Enzyme Affinity Assays Involving a Single-Use Electrochemical Sensor. Applications to the Enzyme Immunoassay of Human Chorionic Gonadotropin Hormone and Nucleic Acid Hybridization of Human Cytomegalovirus DNA

Abstract

A disposable electrochemical sensor based on an ion‐exchange film‐coated screen‐printed electrode adapted to the bottom of a polystyrene microwell has been developed and applied to two bioaffinity assays, i.e., a two‐site heterogeneous enzyme immunoassay of human chorionic gonadotropin hormone (hCG) and a DNA enzyme hybridization assay of an oligonucleotide sequence related to the human cytomegalovirus (hCMV). In each case, the alkaline phosphatase label was used to hydrolyze the monoester phosphate salt of [(4‐hydroxyphenyl)aminocarbonyl]‐cobaltocenium. This anionic substrate is transformed into a cationic electroactive product, which is then accumulated by ion‐exchange at the electrode surface to give an amplified electrochemical response. Detection limits of 100 mIU mL–1 and 10 amol mL–1 were thus achieved for hCG and amplified hCMV DNA, respectively. The electrochemical approach competes favorably with the conventional absorption spectrophotometry using p‐nitrophenyl phosphate as the substrate, since it was 10 to 40‐fold more sensitive.

Electrochemical DNA Hybridization Biosensors

“…Attaching an enzyme to the target strand of DNA followed by electrochemical transduction has also been demonstrated using alkaline phosphatase as the enzyme [48,49]. In this recent work by Wang et al [49] not only is an enzyme label used, but magnetic separation is employed to improve selectivity with the enzyme label.…”

Current strategies for electrochemical detection of DNA with solid electrodes

“…One is interaction of the immobilised (captured) probe with an unknown sample in which its complementary sequence (analyte or target sequence) might be present. Occasionally roles are reversed -the analyte is immobilised and the probe is in solution [75,76,77,78,79]. The second format is the "sandwich-type" assay, socalled because the target sequence is hybridised both with the immobilised probe and, in another region, with a second probe (reporter probe), which is commonly labelled for detection [43,46,50,52,56,80,81,82,83,84,85,86,87].…”

Evaluation of the analytical performances of avidin-modified carbon sensors based on a mediated horseradish peroxidase enzyme label and their application to the amperometric detection of nucleic acids

“…A detection limit of 30 pM can be estimated, which corresponds to an absolute amount of 1.8 × 10 8 copies HCMV DNA per electrochemical cell (i.e., 10 L of a 30 pM HCMV DNA sample incubated on the electrode surface during hybridization step). This detection limit is 500 and 300-fold higher than in our previous enzyme-amplified electrochemical HCMV DNA assays involving an HRP label with the o-phenylenediamine cosubstrate (3.6 × 10 5 copies per electrode) and an alkaline phosphatase label (6 × 10 5 copies per well) (Bagel et al, 2000), respectively. There are a least two reasons to explain such differences.…”

“…Because of their high amplification of the signal through catalytic generation of a large number of electroactive molecules (i.e., high turnover number), enzyme labels have led to the most sensitive electrochemical detection schemes of DNA hybridization. This has been demonstrated with various enzymes labels such as the horseradish peroxidase (De Lumley-Woodyear et al, 1996;Azek et al, 2000;Campbell et al, 2002;Dequaire and Heller, 2002;Marchand et al, 2005), soybean peroxidase (Caruana and Heller, 1999), alkaline phosphatase (Bagel et al, 2000;Aguilar and Fritsch, 2003;Carpini et al, 2004;Hernández-Santos et al, 2004;Nebling et al, 2004), PQQ-dependent glucose dehydrogenase (Ikebukuro et al, 2002), bilirubin oxidase (Kim et al, 2004), and glucose oxidase (Kavanagh and Leech, 2006). Among these enzyme labels the HRP is undoubtedly an excellent choice because of its high turnover, commercial availability, low cost, high stability and easy coupling with many biomacromolecules without significant loss of its catalytic activity.…”