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

Abstract

A carbon nanofiber enriched 8-channel screen-printed electrochemical array was used for the impedimetric detection of SNP related to Factor V Leiden (FV Leiden) mutation, which is the most common inherited form of thrombophilia. FV Leiden mutation sensing was carried out in three steps: solution-phase nucleic acid hybridization between zip nucleic acid probe (Z-probe) and mutant type DNA target, followed by the immobilization of the hybrid on the working electrode area of array, and measurement by electrochemical impedance spectroscopy (EIS). TArzhe selectivity of the assay was tested against mutation-free DNA sequences and synthetic polymerase chain reaction (PCR) samples. The developed biosensor was a trustful assay for FV Leiden mutation diagnosis, which can effectively discriminate wild type and mutant type even in PCR samples.

Keywords: 8-channel screen-printed electrochemical arrays; SNP; electrochemical impedance spectroscopy; zip nucleic acids.

Scheme 1

The representative experimental scheme related to electrochemical impedance spectroscopy based sensing of the FV Leiden mutation. (a) Electrode control, (b) Z-probe in the absence of target, (c) the hybrid form of Z-probe and mutant type DNA target.

Figure 1

The Nyquist diagrams obtained after the hybridization of the Z-probe and mutant type DNA target under optimized conditions. (a) Electrode control, (b) Z-probe in the absence of target, (c) the hybrid form of Z-probe and mutant type DNA target.

Figure 2

Nyquist diagrams related to (a) electrode control, (b) 1.0 µg mL⁻¹ Z-probe in the absence of target, the hybrid form of Z-probe and (c) 2.0, (d) 4.0, (e) 6.0, (f) 8.0, (g) 10.0, (h) 12.0, (i) 14.0, (j) 16.0 µg mL⁻¹ mutant type DNA target.

Figure 3

The hybridization between 1.0 µg mL⁻¹ (A) Z-probe, or (B) DNA probe and 12.0 µg mL⁻¹. mutant type DNA or wild type DNA target. Nyquist diagrams related to (a) Z-probe or DNA probe in the absence of target, the hybrid form of Z-probe, or DNA probe with (b) mutant type DNA target, (c) wild type DNA target.