Microvolume Analysis Since the Turn of the 21st Century

Back in the ancient days of science known as the 1990s, researchers who wished to analyze and quantify nucleic acids or proteins were relegated to time-consuming, labor-intensive practices. To take UV-Vis measurements of DNA or RNA solutions required multiple steps just to prepare samples for analysis. Highly concentrated samples needed to be diluted to achieve accurate measurements; these dilutions involved multiple manual steps like adding precisely measured amounts of water and/or buffer solution, with each step giving rise to the possibility of an error. With so many individual steps, generating accurate data was also a challenge in these early days before the turn of the 21^st century.

Traditionally, these quantification measurements were made on an ultraviolet-visible (UV-Vis) spectrophotometer. Taking advantage of the aromatic compounds that are intrinsic in the bases of nucleic acids or the amino acid residues of proteins, UV-Vis spectrophotometers are able to measure the absorbance of specific wavelengths of light by such compounds and thus generate information about the amounts of the substances of interest. These instruments typically use a 1 cm wide cuvette to hold a volume of 3 to 4 mL of a sample. While this method of analysis is completely viable and is still used in some places today, it is far from ideal. A desire to avoid painstaking sample dilutions and provide a much simpler workflow led, as such desires often do, to research and development of new technologies.

In 2001 a breakthrough instrument was launched: the microvolume spectrophotometer. The original instrument could perform an accurate quantitative analysis of biomolecules using just a single microliter of sample. The technique worked because the path length could be adjusted to ensure that the absorbance values stayed within the detection limits of the instrument. This meant that time-consuming dilutions could be avoided, and with that the chances for sample preparation errors could be reduced.

The microvolume spectrophotometer easily gained acolytes and its adoption by labs spread quickly. Why wouldn’t it? With sample analyses being performed faster and without the need for dilutions, the instrument gave researchers the ability to accomplish more tasks, more accurately. Later developments continued to enhance and expand on the ideas first developed in 2001.

The principles used at the turn of the 21^st century to quantitate nucleic acid have not changed, nor has the workflow: once a customer pipettes 1-2 microliters of their sample onto a metal pedestal within an instrument, another instrument pedestal is lowered, which touches the sample and creates a surface-tension bridge. By shining light through the sample and comparing it with a “blank,” an absorbance measurement can be calculated.

In 2008, a multi-sample analyzer was introduced, able to assess up to 8 multiple samples simultaneously. This offered higher throughput than anything that came prior. The particular instrument with this expanded capability allowed researchers to efficiently and accurately process and analyze up to eight protein and nucleic acid samples at a time.

Before more sophisticated software was developed, users had to manually compare two different purity ratios of a sample against set guidelines to infer the purity of their nucleic acid sample. With the advent of advanced mathematical algorithms (i.e., software programming), researchers were given the ability to go beyond simple assessment of purity to actually identify what contaminant might be present in a sample. Advanced software was developed that could provide four important advantages: 1) it could identify bubbles in a sample that might adversely affect readings; 2) the software could recognize when the liquid column was no longer in contact with the instrument’s top pedestal; 3) it provided on-board tech support and guided troubleshooting; and 4) it not only identified but it corrected for the presence of a contaminant. These advances provided customers with more knowledge about their sample composition, which allowed them to be more successful in downstream experiments.

Meeting Guidelines

The software as it exists today has the ability to adhere to a set of guidelines that help ensure reproducibility and or data security regulations like 21 CFR Part 11. Some users require that their research meets these guidelines, which codify criteria that the information researchers should provide when publishing data, to prove their research is accurate and reproduceable. That the software can do this saves significant time and effort on the part of the researchers, and gives confidence that the results are compliant with regulations.

The ability to identify and correct for impurities using chemometrics is one of the most important improvements throughout the two decades of ongoing development of microvolume analysis. Beyond that, enhanced software capabilities have helped drive the evolution of microvolume spectrophotometers’ capabilities to measure undiluted protein samples and provide regulatory-compliant data. As of 2020, the software algorithms even incorporated the ability to differentiate mammalian DNA from mammalian RNA. Advances like this will surely lead to even more beneficial developments down the line.

It may be difficult to keep track of all these historical developments across the years, but don’t worry: it’s all available in one helpful infographic.

The popularity of advanced microvolume analysis is readily apparent, and microvolume spectrophotometers are used throughout academia and across multiple industries.

What does the future hold? Nobody knows for sure, but if the goal is to make scientists’ lives easier, accelerate their research, and enable users to publish scientific papers, then fast, efficient and dependable microvolume analysis is sure to be a part of it.

Additional resources

Editor’s Note: This month, the Thermo Scientific™ Nanodrop™ Spectrophotometer, the first microvolume spectrophotometer to perform an accurate quantitative analysis of biomolecules using just a single microliter of sample, celebrates over 100,000 units built and used throughout academia and across multiple industries since its introduction in 2001.

Meeting Guidelines

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