Near real-time immuno-optical sensor for diagnosing single point mutation

Abstract

Factor V leiden (FVL) is an abnormality of factor V (FV), a blood coagulation factor. It is a hereditary blood coagulation disorder with a high frequency (3-7% of general population). The most common type of FVL is caused by a single amino acid mutation and, therefore, its diagnosis is currently done only by DNA analysis, which takes a long time and is expensive. We have developed a rapid, accurate, and cost-effective, sandwich immuno-optical sensing method. To produce monoclonal antibodies against FV or FVL, having minimal cross-reactivity with the other molecule, a 20 amino acid sequence (20-mer) of FV or FVL at around the mutation site was utilized. The antibodies were screened first with the 20-mers and then with native FV or FVL molecules and they showed some cross reactivity. Using two antibodies having strongest affinity to either FV or FVL molecule, a FV and a FVL preferred sensors, were produced. After verifying that the levels of the antibody affinity to the two different molecules remained constant with changes in analyte concentration, a two-sensor system is developed to quantify FV and FVL in plasma samples. The system quantified the levels of FV and FVL at the maximum error of 0.5 μg/ml-plasma, in their physiological concentration range of 0-12 μg/ml-plasma. The levels of both molecules may provide us whether the patient has FVL or not but also the seriousness level of the disease (homozygous and different level of heterozygous).

Electrochemical Detection of Solution Phase Hybridization Related to Single Nucleotide Mutation by Carbon Nanofibers Enriched Electrodes

“…In the present study, the impedimetric detection of the FV Leiden mutation was performed in contribution of the ZNA probe which resulted in a relatively shorter time (i.e., 30 min) and selectively in comparison to earlier studies in the literature without using any type of labeled probe [23,24,25,36,37,38,50,51,52,53,54,55,56,57]. Some of these studies related to the electrochemical detection of FV Leiden mutation was presented in Table S6.…”

“…FV Leiden is a specific gene mutation as G-to-A substitution at nucleotide 1691 that predicts a single-amino acid replacement (R506Q) which results in thrombophilia [19,20]. The quantitative analysis of FV Leiden was performed by different techniques, such as, an immunosorbent assay [21], a fluorescent assay [22], the sandwich-optical sensing method [23] or a voltammetric assay [24,25]. Along with these conventional techniques, the impedance spectroscopy has an inherent potential for label-free detection, which is of special interest in bioanalysis since it circumvents the need to modify biomolecules with fluorescence dyes, enzymes, redox or radioactive labels [14].…”

Fluorescence based fiber optic and planar waveguide biosensors. A review

“…Chou and co-workers [64,116,117] developed another group of immunosensors that combine localized surface plasmon techniques, to increase the sensitivity of protein immunoassays, and EW excitation, to perform protein binding kinetics. Lastly, the group of K. Kang and colleagues [118] have determined several analytes of clinical interest and have studied the surface plasmon enhancement of cardiac marker detection.…”

Impedimetric Sensing of Factor V Leiden Mutation by Zip Nucleic Acid Probe and Electrochemical Array

“…Due to the importance of FV Leiden mutation sensing, there is an urgent need to develop sensitive, reliable, and fast detection protocols for the FV Leiden mutation. Under this scope, the quantitative analysis of Factor V Leiden was carried out by different methodologies such as the immunosorbent assay [ 18 ], fluorescent assay [ 19 ], and sandwich-optical sensing method [ 20 ]. However, these methods require expensive facilities and complex procedures in combination with the use of radioactive/fluorescent tags.…”