Direct Detection of Biomolecules in a Capillary Electrophoresis−Chemiluminescence Detection System

Abstract

Direct detection of biomolecules, such as alpha-amino acids, peptides, and proteins, was accomplished using a capillary electrophoresis-chemiluminescence detection system, in which a luminol-hydrogen peroxide-Cu(II)-catalyzed chemiluminescence reaction was utilized. Biomolecules migrated in the capillary, where they mixed with luminol and the Cu(II) catalyst included in the running buffer. The capillary outlet was inserted into a batch-type chemiluminescence detection cell with hydrogen peroxide-supplemented electrolyte solution. Chemiluminescence was observed at the tip of the capillary outlet. The chemiluminescence peak from biomolecules appeared due to the enhancement of Cu(II) catalytic activity for luminol-hydrogen peroxide chemiluminescence. The Cu(II) was more catalytically active when it interacted with biomolecules forming Cu(II)-biomolecule complexes. In this study, biomolecules were directly separated and detected in a capillary electrophoresis-chemiluminescence detection system. Twenty alpha-amino acids, 4 peptides, and 11 proteins were examined. Most of them were detected with satisfactory CL intensity response. Glutamic acid, an alpha-amino acid, was detected at concentrations ranging from 2.0 x 10(-7) to 1.2 x 10(-5) M with a detection limit (S/N = 3) of 1.0 x 10(-7) M (0.6 fmol). Glycylglycine, a peptide, was detected at concentrations ranging from 1.7 x 10(-7) to 1.2 x 10(-5) M with a detection limit (S/N = 3) of 1.7 x 10(-7) M (0.9 fmol). Hemoglobin, a heme protein, in which the heme structure was independently catalytically active, was detected at concentrations ranging from 1.2 x 10(-7) to 1.0 x 10(-5) M with a detection limit (S/N = 3) of 1.2 x 10(-7) M (0.6 fmol). Representative mixtures of alpha-amino acids and peptides were well detected with superior separation.

Capillary electrophoresis of proteins 2003–2005

“…Chemiluminescent detection of peptides and proteins labeled with luminol was applied to CZE [123]. For hemoglobin, a detection limit of 1.2610 27 M (0.6 fmol) was achieved.…”

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

“…The increased CL intensity was probably caused by the increased catalytic ability of Cu (II) after formation of Cu (II)-analyte complex. Tsukagoshi (37) suggested that the catalytic ability of Cu (II) is maximal when two of its coordination sites are occupied by a ligand, and the chelation reactions between Cu (II) and isoniazid (38)/p-aminosalicylic acid (39,40) have been reported in the literature.…”

“…When running buffer was used to prepare the working solution, the CL intensities of the analytes decreased with time gradually after preparation. The reason was probably that Cu (II) formed a complex with four occupied sites prior to injection (37). To overcome this problem, H 3 BO 3 buffer only with luminol was used to prepare working solution, and no significant decrease of CL intensity was found.…”

Enhancing Effect of Phenylboronic Acid Compounds and Their Interactions with the Diol Groups of Saccharides in a Capillary Electrophoresis-Chemiluminescence Detection System

“…[1][2][3][4] We also analyzed many types of sample, including metal ions, metal compounds, amino acids, peptides, proteins, nucleic acids, saccharides, phenolic compounds, fluorescence compounds, and liposomes, using these systems. [5][6][7] We have determined the significant features of CE with CL detection systems through our previous research. 8,9 Naturally, while absorption and fluorescence detection in CE are carried out in an on-capillary manner, CL detection is brought about by endcapillary (postcolumn) detection.…”