Electrochemical Genosensor Based on Colloidal Gold Nanoparticles for the Detection of Factor V Leiden Mutation Using Disposable Pencil Graphite Electrodes

Abstract

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.

Electrochemical DNA sensors

“…In one study, the labeled target is captured by probe strands immobilized on a pencil graphite electrode, and hybridization is detected electrochemically with the appearance of a characteristic gold-oxidation signal 33 . The signal is greatly enhanced because of the large electrode surface area and the availability of many oxidizable gold atoms in each nanoparticle label.…”

Electrochemical Deposition of Gold Nanoparticles on Pencil Graphite by Fast Scan Cyclic Voltammetry

“…It has been used as a sorbent in solidphase microextraction of alcohols, 33 as a substrate to study nucleation mechanisms 34 and for coating polymer films by electropolymerization 35 and as an electrode for electrodeposition metals for analytic and catalytic purposes. [36][37][38][39] The aim of the present study is to achieve smaller sizes of deposited AuNPs by direct electrodeposition on the disposable pencil graphite by varying the deposition time via fast scan cyclic voltammetry.…”

Determination of Surface-Immobilized Double-Stranded DNA Using a Metallointercalator and Catalytic Voltammetry

“…Another group of frequently used covalent labels is that of enzymes, which have produced the most sensitive DNA assays to date (1 fmol L À1 ) thanks to the power of enzymatic amplification [25][26][27]. Apart from molecular labels, various nanoparticles have also been shown to be promising materials in DNA detection [28][29][30].…”