Design of luminescent biochips based on enzyme, antibody, or DNA composite layers

摘要

The use of beads bearing bioactive molecules to develop generic biochips based on chemi- and electro-chemi-luminescent deteciton was evaluated. The biochips were composed of arrayed biosensors, including enzyme-charged beads, antigen-charged beads, or oligonucleotide-charged beads, entrapped in poly (vinyl alcohol) (PVA-SbQ) photopolymer. In each case the sensing layers were spotted at the surface of a glassy carbon electrode as 0.3 μL drops, generating 500-800 μm spots. The luminescent reactions were either catalysed by horseradish peroxidase or triggered by application of a +850 mV potential between the glassy carbon electrode and a platinumpseudo-reference. Enzyme biochips were esigned for the concomitant detection of choline, glucose, glutamate, lactate, lysine, and urate, based on the corresponding oxidase-charged beads and the electro-chemiluminescent (ECL) reaction with luminol-immobilised beads of the hydrogen peroxide produced. Limits of detection of 1 μmol L~(-1) for glutamate, lysine and uric acid, 20 μmol L~(-1) for glucose, and 2 μmol L~(-1) for choline and lactate were found with detection ranging over three decades at least. Use of the electro-chemiluminescent biochip was extended to a tri-enzymatic sensing layer based on kinase-oxidase activity for detection of acetate. A reaction sequence using acetate kinase, pyruvate kinase, and pyruvate oxidase enabled the production of H_2O_2 in response to acetate injection in the range 10 μmol L~(-1) to 100 mmol L~(-1). Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realised for the model detection of human IgG. Biotin-labelled anti-human IgG were used in a competitive assay, in conjunction with peroxidase-labelled streptavidin. Free antigen could then be detected with a detection limit of 25 pg (10~8 molecules) and up to 15 ng. In a similar way, the use of oligonucleotide-immobilised beads enabled the realisation of DNA-sensitive biochips which could be used to detect a biotin-la-belled sequence al a level of 5 * 10~8 molecules.