Bioanalysis: Its past, present, and some future

Abstract

An overview of about 100 years of bioanalysis is here disastrously attempted. The beginning of rigorous analytical systems can perhaps be traced back to the building and testing of the analytical ultracentrifuge by Svedberg and the apparatus for moving-boundary electrophoresis of Tiselius, both systems relying on expensive and hard to operate machines. In the sixties, Porath discovered porous beads for the determination of relative molecular mass (Mr) of proteins, based on the principle of molecular sieving. Concomitantly, Svensson and his pupil Vesterberg described a revolutionary principle for fractionating proteins in a nonisocratic environment, based on generation of stable pH gradients in an electric field, a technique that went down to history as isoelectric focusing (IEF). Polyacrylamide gel electrophoresis (PAGE), with the brilliant idea of discontinuous buffers, was brought to the limelight and in 1967, sodium dodecyl sulfate (SDS)-PAGE was described, permitting easy assessment of protein purity and reasonable measurements of Mr values of denatured polypeptide chains. By the mid seventies, another explosive concept was realized: orthogonal combination of two unrelated techniques, based on surface charge and mass fractionation, namely, two-dimensional (2-D) PAGE already in the very first papers by O'Farrell elaborated to its utmost sophistication. The eighties saw the systematic growth of 2-D PAGE, accompanied by systematic efforts to develop instrumentation for large-scale production of 2-D maps and computer evaluation for 2-D map analysis, based on the sophisticated algorithms adopted by astronomers for mapping stars in the sky. Another fundamental innovation in the field of IEF was the discovery of immobilized pH gradients (IPGs) that brought the much needed reproducibility in 2-D maps while allowing exquisite resolution in very narrow pH ranges. The nineties were definitely the decade of capillary zone electrophoresis, with the concomitant concept of automation and miniaturization in electrokinetic methodologies. Also 2-D map analysis witnessed a big revival, thanks to the adoption of IPGs for the first dimension. The enormous progress of mass spectrometry resulted in first reports on the analysis of macromolecules and the building of data bases on gene and protein banks. The third millennium is, perhaps, exasperating the concept of miniaturization at all costs, while not disdaining increasingly larger maps for 2-D analysis of complex protein mixtures.

Optimization of the porous structure and polarity of polymethacrylate‐based monolithic capillary columns for the LC‐MS separation of enzymatic digests

“…First, the intact proteins are separated by means of twodimensional gel electrophoresis, a process introduced by O'Farell more than 30 years ago [3]. The first dimension-isoelectric focusing-is based on differences in isoelectric point, followed by the second dimension -separation via polyacrylamide gel electrophoresis (PAGE) -involving differences in molecular size [4]. The separated proteins are then stained to visualize them, the spots excised, destained, digested, desalted, and finally identified by mass spectrometry.…”

CE microchips: An opened gate to food analysis

“…Bioanalytical and clinical analyses have been the preferred fields of application of miniaturized CE [24][25][26][27][28]. Indeed, the birth of the m-TAS "lab-on-a-chip" concept is intimately linked to these areas because of the unique suitability in the decentralization of analysis ("point-of-care-testing").…”

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

“…To achieve better separation, a relatively complicated procedure based on 2-D PAGE has been used for the separation of these enzymes in the extracting cytosol from rat hepatocytes [2], based on the fact that the enzymes coexist with many other proteins in the cited hepatocytes. However, 2-D PAGE requires higher protein concentrations or higher loading sample volumes when the proteins are detected with the CBB-R250 staining method, the experimental operation being tedious [14,15]. Furthermore, the lack of a highly selective staining method especially for the detection of the bands of the enzymes from complicated matrices limits its application.…”