregates is desired, the simple act of transferring these samples through capillaries can—and will—destroy the very aggregates the study has been initiated to characterize.
Next generation plate readers now feature non-invasive technology to measure samples directly within industry-standard microplates (96-, 384-, or 1536-well plates) using as little as 2 microliters of sample per well. No sampling system is required. No transfer or perturbation of the sample is necessary. No washing of robotic needles, flushing of solvent or sample-handling is ever needed.
After the plate is loaded into the instrument, the data are collected automatically—unattended—by the software. Once the data are collected, the samples can be recovered and the plates discarded.
Because no sample transfer is needed, an automated plate reader enables the user to perform completely non-invasive, non-perturbing experiments. Using the instrument eliminates cross-contamination from use of the same cuvette for multiple samples or from transferring samples. Additionally, sample measurements can be completely automated, which simplifies measurements of several aliquots of the sample, formulation buffers and repeat measurements. The samples can be easily recovered, or the plate can be used for further screening.
It is also possible to use industry standard disposable microwell plates. As a result, there is no need to clean the optical cell, nor the associated need for suitable wash liquids and a liquid waste reservoir. The technology has the same sensitivity and flexibility to act as a conventional DLS system. The user simply decides which mode to use to perform the measurements. Finally, the world’s most popular robotic plate handling systems can integrate this new DLS technology directly, making the system even more productive and automated.
Benefits
Batch DLS is a complementary biophysical tool for separation techniques such as size exclusion chromatography (SEC), analytical ultracentrifugation (AUC) and field flow fractionation (FFF). Compared with separation methods, DLS measurements with an automated plate reader can be done in seconds to minutes: a 96-well plate can be easily screened in an hour or less, whereas the same number of samples in SEC would require more than two days worth of measurements. Some laboratories use an automated plate reader to screen a multitude of samples and conditions rapidly and then pass promising candidates onto SEC, AUC or FFF as a secondary screen.
Additionally, this technique is non-perturbing, without interactions with any separation matrix or dilution. Therefore the aggregation state of a molecule can be detected accurately, which is imperative when looking at reversible aggregation. Additionally, the sample is easily recoverable or the microwell plate can be directly used for further analysis.
FFF-MALS application
A laboratory wanted to evaluate a large number of antibodies for their formation of aggregates prior to binding the antibodies to antigens. They were using FFF-MALS (a gentle separation method) to determine absolute molar mass of antibodies, fragments and large aggregates. Each FFF experiment, however, was time consuming. Samples are diluted during the separation. So, the laboratory used the Wyatt DynaPro plate reader to screen more than 80 different antibody formulations for the presence of aggregates in one afternoon – FFF measurements on the same number of samples would have taken more than a week.
The analysts then identified those samples with little or no aggregates present as the most promising candidates for additional analysis. Those samples were used for the binding studies and then analyzed by FFF-MALS. It would not have been possible to analyze all the antibody formulations by FFF because it would have been too time-consuming and the sample quantities were limited.
Conclusion
Next generation plate readers are suitable for any application where molecular size, stability and aggregation are of interest. Applications include characterizing proteins for size, homogeneity, aggregates and thermal stability over a wide range of formulation conditions; measuring the stability and size of liposomes, viral particles and drug delivery particles; determining aggregations constants and kinetics; optimizing protein crystallization conditions; monitoring folding of proteins and nucleic acids; and detecting and analyzing compound aggregates.
Even if only a few samples need to be analyzed at a time, the ease of use of the high-throughput plate reader encourages users to perform triplicate measurements of the same sample, repeat experiments and screen buffers in a rapid and automated fashion to ensure the most reliable data. The microwell plate can then simply be stored and unused wells can be filled for the next set of samples to be measured.
DLS technologies are evolving as critical automated tools. Compared to other techniques and tools, automated dynamic light scattering is a non-invasive, reproducible and sensitive method for quick and easy detection of aggregate-based inhibitors in a wide variety of solvents and additives.
For more information, contact: Wyatt Technology, www.wyatt.com.
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