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

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Abstract

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 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 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 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 H2O2 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 (108 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 5×108 molecules.

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Acknowledgements

The authors would like to gratefully thank Jean-Marc Fanjat for his skilful technical assistance during his time in our laboratory.

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Authors and Affiliations

  1. 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 A. Marquette, Daphné Thomas, Agnès Degiuli & Loïc J. Blum

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  1. Christophe A. Marquette
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  2. Daphné Thomas
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  3. Agnès Degiuli
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  4. Loïc J. Blum
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Correspondence to Christophe A. Marquette.

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Marquette, C.A., Thomas, D., Degiuli, A. et al. Design of luminescent biochips based on enzyme, antibody, or DNA composite layers. Anal Bioanal Chem 377, 922–928 (2003). https://doi.org/10.1007/s00216-003-2112-x

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  • DOI: https://doi.org/10.1007/s00216-003-2112-x

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