Editorial Article
Wednesday February 03, 2010
by Catherine Shaffer
Spectrophotometry is a technology that has long been considered mature, and advances in instrumentation tend to focus on fine-tuning the technical details, such as light source or detector, without changing the basic technology. Today’s life science laboratories throughout industry and academia are continually developing methods that require less material for analysis. Quality control tools that can take measurements with only minimal amounts of sample are essential. In early 2000, the introduction of the first microvolume spectrophotometer, the Thermo Scientific NanoDrop 1000, met this demand by enabling laboratories to perform quality control checks with sample volumes as small as 1 uL. Since that time, NanoDrop™ products have been integrated into more than 24,000 sites throughout the scientific community.
NanoDrop technology is a patented sample retention system that uses inherent surface tension to capture and hold small amounts of sample between two optical pedestals during measurement. Since there is no cuvette or capillary containment device required to hold the sample, the path length is determined by the distance of the liquid sample column formed between the two optical surfaces. The auto-ranging capability of the newly introduced NanoDrop 2000 and 2000c models, optimizes the path length automatically for any given sample, resulting in an extensive dynamic range (2 ng/uL to 15,000 ng/uL for dsDNA), essentially eliminating the need to perform dilutions.
Applications range from expression studies that use microarray and quantitative RT-PCR, to HLA typing for organ transplantation. Although NanoDrop spectrophotometers are full UV-Vis instruments that can measure any common biomolecule that absorbs light in this range, quantification of DNA, RNA, and protein are most common. Philippe Desjardins, scientific marketing manager for NanoDrop products, states, “One big misperception of NanoDrop technology is that it is used exclusively for applications that have limited sample, such as laser-capture microdissection, needle biopsies, and forensics. Although NanoDrop is an enabling technology for such cases, the vast majority of our instruments are used for taking measurements when sample is plentiful.” That is because NanoDrop is often integrated into larger scale workflows to increase efficiency without compromising accuracy. In fact, the NanoDrop 2000c model has both the microvolume pedestal system as well as a cuvette option. “Why did we put a cuvette into the system?” says Desjardins. “There are some special circumstances where a cuvette is actually preferred, such as for volatile samples, microbial cell cultures, or kinetic studies, making this an ideal 2-in-1 compact spectrophotometer.”
The NanoDrop 8000 is an eight-sample version of the original NanoDrop microvolume spectrophotometer and is increasingly being integrated into bioproduction facilities to increase efficiency. Desjardins comments, “For example, protein facilities that produce recombinant proteins and antibodies for human use previously required cuvette-volumes of material to be drawn off of a production batch and taken to a separate lab for quality assurance analysis. Now, with a NanoDrop 8000 spectrophotometer, you can perform those quality assurance checks in real-time right there on the production floor. The samples can be drawn at-line and tested without time delays. Also, the samples are not compromised by dilutions and are therefore truly representative of the production batch. This provides greater confidence in testing results, which is also advantageous from a regulatory perspective.”
Playing with path length has another advantage in that there is no need to build a standard concentration curve to calibrate the instrument. This is usually done by scanning multiple samples with different concentrations. The SoloVPE from C Technologies Inc., uses a fiber optic probe lowered into the sample to take absorbance readings. The light path in this case is vertical, running from the probe through the bottom of the cuvette. The probe can then be lowered in five micron increments, taking an absorbance reading or scan at each stage. Older techniques rely on changing the concentration variable in Beer's Law, introducing dilution errors, and raising the question as to whether the absorbance value is within the linear range of Beer's Law. Instead, by varying path length, a graph of absorbance versus path length is obtained whose slope is used to calculate concentration. This is known as slope spectroscopy. The instrument adjusts the path length automatically until the linear range is identified for the sample, eliminating any need to make dilutions of the sample. Like the Nanodrop instrument, the SoloVPE uses a vertical beam and an adjustable light source to create an adjustable space between source and detector. A main difference, however, is that with slope spectroscopy, the SoloVPE does not rely on absolute absorbance, and can make measurements using relative absorbance changes.
Says Craig Harrison, president of C Technologies, Inc, “What we did was we made the equivalent of several thousand cuvettes.... It's a very sophisticated yet simple way to get accurate measurements ... the comment we get more often than not is 'Why hasn't anyone done this before?' and 'Wow, this is so simple.'”
Picodrop has developed a different approach to microliter spectrophotometry. The Picodrop spectrophotometer uses a p10 pipette tip as the container for the sample volume. Light passes directly through the tip, which remains attached to the pipette. The sample is then removed the same way and transferred to the next step in the procedure. Says Jonathan Redfern, founder and CEO of Picodrop, “Our main advantage is complete recovery of the sample and complete containment of the sample. We like to think it's easier to use.”
The Picodrop is very effective for samples as small as 1 to 2 ul, or samples that are too weak to dilute further. In addition to conventional spectrophotometry applications like nucleic acid or protein quantification, the small volume offers novel applications. For example, a Picodrop scan can be used to quickly determine the ratio of two different colored dyes in a microarray experiment, and make a correction if necessary.
Because the sample does not contact any surface inside the instrument, Picodrop can be used for applications where contamination is a concern, such as cell culture samples or radioactive samples. In addition to the barrier formed by the walls of the pipette tip, Picodrop's electronic pipette will draw air into the bottom of the tip, to create an additional buffer space preventing contamination. A new liquid handling deck called the Seadragon, developed in partnership with Gilson, extends compatibility of the Picodrop to automated platforms.
Most people are aware of microvolume spectrophotometers, but may not be aware that these are devices that compare favorably with conventional spectrophotometers. There is no loss of accuracy in reducing the size of the cuvette or eliminating it entirely, and these new, smaller scale devices can be useful even in very large scale processes.