Biomolecules quantification

Sol-gel sensors can be easily prepared to function as site selectively templated and tagged xerogels (SSTTXs). The resulting platform is completely self-contained, and it achieves analyte recognition without the use of biomolecules,10 as shown for example by the selective detection and quantification of model compound 9-anthrol (Figure 6.11). 

The fast, sensitive, reliable, and reproducible detection of (bio)molecules including quantification as well as biomolecule localization, the measurement of their interplay with one another or with other species, and the assessment of biomolecule function in bioassays as well as in vitro and in vivo plays an ever increasing role in the life sciences. The vast majority of applications exploit extrinsic fluorophores like organic dyes, fluorescent proteins, and also increasingly QDs, as the number of bright intrinsic fluorophores emitting in the visible and NIR is limited. In the near future, the use of fluorophore-doped nanoparticles is also expected to constantly increase, with their applicability in vivo being closely linked to the intensively discussed issue of size-related nanotoxicity [88]. 

Among the many microscopy-based techniques for the study of biomolecules immobilised on surfaces, scanning probe microscopies (SPM) and especially atomic force microscopies (AFM) are arguably the most used techniques because of their molecular and sub-molecular level resolution and in situ imaging capability. Moreover, the invasiveness of AFM, which is less of a problem for the DNA molecules, is essential for another two functions, apart from the mapping of surface nanotopographies, namely the quantification and visualisation of the distribution of chemistry, hydrophobicity and local mechanical properties on surfaces and the fabrication of nanostructures. 

CAE employing antibodies or antibody-related substances is currently referred to as immunoaf-hnity capillary electrophoresis (lACE), and is emerging as a powerful tool for the identification and characterization of biomolecules found in low abundance in complex matrices that can be used as biomarkers, which are essential for pharmaceutical and clinical research [166]. Besides the heterogeneous mode utilizing immobilized antibodies as described above, lACE can be performed in homogeneous format where both the analyte and the antibody are in a liquid phase. Two different approaches are available competitive and noncompetitive immunoassay. The noncompetitive immunoassay is performed by incubating the sample with a known excess of a labeled antibody prior to the separation by CE. The labeled antibodies that are bound to the analyte (the immuno-complex) are then separated from the nonbound labeled antibody on the basis of their different electrophoretic mobility. The quantification of the analyte is then performed on the basis of the peak area of the nonbonded antibody. 

The detection and quantification of the presence of biomolecules at the surface is based on specific interactions taking place in the evanescent field, generated by the total internal reflectance or by the surface plasmon resonance. The latter is the key transduction principle in the optical bioanalysis and biosensing area (Narayanaswamy and Wolfbeis, 2004). Launched in the early 1980s in Sweden,... 

To comprehend and apply knowledge of key parameters in the quantification of biomolecules. 

There is often some confusion regarding the use of the terms accuracy and precision with respect to the quantification of biomolecules. While accuracy is defined by how closely a measured value matches the real or true value, precision describes how reproducible the measurements (repeated measurements) are with respect to each other, in other words how closely they match each other (see Figure 2.1). 

Each test method relies on one or more chemical and/or physical attributes of a biomolecule for detection and/or quantification. It is obviously impossible to captiue the sheer number of diverse methods that are used in biological, biomedical and environmental testing in this textbook. However, many of these methods... 

As mentioned above, spectroscopy is an umbrella term for a range of techniques that allow identification, quantification and determination of molecular stmcture of biomolecules. This technology also allows the bioanalytical chemist to follow key reaction pathways including, for example, the rate of an enzyme-catalysed reaction. 

High-performance liquid chromatography (HPLC) has emerged as one of the most useful tools for the bioanalytical laboratory. This is a powerful stand-alone method that can be used for the purification, separation and quantification of biomolecules. Notably HPLC analysis can be combined with other high-tech methods such as mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy to enable the identification of biomolecules. 

While straight MS analysis can yield important information in terms of identification, characterization and quantification of biomolecules, it becomes a much more powerful tool with further MS or when combined with other separation technologies. As noted earlier, these approaches include MS/MS, GC-MS, LC-MS and CE-MS. These methods have been extensively exploited in virtually all aspects of bioanalysis, and while fundamentally useful for peptide and protein analysis, these methods have also been used in the analysis of lipids, nucleic acids and a wide range of small molecules and drugs. The range of applications is obviously outside the scope of a book like this, but some indications of the uses of each of these techniques are given below. 

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