New possibilities for measurement

1st April 2013

A new system configured for both microvolume pedestal measurements as well as cuvette measurements gives scientists the flexibility to use the unique capabilities of a novel microsample retention system as well as those of traditional cuvettes. This dual functionality is ideal as microsample analyses continue to push the limits of detection and molecular techniques continue to evolve that require the flexibility to perform conventional and unconventional quantitation methods.

Devices are being developed that integrate microsample technologies with simple 'sample-in answer-out' capability. As sequencing, PCR (polymerase chain reaction), microarrays and other molecular techniques continue to use ever smaller amounts of sample, the quality control of these samples is essential.

In order to keep pace with the miniaturisation of sample volume throughout all of molecular biology, new quantification methods had to be developed. To meet this challenge, Dr Charles Robertson, then a physicist at the E.I DuPont Experimental Station, developed an elegant solution - remove the containment device altogether (ie cuvettes). The central idea is based on the concept that the containment device itself was a limiting factor in reducing volume. Removing the containment device in effect removes the limitation of having to fill a certain minimum volume in order to take a measurement. But how is this done? The answer lies in using the physical property of the sample itself, namely surface tension, to hold itself in place during the measurement cycle. Combining fiber optic technology with the inherent surface tension of liquids resulted in the development of a unique sample retention system that is the basis of NanoDrop technology.

The Thermo Scientific NanoDrop 2000c Spectrophotometer employs this sample retention system by using a pair of optical pedestals to hold the sample during measurement. The user places a droplet of sample (usually 1ul of sample for aqueous solutions of nucleic acids) onto the lower optical pedestal (Fig.1) and lowers the lever arm. The sample makes contacts with both optical surfaces, forming a vertical liquid bridge (Fig.2). Light from a xenon flash lamp fires through an optical fibre embedded in the upper pedestal, then passes through the sample, and is collected by another optical fiber embedded in the lower optical pedestal. The light then continues to an internal CCD detector to provide the requisite data. The software displays a full UV-Vis spectrum as well as a calculated concentration of the sample being measured.

The absence of a solid containment barrier allows the distance between the optical surfaces to change in real time. The system finds the correct transmittance of light through the sample column by rapidly changing the distance or path length from 1 mm to 0.2mm, 0.1mm, and 0.05mm (Fig.3). According to the basic laws of spectroscopy, shorter path lengths allow for higher concentrations to be measured. By having four incrementally shorter path lengths, the new system has the broadest dynamic measurement range of any spectrophotometer, essentially eliminating the need to perform sample dilutions. The system automatically finds the optimal path in less than five seconds.

Greatly reducing the volume necessary to perform measurement has several advantages. Conserving limited, often highly valuable samples is paramount in situations that involve extraction of biomolecules such as DNA, RNA, and proteins, from a limited cell mass. There are a wide variety of circumstances in which the sample mass is very limited and require microsample quantitation post extraction. These include samples derived from laser-capture microdissection, needle biopsies, tumor subtyping, surgical resections, forensic specimens, extremophiles and many more. Virtually any situation in which the sample is derived from limited cell mass is applicable for NanoDrop technology quantitation. Even though microsample pedestal measurements are ideal for limited cell mass scenarios, the speed of the system also makes it an attractive alternative to traditional quantitation methods for situations in which the quantity of sample is plentiful.

The microsample quantitation capability has greatly increased the efficiency of many molecular workflows throughout the life sciences. These workflows range from expression studies including microarrays and quantitative real-time PCR, to clinical workflows such as HLA (human lymphocyte antigen) typing for organ transplantation. Scientists would often forgo taking measurements at various time points throughout a workflow due to limited sample, limited time, or both. Reducing the amount of sample required for measurement as well as increasing the speed of the measurement itself, allows for more quality control steps to be performed. By allowing measurements to be performed at crucial steps throughout a given workflow, microsample quantitation technology greatly increases the chances for success.

For example, expression studies often require extracted biomolecules such as RNA to be treated at various steps in preparation for a downstream assay such as microarray analysis. Prior to microsample quantitation, many quality control steps including the initial RNA extraction as well as checking the fluorescence labelling efficiency of the final probe were simply not performed - often resulting in failed arrays. Providing a fast, reliable way to check samples at various stages of microarray probe development, from extraction through labeling, greatly improves the chance for a successful array.

NanoDrop instruments have been widely accepted in common research environments, but are increasingly being used for clinical applications. The differentiating factor between molecular biology techniques performed in basic research environments and the same techniques performed in medical settings is simply that the results are used for clinical purposes. The techniques themselves remain the same. Due to the often limited amounts of material acquired from clinical samples, reducing the amount of volume required for quality control steps is important. This is the main reason NanoDrop microsample quantitation is being adopted in several areas of molecular diagnostics.

One example is sequence-based genotyping in which microsample technology is being used to quantify critical biomolecules at several steps during the diagnostic workflow. After a clinical specimen is acquired, DNA extraction is performed. Using a minute amount of elution, the NanoDrop 2000c determines the concentration and purity of the extracted sample. This information is critical for optimising the next step in the process - DNA amplification by PCR (polymerase chain reaction). The microsample quality control measurement not only conserves the maximum amount of the original genetic material, it allows the clinician to determine the smallest amount of template DNA that can be used for a successful PCR reaction. Post amplification, the instrument can also be used to measure the final concentration of PCR product. This measurement is used to optimise the sequencing reaction, which requires a specific ratio of DNA to primer concentration. By using microsample quantitation instrumentation, quality control steps are easy to perform throughout the process, without compromising accuracy or consuming large portions of sample. The same is true for many molecular diagnostic workflows, such as microarray-based diagnostics and tissue typing for patient-donor crossmatching.

Furthermore, laboratories involved in medical research are continually developing new clinical tests that use molecular biology techniques. For example, the development of solid tumor testing is often extremely difficult due to the small amounts of available tumor cell mass. The samples are often difficult or simply impossible to reacquire. The amount of genetic material extracted from a specific solid tumor may be so limited that the only possible method of measuring the sample is by microsample quantitation.

As more molecular biology techniques are integrated into the clinical setting, microvolume quality control steps that are successful in the research environment are applicable to molecular diagnostics. By consuming the least amount genetic material derived from precious medical samples, NanoDrop technology is proving to be an important tool for quantifying biomolecules.

Although the sample retention system represents a fundamental paradigm shift away from traditional containment devices, conventional devices do have their benefits. Temperature controlled experiments and samples that are volatile lend themselves to traditional containment devices. The NanoDrop 2000c combines both the microsample retention system with a traditional cuvette mechanism (Fig.4). The light path is redirected from the vertical orientation of the sample retention system to the horizontal orientation of the cuvette holder mechanism. The dual arrangement allows flexibility to take advantage of the broad dynamic range and microvolume capability of pedestal measurements as well as methods in which traditional cuvette measurements may be more suitable.

Spectrophotometers have long been used to estimate the growth stage of a microbial cell culture by using the empirical observation that light scattering caused by the cell culture turbidity can be measured. Although this observation is empirical rather than true absorbance, most scientists are familiar with the absorbance values using a standard one centimeter cuvette and closely correlate those values with the growth stage of their cultures. The dual capability of the NanoDrop 2000c allows investigators to measure cell cultures in vivo, then subsequently measure extracted biomolecules using the microsample pedestal method. Due to the potential evaporation of microvolume samples, kinetic studies and other assays that also require several time point readings are more amenable to cuvette measurements. The NanoDrop 2000c cuvette mechanism contains a temperature control option for common time point assays such as those used for enzymatic kinetics studies.

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Philippe Desjardins is scientific marketing manager for NanoDrop instruments at Thermo Fisher Scientific, Wilmington, USA.






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