Abstract
Nucleic acids, proteins, and polysaccharides are the most important classes of biopolymers. The inherent properties of biomacromolecules are contrary to those of well-defined small molecules consequently raising a number of specific challenges which become particularly apparent if biomacromolecules are treated as objects in quantitative analysis. At the same time, their specific functional ability of molecular recognition and self-organization (e.g., enzymes, antibodies, DNA) enables us to make biomacromolecules serving as molecular tools in biochemistry and molecular biology, or as precisely controllable dimensional platforms for nanometrological applications. Given the complexity of biomacromolecules, quantitative analysis is not limited to the measurement of their concentration but also involves the determination of numerous descriptors related to structure, interaction, activity, and function. Among the biomacromolecules, glycans set examples that quantitative characterization is not necessarily directed to the measurement of amount-of-substance concentration but instead involves the determination of relative proportions (molar ratios) of structural features for comparison with theoretical models. This article addresses current activities to combine optical techniques such as Raman spectroscopy with isotope dilution approaches to realize reference measurement procedures for the quantification of protein biomarkers as an alternative to mass spectrometry-based techniques. Furthermore, it is explored how established ID-MS protocols are being modified to make them applicable for quantifying virus proteins to measure the HIV viral load in blood samples. As an example from the class of carbohydrates, the challenges in accurate determination of substitution patterns are outlined and discussed. Finally, it is presented that biomacromolecules can also serve as tools in quantitative measurements of dimensions with an example of DNA origami to generate defined dimensional standards to be used for calibration in super-resolution fluorescence microscopy.Graphical abstractQuantitative analysis of biomacromolecules is accompanied with special challenges different from low molecular weight compounds. In addition, they are not only objects but also tools applicable for quantitative measurements
Highlights
Biomacromolecules are naturally occurring macromolecular compounds that make up the essential building blocks of most types of biological systems
The methods and applications discussed in this perspective are developing strategies for the analysis of biomacromolecules at the nanoscale
Quantification of biomacromolecules that serve as diagnostic markers is a challenging task in medical diagnostics
Summary
Biomacromolecules are naturally occurring macromolecular compounds that make up the essential building blocks of most types of biological systems. This paper points out how Raman spectroscopy is currently being combined with isotope dilution approaches for realizing optical techniques as a possible alternative to ID-MS It addresses current activities aiming at modifying ID-MS protocols to make them applicable for quantifying virus proteins to measure the viral load of HIV in blood samples. It is shown that and how biomacromolecules can serve as tools in quantitative measurements of dimensions is presented on the example of DNA origami to generate defined dimensional test samples that can be used for calibration in super-resolution fluorescence microscopy. While most established reference methods in clinical chemistry are based on mass spectrometry [2], recent developments have demonstrated the capability of optical techniques such as Raman spectroscopy for this purpose [8] Both techniques unite molecular specificity with the advantages of reliability, accuracy, and repeatability during quantification when applied in conjunction with the concept of isotope dilution (ID). The DNA origamis can be modified to test for different distances as well as different wavelengths
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