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

Abstract

In this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H(2)O(2) as substrate and the single electron donor [Os(III)(bpy)(2)pyCl](2+) as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6 pmol cm(-2) was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200 pM and 1 nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [Os(II)(bpy)(2)pyCl](+), thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment.

Electrochemical Sensors for Clinic Analysis

“…They can be subdivided in two groups: electroactive, such as metal complexes [182, 183] and metal nanoparticles [184], and non-electroactive such as enzymes [185]. …”

Development of a disposable glucose biosensor using electroless-plated Au/Ni/copper low electrical resistance electrodes

“…These electrodes are widely used, mainly for voltammetric measurements. Carbon paste-based sensors (Yabuki et al, 1992;Ge et al, 1998;Boujtita et al, 2000;Hong et al, 2002;Hart et al, 2002;Fähnrich et al, 2003;Darain et al, 2003;Gao et al, 2003;Honeychurch et al, 2003;Darain et al, 2005;Díaz-González et al, 2005;Ledru et al, 2006;Corgier et al, 2007;Piermarini et al, 2007;Tangkuaram et al, 2007;Honeychurch et al, 2007;Djellouli et al, 2007) are also applicable in coulometry (both amperometry and potentiometry). The wide application of a carbon paste as the matrix of an enzyme electrode is attributed to the simple preparation procedure, which facilitates mass production and therefore, is economical.…”

Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay

“…In recent years, numerous PCR-coupled electrochemical genosensors have been developed to detect infectious agents, including hepatitis B virus (Erdem et al, 2005), Salmonella enteric serovar Typhimurium (Lai et al, 2006), human cytomegalovirus (Djellouli et al, 2007), and Mycobacterium tuberculosis (Zhang et al, 2012). The functionality of these electrochemical genosensors is usually established with short synthetic oligonucleotides, and the actual performance of these genosensors with real clinical specimens containing complex biological molecules or repertoires of nontarget nucleic acid sequences has not been explored in depth (Pedrero et al, 2011).…”