Functional bioassays proteins

The applications of nanoparticles in biosensors can be classified into two categories according to their functions (1) nanoparticle-modified transducers for bioanalytical applications and (2) biomolecule-nanoparticle conjugates as labels for biosensing and bioassays. We intend to review some of the major advances and milestones in biosensor development based upon nanoparticle labels and their roles in biosensors and bioassays for nucleic acids and proteins. Moreover, we focus on some of the key fundamental properties of certain nanoparticles that make them ideal for different biosensing applications. 

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]. 

The quantitation of a protein that has a specific biological function, a hormone, for instance, may not give a true indication of its biological activity owing to the inactivation of some of the protein. For proteins that have definite biological functions the choice is between chemical quantitation and bioassays. For this reason the catalytic activity of an enzyme is more frequently measured than is its protein concentration. 

Fig. 5.17 Demonstration of MS-based bioassay functionality using a plant extract. MS instrument Ion-trap mass spectrometer (LCQ Deca, Thermo Electron), (a) MS analysis of pure extract by direct injection onto restricted-access column 2 in the absence of affinity protein, (b) Analysis of the same natural extract spiked with digoxin using the label-free MS assay method as shown in Fig. 5.15.

Cytokines, which are protein mediators produced by immune cells, are involved in the regulation of cell activation, growth and differentiation, inflammation, and immunity. Measurement of cytokine production, as determined by techniques such as bioassay, radioimmunoassay, and enzyme-linked immunosorbent assay, has been used to examine various immune functions. 

Methods to determine the potential biological activity of products obtained through recombinant DNA techniques are of fundamental importance. Despite the existence of numerous physicochemical techniques to characterize the protein product structure and the presence of contaminants, they provide little, if any, information about its biological potency. A bioassay is defined as a functional test, and no physicochemical test can measure the function. However, for some peptide hormones, which are less complex in structure than most cytokines, well defined physicochemical tests may be used to estimate biological activity for instance, the capillary electrophoresis analysis of a protein s isoform content if the specific activity of each one is known. 

To investigate whether bevirimat functions as a PR inhibitor, several bioassays were conducted, including a cell-free fluorometric assay using a synthetic peptide substrate of PR and experiments using a recombinant form of the Gag precursor protein Pr55Gag. Both assays showed no effect of bevirimat on PR function compared with the PR inhibitor indinavir. Indeed, bevirimat at nanomolar concentrations could inhibit the replication of HIV-1 isolates resistant to PR inhibitors. 

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