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

In the present study, a sensitive and selective impedimetric detection of solution-phase nucleic acid hybridization related to Factor V Leiden (FV Leiden) mutation was performed by carbon nanofibers (CNF) modified screen printed electrodes (SPE). The microscopic and electrochemical characterization of CNF-SPEs was explored in comparison to the unmodified electrodes. Since the FV Leiden mutation is a widespread inherited risk factor predisposing to venous thromboembolism, this study herein aimed to perform the impedimetric detection of FV Leiden mutation by a zip nucleic acid (ZNA) probe-based assay in combination with CNF-SPEs. The selectivity of the assay was then examined against the mutation-free DNA sequences as well as the synthetic PCR samples.

After placing a 35 µL drop of the corresponding solution to the working area of these electrodes, each measurement was performed using CNF-SPEs similiarly introduced in our earlier reports [36][37][38].

The optimization of experimental parameters for impedimetric detection of FV Leiden mutation by CNF-SPEs:
The comparison of 5'ZNA probe, 3'ZNA probe and DNA probe The selectivity of ZNA probe was firstly investigated. For this purpose, the impedimetric detection of mDNA target in the full match ZNA:DNA hybridization was performed in the presence of 3'ZNA probe, 5'ZNA probe, DNA probe, or spermine alone ( Figure S1). After the pseudo-hybridization of 3' ZNA probe, 5'ZNA probe, DNA probe the Rct values were recorded as 290 Ohm, 228 Ohm, 1213 Ohm and 2.1 µA, respectively. On the other hand, after the hybridization of 3'ZNA probe, 5'ZNA probe and DNA probe with mDNA target, the Rct values were recorded as 1215 Ohm, 1038 Ohm and 1564 Ohm, respectively. After the interaction of spermine and mDNA target, the Rct value was 92% higher than the one obtained by the spermine in the absence of mDNA target. The highest Rct value was obtained in the presence of full match hybridization of 5'ZNA probe with mDNA target of all (shown in Figure S1-h), and the Rct value was measured 355% higher than the ones recorded after the pseudo hybridization of 5'ZNA probe. It was concluded that the 5'ZNA probe presented more selective behavior than the one of 3'ZNA probe and DNA probe. Figure S1. Nyquist diagrams obtained by (a) CNF-SPE, after pseudo hybridization of 2 µg/mL (b) 3'ZNA probe, (c) 5'ZNA probe, (d) DNA probe, (e) spermine, after hybridization of (f) 3'ZNA probe (g) 5'ZNA probe, (h) DNA probe, (i) spermine with 10 µg/mL mDNA target. Inset was the equivalent circuit model used for fitting of the impedance data.

The effect of temperature at hybridization process occurred between ZNA probe and mDNA target
The hybridization of 2 µg/mL ZNA probe and 10 µg/mL mDNA target was performed at 25 °C, or 50 °C during 10 min in PBS (pH 7.40) ( Figure S2). Pseudo-hybridization of ZNA probe at 25 °C or 50 °C was also performed the Rct value was measured as 246.50 Ohm and 344 Ohm. After the hybridization of 5'ZNA probe and mDNA target at 25 °C or 50 °C, the Rct value was recorded as 1025.50 ± 17.68 Ohm (RSD%, 1%, n=2) and 1383.50 ± 89.80 Ohm (RSD%, 6%, n=2) respectively. The higher increase (as 316%) at Rct value in contrast to the one obtained by pseudo hybridization was obtained in the case of the hybridization occurred at 25 °C. Thus, 25 °C was chosen as optimum temperature for hybridization. Figure S2. Nyquist diagrams obtained by (a) CNF-SPE, (b) pseudo hybridization of ZNA probe at 25 °C, (c) hybridization between 2 µg/mL ZNA probe and 10 µg/mL mDNA target at 25 °C, (d) pseudo hybridization of 5'ZNA probe at 50 °C, (e) hybridization between 2 µg/mL ZNA probe and 10 µg/mL mDNA target at 50 °C. Inset was the equivalent circuit model used for fitting of the impedance data.

The effect of Mg +2 concentration at hybridization process
The effect of MgCl2 concentration upon the hybridization of ZNA probe with mDNA target was studied ( Figure S3). For this purpose, the hybridization was performed during 10 min in the solution of PBS (pH 7.40), or PBS containing 0.5 mM Mg +2 . As seen in Figure S3, , the highest Rct value was measured as 1025.50 ± 17.68 Ohm (RSD%, 1%, n=2) in PBS (pH 7.40) without Mg +2 .

The effect of pH at hybridization process
The effect of pH upon the hybridization process was evaluated and the results were shown in Figure S4. The hybridization between 2 µg/mL ZNA probe and 10 µg/mL (equals to 1.4 µM) mDNA target was done in ABS (pH 4.80), PBS (pH 7.40), or CBS (pH 9.50). The pseudo-hybridization of ZNA probe in ABS (pH 4.80), PBS (pH 7.40), or CBS (pH 9.50) was also studied and the Rct values were measured as 271 Ohm, 248 ± 18.68 Ohm (RSD%, 7.53%, n=3) and 440 Ohm, respectively (shown in Figure S4). After hybridization in ABS (pH 4.80), PBS (pH 7.40), or CBS (pH 9.50), the Rct values were measured as 702 Ohm, 1132 ± 184.89 Ohm (RSD%, 16.33%, n=3) and 473 Ohm, respectively. It was concluded that the hybridization could be occurred efficiently in the medium of PBS with pH 7.40 since the highest increase (356%) at the Rct was obtained in hybridization of ZNA:DNA performed in PBS medium.

The effect of hybridization time at hybridization process
The hybridization between ZNA probe and mDNA target was performed during 5, 10 and 15 min. The highest increase at the Rct value was calculated as 356% according to the changes at Rct after hybridization of ZNA:DNA in 10 min ( Figure S5). Thus, 10 min was used as the optimum hybridization time in further studies.

The effect of ZNA probe concentration at hybridization process:
In order to find optimum concentration of ZNA probe, the hybridization was performed in various concentrations of ZNA probe (0.5, 1, 2 and 4 µg/mL) and 10 µg/mL (equals to 1.4 µM) mDNA target and accordingly, the Rct value was measured (shown in Figure S6). The highest Rct value was recorded as 1355 ± 4.24 Ohm (RSD%, 4.24%, n=2) in the presence of 1 µg/mL ZNA probe. Therefore, 1 µg/mL was chosen as optimum ZNA probe concentration for the further studies. Figure S6. Nyquist diagrams of (a) CNF-SPE, after the hybridization of (b) 0.5 µg/mL, (c) 1 µg/mL (d) 2 µg/mL (e) 4 µg/mL ZNA prob and 10 µg/mL mDNA target. Inset was the equivalent circuit model used for fitting of the impedance data. The hybridization efficiency (HE%) was calculated for each hybridization occurred between DNA probe/ZNA probe with mDNA/wDNA, mPCR-1/wPCR-1, or mPCR-2/mPCR-2 according to the equation 1.

The hybridization efficiency (HE%) = [ΔRct / Rct hybrid] × 100
(ΔRct = Rct hybrid -Rct probe) (1) The hybridization efficiency (HE%) was calculated based on the results obtained by CNF-SPE related to each experiment on possible hybridization between DNA probe/ZNA probe with mDNA/wDNA target or mPCR/wPCR (Equation 1). The higher HE% value is expected in the case of hybridization of ZNA probe with mDNA/mPCR comparison to the one with wDNA/wPCR. In addition, the higher HE% is expected in case of hybridization between ZNA probe and its target DNA in contrast to DNA probe. Therefore, we can consider that ZNA probe can recognize SNP in NA hybridization more selectively than DNA probe. Table S1. The Rct values measured in the presence of the hybridization occurred between 1 µg/mL ZNA probe and 10 µg/mL mDNA target by single-use CNF-SPEs for three different days with the values of the average Rct and the standard deviation with the RSD% for presenting the intra-day reproducibility.