NanoDrop Microvolume Spectrophotometer Applications

The Acclaro reference libraries currently support detection of protein, phenol, guanidine HCl, and RNA in dsDNA samples. They also support detection of protein, phenol, guanidine isothiocyanate, and dsDNA in RNA samples. Finally, they allow detection of DNA in protein samples. Additional contaminants will be added to the libraries in the future.

Contaminant identification gives researchers two important pieces of information. First, it identifies specific contaminants that are likely to be present in the sample. This information can help scientists troubleshoot difficult extractions or purifications and make decisions regarding sample use in downstream experiments. Second, it provides a corrected concentration of the analyte. When setting up downstream reactions such as PCR where DNA concentration is a critical parameter, the corrected concentration will help scientists ensure the success of downstream experiments.

For example, RNA contamination in genomic DNA preparations is a common issue in molecular biology workflows. In traditional spectrophotometry, the presence of copurified RNA can artificially inflate the concentration of DNA. By using the full spectral data and multivariate mathematical algorithms available in the Acclaro software, researchers can identify RNA contamination in a DNA sample and see the corrected DNA concentration results.

The MIQE guidelines—minimum information for publication of quantitative real-time PCR experiments—require that input RNA quantity and purity are reported in publications that use qPCR.

Purified protein samples can be accurately measured using direct absorbance at 280 nm, which is mostly due to the aromatic chains on the Trp and Tyr amino acids. Protein A280, a preconfigured application in our software, is the most popular quantification method because it is fast and simple, requires no reagents or standard curves, and consumes very little sample.

Unlike nucleic acids, each pure protein has a unique Beer-Lambert extinction coefficient based on its amino acid sequence. For accurate results, select the correct sample type from the menu or enter its protein extinction coefficient manually. The NanoDrop One and NanoDrop Eight Protein Editor feature allows you to save the extinction coefficients of specific proteins so that you can customize your Sample Type options.

In addition to concentration, NanoDrop spectrophotometers can provide information about contaminants in the sample. Acclaro Sample Intelligence Technology (included on the NanoDrop One/One^C and NanoDrop Eight instruments) uses mathematical algorithms to detect nucleic acids in protein samples and correct the concentration result as needed. Sample purity can also be assessed by measuring its A260/A280 ratio; a value >1 may indicate nucleic acid contamination in the protein sample.

What if your protein or peptide lacks Trp and Tyr residues and thus cannot be measured using the A280 application? Since a protein’s peptide backbone absorbs light at 190–220 nm, peptides that lack Tyr or Trp residues can be quantified using absorbance at 205 nm. Protein A205 is a preconfigured application in NanoDrop software.

Proteins that have significant amounts of Trp and Tyr can also be quantified using the A205 application by selecting the Scopes method option. In fact, the A205 method has some advantages over the A280 protein method such as lower protein-to-protein variability (because A205 extinction coefficients are not based on amino acid composition) and higher sensitivity (because of proteins’ high molar absorptivity at 205 nm). Although technical limitations made these measurements difficult in the past, NanoDrop sample-retention technology and low stray light performance have simplified quantification of small amounts of protein by A205 methods.

One limitation of the A205 method is that many commonly used protein buffers have absorbance at 205 nm. Before using this technique, we recommend checking the protein buffer for any contribution to the absorbance at 205 nm.

Proteins in complex mixtures such as cell extracts or lysates are best measured using a protein colorimetric assay such as the Bradford, BCA, Lowry, or Pierce 660nm assay. These assays provide protein-specific concentrations, avoiding absorbance from cell components that absorb in the UV range and would inflate A280. These and other applications are preconfigured on selected NanoDrop instruments.

Labeled antibodies or other fluorescently labeled proteins and metalloproteins can be quantified using the Proteins & Labels preconfigured software application that delivers the protein as well as the label concentration (up to 2 dyes). This application is available as a preconfigured application on NanoDrop One/One^C and NanoDrop Eight Spectrophotometers.

The mRNA vaccine against COVID-19 was the first mRNA vaccine to get FDA approval. Its success has re-ignited interest in using mRNA to deliver vaccines against a myriad of diseases including cancers. In vaccine production, quality checks of sample concentration and purity are useful at several steps in the process.

• In the sequencing stage, both the quality and quantity of the starting material are crucial for accurate results.
• In plasmid production, the purified plasma must be checked for genomic DNA and other contaminants.
• In in vitro transcription, the purified mRNA must be checked for purity before the mRNA solution is filled into the vaccine vials.

View our on-demand webinar to see how the NanoDrop One/One^C and NanoDrop Eight spectrophotometers can readily be deployed for all these tasks. And, with software available to aid compliance with FDA 21 CFR Part 11 regulations, these instruments are ready to be implemented into the mRNA vaccine workflow at a GMP laboratory.

Our trusted, premier NanoDrop One/One^C and NanoDrop Eight spectrophotometers can be used in regulated environments such as pharmaceuticals and biotechnology. These models can help you quantify and qualify DNA, RNA, and protein samples of only 1–2 µL in seconds and obtain full-spectrum data to confirm that samples are qualified for downstream applications.

In addition, optional Thermo Scientific Security Suite or Thermo Scientific SciVault software helps your lab stay compliant with US FDA data regulations every step of the way. These software modules enable regulatory compliance with part 11 of the US FDA’s Title 21 of the Code of Federal Regulations pertaining to electronic records and signatures (21 CFR Part 11).

The following sections show selected features of Security Suite software for the NanoDrop One/One^C spectrophotometer, which include user account access, audit trail capability, electronic signatures, automated data back-up, and more. SciVault software for the NanoDrop Eight instrument provides similar functionality.

The ever-changing regulatory environment faced by manufacturers in pharmaceuticals and other regulated industries can cause confusion and doubts about compliance. Instrument systems designed to meet regulatory requirements can relieve the burden on manufacturers. How do they do it? Watch our webinar on maintaining compliance in pharma QA/QC labs to see how two different instrument platforms—the Thermo Scientific Nicolet Summit FTIR Spectrometer (for raw and finished material QA/QC) and the NanoDrop One Spectrophotometer (for DNA, RNA, and protein quantification)— help labs assure compliance in their processes.

The growth of bacterial cultures can be monitored by measuring their optical density at 600 nm (OD₆₀₀). Although this is a central technique in microbiology, these optical density measurements often contain very little true chemical absorbance. Instead, they are representative of the amount of light scattered away from the instrument’s detector by the bacterial suspension.

You can measure OD₆₀₀ on the NanoDrop One/One^C and NanoDrop Eight spectrophotometers using the OD600 preprogrammed application. The software uses the Beer-Lambert equation and a user-entered cell number conversion factor to automatically convert the OD₆₀₀ value into the number of cells per milliliter.

Note that on the NanoDrop One^C instrument, the pedestal and cuvette options will give different OD₆₀₀ values. You can use a conversion factor to compare between pedestal and cuvette measurements.

On the NanoDrop One^C instrument, you can make time-based kinetic measurements on samples in cuvettes. You can designate up to three wavelengths from 190–850 nm for continuous absorbance monitoring at user-defined intervals in up to 5 stages. Cuvette measurements offer an extended lower detection limit and an optional 37°C heater and micro-stirrer.

You can create, edit, and save kinetics experiments as custom methods.

NanoDrop spectrophotometers come with pre-loaded methods for nucleic acid quantification (like dsDNA, ssDNA, and RNA) and protein quantification (like A280, BCA, and Bradford) as described in the sections above. You can also create custom methods on the NanoDrop One/One^C or NanoDrop Eight spectrophotometer to perform UV-Vis or other user-defined measurements to suit your needs, allowing the instrument to function as a conventional spectrophotometer. Custom methods can monitor and report up to 40 wavelengths from 190–850 nm and can be set up directly from the touchscreen or PC control software. Custom methods provide the flexibility to use your instrument for additional applications.

• Use predefined custom methods to analyze samples such as nanoparticles, chlorophyll, hemoglobin, glucose, and proteins via BCA assays.
• Create new custom methods to analyze your special samples and save the methods for future use.

As a case example, a spectrophotometer may be used to quantitate various forms of hemoglobin using the appropriate extinction coefficient and peak absorbance value.

• Oxyhemoglobin contains bound oxygen and exhibits absorbance peaks at 414 nm, 541 nm, and 576 nm.
• Deoxyhemoglobin does not contain bound oxygen and exhibits an absorbance peak at 431 nm.
• Methemoglobin cannot bind oxygen due to containing iron in the ferric state and exhibits an absorbance peak at 406 nm.

We created a custom method for the NanoDrop One/One^C instrument to measure the absorbance of hemoglobin at 406, 414, 431, 541, and 576 nm—the five wavelengths needed to differentiate oxyhemoglobin, deoxyhemoglobin, and methemoglobin. This result graph shows a typical absorbance spectrum for methemoglobin.

Chemometrics is the use of multivariate mathematical models and statistical techniques to determine quantitative concentration information of many components simultaneously. When used to analyze UV-Vis data from complex chemical samples (mixtures), chemometrics can provide a powerful approach to determining the concentration of chemical species that have overlapping spectra. Historically, the sophistication of its multivariate calibration models limited its use to those with deep knowledge of the field, so most data had to be transmitted to an experienced chemometrics expert for post-collection analysis.

Thermo Scientific NanoDrop QC Software brings powerful chemometric analysis to the NanoDrop One^C microvolume measurement platform. This combination is an attractive solution for a wide range of applications including petrochemical analysis, drug purity, polymer manufacturing, food dye applications, and many more applications where chemometric analysis is required.

NanoDrop QC Software simplifies and streamlines the development and deployment of chemometric methods. Scientists developing methods can collect spectra on the compounds of choice and import them into Thermo Scientific TQ Analyst Software to build the method. Methods can then be deployed directly to NanoDrop One^C UV-Vis Spectrophotometers around the world, allowing QC technicians to achieve pass/fail and other quantitative results on the factory floor.

Cuvette-based UV-Vis technology and PC-based chemometric software are staples in the quality analysis of liquids such as dyes, lubricants, and adhesives. The sample preparation and analysis of these solutions may include multiple time-consuming processes impacting the release of high-value products. Our innovative, auto-ranging pedestal UV-Vis spectrophotometer eliminates time-consuming steps such as dilution of highly concentrated samples and simplifies chemometric method development data analysis.

In our chemometrics application note, we demonstrate a step-by-step approach to create and validate a chemometric method that determines concentrations of individual azo dyes in a complex mixture. We show how NanoDrop QC Software can be used to obtain quantitative information in samples that have multiple components with highly overlapping UV-Vis spectra.

Access to real-world instrumentation is key to preparing and inspiring the next generation of scientists. Hands-on experience helps make science more approachable and scientific principles more memorable.

In particular, UV-Vis spectroscopy is a valuable tool for studying macromolecules such as proteins and nucleic acids in the biology classroom. The simplicity and affordability of NanoDrop instruments makes them highly suitable for use in undergraduate and even secondary science curricula. Experience with NanoDrop spectrophotometers can support lessons in genomics, immunology, microbial cell growth, enzyme kinetics, and the structure and function of the fundamental components of life.

This webinar documents how two high school teachers have integrated NanoDrop One Spectrophotometers into their classes and workshops.