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

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 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.

Mercaptoethylpyrazine promoted electrochemistry of redox protein and amperometric biosensing of uric acid

“…Various methodologies based on UV-vis spectrophotometry, chromatography, voltammetry, amperometry etc. have been developed for the accurate determination of UA (Thrasher and Abadie, 1978;Strochkova et al, 1997;Marquette et al, 2003;Perello et al, 2005;George et al, 2006;Popa et al, 2000). The electrochemical techniques are promising because of their fast response and high sensitivity.…”

Imaging Analysis Based on Electrogenerated Chemiluminescence

“…Furthermore, the detection of glucose, lactate, and uric acid in human serum was performed on this ECL array, and a good agreement was achieved between measured and reference values [85]. Based on the strategy of co-immobilized luminol and enzyme, a multi-parametric ECL microarray consisting of nine screen-printed (SP) graphite electrodes was fabricated to detect the enzymatically generated H 2 O 2 [86].…”

Existing Biosensing Platforms for the Nucleotide Markers Detection

“…Zero background glows and no need to light excitation are the advantages of such systems in comparison with the fluorescent methods. Often in chemiluminescent assay, there are applied the enzyme mediated detection system using enzymes such as horseradish peroxidase, [34], alkaline phosphatase, [19], glucose oxidase [35], etc. generating a chemiluminescent signal because of enzymatic conversion of specific substrates.…”

“…generating a chemiluminescent signal because of enzymatic conversion of specific substrates. For example, the chemiluminescent oxidation of luminol catalyzed by horseradish peroxidase in composition of conjugate with streptavidin was used for the detection of biotinylated DNA in the paper [34] (Figure 5). The detection limit of the DNA model sequence was 5 × 10 -11 M. One should specially focus on the system detection of biomolecules based on the bioluminescence.…”