Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers.

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF6)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF6 which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2×10(5)±0.3 L mol(-1) and 0.8×10(4)±0.4 L mol(-1) for L- and D-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers-HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that L-fenoprofen replaced D-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.

Comparative signature diagrams to evaluate biophysical data for differences in protein structure across various formulations.

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher-order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8-anilino-1-naphthalene-sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high-resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.