Design of luminescent biochips based on enzyme, antibody, or DNA composite layers.
The use of beads bearing bioactive molecules to develop generic biochips based on chemi- and electro-chemiluminescent detection 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 microL drops, generating 500-800 microm 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 platinum pseudo-reference. Enzyme biochips were designed 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 micromol L(-1) for glutamate, lysine and uric acid, 20 micromol L(-1) for glucose, and 2 micromol 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 H2O2 in response to acetate injection in the range 10 micromol 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-labelled sequence al a level of 5x10(8) molecules.
Marquette
Christophe A
CA
Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013 CNRS, Université Claude Bernard Lyon 1 Bât. CPE, 43 Bd. 11 Nov. 1918, 69622, Villeurbanne Cedex, France. Christophe.marquette@univ-lyon1.fr
Thomas
Daphné
D
Degiuli
Agnès
A
Blum
Loïc J
LJ
D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002794:Choline / Q000032:analysis; D004247:DNA / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry*; D005947:Glucose / Q000032:analysis; D005973:Glutamine / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000032:analysis; D008163:Luminescent Measurements; D008239:Lysine / Q000032:analysis; D014527:Uric Acid / Q000032:analysis
D000906:Antibodies; D004798:Enzymes; D005973:Glutamine; D014527:Uric Acid; D019344:Lactic Acid; D004247:DNA; D005947:Glucose; D008239:Lysine; D002794:Choline