Magnetic beads assay based on Zip nucleic acid for electrochemical detection of Factor V Leiden mutation

Accepted Manuscript Magnetic beads assay based on zip nucleic acid for electrochemical detection of factor V Leiden mutation

Arzum Erdem, Ece Eksin PII: DOI: Reference:

S0141-8130(18)35142-0 https://doi.org/10.1016/j.ijbiomac.2018.12.107 BIOMAC 11266

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

26 September 2018 10 December 2018 12 December 2018

Please cite this article as: Arzum Erdem, Ece Eksin , Magnetic beads assay based on zip nucleic acid for electrochemical detection of factor V Leiden mutation. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.12.107

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ACCEPTED MANUSCRIPT Magnetic beads assay based on Zip nucleic acid for electrochemical detection of Factor V Leiden mutation Arzum Erdem 1,2 * and Ece Eksin 1,2

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Faculty of Pharmacy, Analytical Chemistry Department,

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Biotechnology Department, Graduate School of Natural and Applied Sciences,

Erdem):

[email protected]

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*Correspondence: (A. Tel: +90-232-311 5131

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Ege University, Bornova, Izmir, 35100, TURKEY

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[email protected];

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Abstract

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation among people. Developm ent of reliable methods for the detection of SNP is crucial in aspects of molecular diagnosis and personalized medicine. In our study, a genomagnetic assay in combination with zip

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nucleic acid (ZNA) for electrochemical detection of SNP related to Factor V Leiden (FV Leiden)

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mutation. For the first time in the literature, a new generation nucleic acid; ZNA was applied

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herein for electrochemical monitoring of nucleic acid hybridization. Streptavidin coated magnetic beads (MBs) were used for preparation of samples containing ZNA-DNA hybrid and accordingly,

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the guanine signal was measured as a response of hybridization related to FV Leiden mutation by carbon nanofibers (CNF) modified screen printed electrodes (SPE) and multi -channel screen

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printed array of electrodes (CNF-MULTI SPEx8). The detection limit (DL) was found to be 3.79 µg/mL (376 nM) and, 11.63 µg/mL (1.624 µM), respectively CNF-SPE and CNF-MULTI SPEx8. The

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selectivity of ZNA probe to mutation-free DNA sequences was also investigated in contrast to DNA probe. The applicability of ZNA based magnetic beads assay to sequence selective

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hybridization related to FV Leiden was also tested in synthetic PCR samples.

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Keywords: Zip nucleic acids; ZNA; Magnetic beads; Factor V Leiden mutation; Electrochemical nucleic acid biosensors; multi-channel screen printed array of electrodes.

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ACCEPTED MANUSCRIPT 1. Introduction Zip nucleic acids (ZNA) are oligonucleotide-oligocation conjugates with multiple cationic spermine moieties attached to the nucleic acid oligomer [1]. ZNAs have ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence that exhibit a high affinity against to target oligonucleotide sequence. In addition, they display a quite selective

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behaviour to the single base of DNA sequence which is different than the target sequence. ZNA

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probes are superior than other new generation nucleic acids; such as PNA and LN A [2]. The

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quantitative analysis of genomic DNA of cervical carcinoma cells infected with human papilloma virus type 16 (HPV 16) was performed by using PCR method and the primers; such as, DNA, LNA

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and ZNA primers [1]. They reported that the efficiency of PCR analysis carried out by using ZNA primers was found higher than the ones of DNA and LNA. In a similar study performed by Paris et

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al. [3] the quantitative analysis of lung carcinoma cell DNA having Factor V Leiden point mutation was carried out by PCR and accordingly it was stated that ZNA primers provided more efficient

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hybridization with more selective analysis in comparison to the ones of LNA primers. The one of the most common forms of genetic variation is the single nucleotide polymorphism (SNP) an d SNP can

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occur in human genome at a frequency of approximately 1 in every 1000 bases [4]. Due to the potential diagnostic and prognostic values of SNPs, the assays for SNP genotyping should be highly

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sensitive, specific, and capable of working with samples without/with minimal pretreatment. It is highly desirable to improve the current technologies and to develop novel assays with high sensitivity, selectivity, and throughput appropriate for routine SNP analysis at point -of-care [5]. A variety of techniques for SNP genotyping have been reported in recent years [6]. Classic methods such as DNA sequencing could be used for the detection of new and unknown SNPs [7], but these methods require complex procedures and long-lasting operation times. Alternatively, other methods monitoring the conformation changes [8,9], o r mass spectroscopy [10] and poly merase chain reaction (PCR) [11,12] techniques could be used. These approaches could specifically detect SNP-containing regions to avoid complicated DNA sequencing, but the intrinsic shortcomings, such as low throughput and 3

ACCEPTED MANUSCRIPT specificity, limit their applications. Recently, DNA microarray and denaturing high performance liquid chromatography have been proposed for fast, efficient, and large-scale analysis of SNPs [13,14]. However, these methods require expensive facilities and radioactive/ fluorescent tags. Factor V Leiden is the name of a specific gene mutation that results in thrombophilia, which is an increased tendency to form abnormal blood clots blocking the blood vessels [15,16]. Factor V

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Leiden is the most common inherited form of thrombophilia. Between 3 and 8 percent of people with

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European ancestry carry the one copy of the Factor V Leiden mutation in each cell, and about 1 in 5,000 people have two copies of the mutation. The diagnosis of Factor V Leiden requires the

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activated Protein C (APC) resistance assay, a coagulation screening test, or DNA analysis of F5 the

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gene encoding Factor V, to identify the Leiden mutation, a specific G-to-A substitution at nucleotide 1691 that predicts a single-amino acid replacement (R506Q) [16]. The quantitative analysis of FV

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Leiden was carried out by different methodologies, such as, immunosorbent assay [ 17], fluorescent assay [18] the sandwich-optical sensing method [19] or voltammetric assay [20,21]. In brief, Ren et al.

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[17] reported a sandwich immuno-reaction based biosensor and developed an immunosorbent assay for quantification of FV Leiden in plasma. Vlachou et al. [ 18] used the triplex PCR for simultaneous

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amplification of three fragments followed by a single primer extension reaction (PEXT) for each locus and accordingly, the products of PEX T reaction were captured on the two zones of the

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biosensor by the interaction of anti-fluorescein antibody and streptavidin. In another study, Kang et al. [19] developed the sandwich-optical sensing method by combining the methods of developing antibodies against to the single-point sites and dual immuno-fiber-optic biosensing system. The voltammetric detection of FV Leiden mutation was performed in the one of studies by our group [20], where, the target DNA was immobilized onto the surface of the pencil graphite electrode after its hybridization with oligonucleotide probes conjugated to gold nanoparticles. In another work of our group [21], the discrimination between the homozygous and heterozygous mutations related to factor V Leiden was explored according to the changes at the peak currents of the guanine signals without any label-binding step. The discrimination between the homozygous and heterozygous 4

ACCEPTED MANUSCRIPT mutations was established by comparing the peak currents of the guanine signals without any label-binding step. Since magnetic particles (MBs) are the one of the efficient tools for development of selective and sensitive biosensing assay for nucleic acid hybridization, numerous electrochemical biosensor applications have been extensively introduced by using MBs [ 22-30]. A major advantage of MBs is

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the easy and rapid recovery of target molecules by the efficient magnetic separation. The

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performance of MBs could be significantly affected by surface modifications. Various molecules could be easily immobilized onto the surfaces of MBs by surface chemistry [ 31].

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In the present study, a genomagnetic assay in combination with ZNA for electrochemical

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detection of SNP related to Factor V Leiden mutation was introduced. To the best of our knowledge, no report presenting ZNA based electrochemical biosensing assay based on magnetic beads (MB)

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has been available in the literature yet. A new generation nucleic acid; ZNA was applied herein for electrochemical monitoring of nucleic acid hybridization related to Factor V Leiden mutation based

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on magnetic beads assay. The samples containing ZNA-DNA hybrid were prepared by streptavidin coated magnetic beads (MBs) and accordingly, the oxidation signal of guanine was measured as a

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response of ZNA:DNA hybridization by using single-use electrodes; carbon nanofibers (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes

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(CNF-MULTI SPEx8).

The selectivity of ZNA probe against to other oligonucleotides having different mutations (G>A, G>C and G>T) was also tested as well as the synthetic single stranded PCR samples containing a single base mutation /without mutation in the medium of PCR template.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Apparatus Electrochemical measurements were performed by µAUTOLAB and AUTOLAB-302 PGSTAT electrochemical analysis system and GPES 4.9.007 software package (Eco Chemie, Th e Net herlands) using differential pulse voltammetry (DPV) technique.

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The raw data were also treated using the Savitzky and Golay filter (level 2) of the GPES

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software, followed by the moving average baseline correction with a “peak width” of 0.03.

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2.2. Chemicals

The biotin linked from 5 ’ end or 3’ end of ZNA probe (5 ’bio ZNA or 3’bio ZNA) and the other

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oligonucleotides; complementary of probe (i.e, mutant type DNA target specific to Factor V Leiden mutation), wild type DNA oligonucleotide, the oligonucleotides having different type of single base

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mutation, non-complementary DNA sequences and PCR products were purchased from (as

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lyophilized powder) TIB Molbiol (Germany).

The base sequences of single stranded oligonucleotides and PCR products were given as below:

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5’bio ZNA probe (Biotin linked, inosine substituted, 23 nt): 5’-biotin-5S-AAT ACC TIT ATT CCT TIC CTI TC-3’ (S: Spermine, I: Inosine)

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3’bio ZNA probe (Biotin linked, inosine substituted, 23 nt): 5’AAT ACC TIT ATT CCT TIC CTI TC-5S-biotin-3’ (S: Spermine, I: Inosine) 3’bio DNA probe (Biotin linked, inosine substituted, 23 nt): 5’-AAT ACC TIT ATT CCT TIC CTI TC-biotin-3’ (I: Inosine) Complementary mtDNA target (23 nt): 5’-GAC AGG CAA GGA ATA CAG GTA TT-3’ Wild type DNA oligonucleotide (wtDNA ODN, 23 nt): 5’-GAC AGG CGA GGA ATA CAG GTA TT-3’ C-mtDNA oligonucleotide (C-mtDNA ODN, 23 nt): 6

ACCEPTED MANUSCRIPT 5’-GAC AGG CCA GGA ATA CAG GTA TT-3’ T-mtDNA oligonucleotide (T-mtDNA ODN, 23 nt): 5’-GAC AGG CTA GGA ATA CAG GTA TT-3’ Noncomplementary DNA oligonucleotide-1 (NC-1, 20 nt): 5’-AAT ACC ACA TCA TCC ATA TA-3’

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Noncomplementary DNA oligonucleotide-2 (NC-2, 23 nt):

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5’-AAT ACC TGT ATT CCT CGC CTG TC-3’

Complementary mutant type PCR-1 (single stranded, 143 nt, mtPCR-1):

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5’-ACC CAC AGA AAA TGA TGC CCA GTG CTT AAC AAG ACC ATA CTA CAG TGA CGT

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GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT GGA CAG GCA AGG AAT ACA GGT ATT TT-3’

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Wild type PCR-1 (single stranded, 143 nt, wtPCR-1):

5’-ACC CAC AGA AAA TGA TGC CCA GTG CTT AAC AAG ACC ATA CTA CAG TGA CGT

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GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT GGA CAG GCG AGG AAT ACA GGT ATT TT-3’

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Complementary mutant type PCR-2 (single stranded, 220 nt, mtPCR-2): 5’-ACC CAC AGA AAA TGA TGC CCA GTG CTT AAC AAG ACC ATA CT A CAG TGA CGT

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GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT G A C AG TGA CGT GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT GGA CAG GCA AGG AAT ACA GGT ATT TT-3’

Wild type PCR-2 (single stranded, 220 nt, wtPCR-2): 5’-ACC CAC AGA AAA TGA TGC CCA GTG CTT AAC AAG ACC ATA CT A CAG TGA CGT GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT G A C AG TGA CGT GGA CAT CAT GAG AGA CAT CGC CTC TGG GCT

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ACCEPTED MANUSCRIPT AAT AGG ACT ACT TCT AAT CTG TAA GAG CAG ATC CCT G GA CAG GCG AGG AAT ACA GGT ATT TT-3’ Streptavidin coated magnetic particles (MB-STR) in 0.94 mm diameter size were purchased from Estapor, Merck (France). The stock solutions of 5’bio ZNA and 3’bio ZNA probe were prepared in 2.5xDPBS (Dulbecco’s

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modified Phosphate Buffer Solution) (pH 7.40) and kept frozen. The stock solutions of other

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oligonucleotides were prepared in ultrapure water (i.e, RNase/DNase free). The diluted solutions of oligonucleotides; 5 ’bio ZNA probe, 3’bio ZNA probe, DNA probe, wtDNA target, mtDNA target,

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C-mtDNA target, T-mtDNA target, NC-1 and NC-2 prepared in 50 mM phosphate buffer solution

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containing 20 mM NaCl (PBS, pH 7.40). The diluted solutions of mt DN A target was prepared in PBS (pH 7.40), acetate buffer solution (ABS, pH 4.80), or carbonate buffer (CBS, pH 9.50), that was

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preferentially used according to the protocol followed herein. The PCR template was purchased from Thermo Fisher Scientific Inc. and contains Taq DNA

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polymerase, dNTPs and all other components required for PCR, except DNA template and primers. Other chemicals were in analytical reagent grade and they were supplied from Sigma -Aldrich and

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Merck.

Carbon nanofibers enriched screen printed electrodes (CNF-SPEs) and multi-channel screen

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printed array of electrodes (CNF-MULTI SPEx8): The single use electrodes; CNF-SPEs and CNF-MULTI SPEx8 were purchased from DropSens (Oviedo-Asturias, Spain).

The planar screen-printed electrode; CNF-SPEs in 3.3 × 1.0 × 0.05 cm (length × width × height) consists of three main parts, which are graphitized carbon nanofiber modified carbon working electrode (4 mm in diameter), a carbon counter electrode and a silver pseudo reference electrode. A specific DropSens connector (ref. DSC) allows the connection of CNF-SPE to the potentiostat. All measurements on SPEs were performed by placing a 35 μL drop of the corresponding solution to the working area. 8

ACCEPTED MANUSCRIPT The CNF-MULTI SPEx8 in 3.3 x 7.8 x 0.1 cm (length × width × height) consists of graphitized carbon nanofiber modified carbon working electrode (2.56 mm in diameter), a carbon counter electrode and a silver pseudo reference electrode. A specific DropSens connector (ref. DR P-CAST8X) allows the connection of the array of 8 electrochemical cells (CNF-MULTI SPEx8) to the potentiostat. All measurements on CNF-MULTI

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SPEx8 were performed by placing a 10 μL drop of the corresponding solution to the working area.

2.3. Electrode preparation

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The working electrode of CNF-SPEs was electrochemically pretreated by applying +0.90 V for

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60 s in ABS (pH 4.80), as well as each working electrode of CNF-MULTI SPEx8 .

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2.4. Procedure

The procedure included (i) immobilization of 3’bio ZNA probe onto MB-STR surface (ii)

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hybridization of 3’bio ZNA probe and complementary mtDNA target, or other oligonucleotides; wtDNA oligonucleotide/ non-complementary ODNs (C-Target, T-Target, NC-1, NC-2), or

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complementary mutant type PCR-1,2/wild type PCR-1,2/ onto the surface of MB-STR and (iii) separation of ZNA:DNA hybrid from MB-STR surface (iv) immobilization of ZNA:DNA hybrid

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onto the CNF-SPE, or CNF-MULTI SPEx8 surface (v) voltammetric measurement. The experimental scheme followed in our study was presented in Fig. 1. Figure 1.

Hybridization procedure between 3’bio ZNA probe/DNA probe and its complementary mtDNA target or other ODNs onto the surface of MB-STRs: The preparation of nucleic acid immobilized MBs and hybridization procedure was performed as reported in earlier studies [26-28,30]. 3 µL of MB-STRs were transferred into a 1.5 mL centrifuge tube and washed with 90 µL of TTL buffer (100 mM Tris-HCl (pH 8.00); % 0.1 Tween20 and 1 M LiCl) followed by second washing step 9

ACCEPTED MANUSCRIPT with 90 µL of 50 mM phosphate buffer (PBS, pH 7.40) during 5 min. After washing steps, 20 μL of PBS (pH 7.40) buffer containing 50 μg/mL of 3’bio ZNA probe was transferred into the tubes and incubated for 30 min at room temperature with gentle mixing. After then, 3’bio ZNA probe immobilized MB-STRs were separated and washed with 90 μL of PBS (pH 7.40). The 3’bio ZNA probe immobilized MB-STRs were resuspended in 20 μL of 50 mM phosphate buffer (PBS, pH 7.40)

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containing required amount of complementary mtDNA target, or other oligonucleotides. The

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hybridization was carried out at room temperature during 60 min. The samples containing ZNA:DNA hybrid with MB-STR conjugates were then washed twice with 90 μL of PBS (pH 7.40)

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and resuspended in 25 μL of 0.05 M NaOH solution during 5 min. Then, 25 μL of the resulted

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sample was transferred into 85 μL of 0.5 M acetate buffer (ABS, pH 4.80). A resulted sample containing ZNA:DNA hybrid was vortexed during 1 min.

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The working electrode of CNF-SPE / MULTIx8 CNF-SPE was covered by a drop of sample containing ZNA:DNA hybrids and kept during 15 min. Then, the electrodes were washed with 0.5

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M ABS (pH 4.80) to eliminate unspecific binding.

DPV measurements were performed in ABS (pH 4.80) and the guanine oxidation signal was

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evaluated as a response of nucleic acid hybridization. Since ZNA/DNA probe sequences were inosine base substitude oligonucleotides (i.e, not contain any guanine bases), this important detail

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brings a benefit to our study in order to direct electrochemical detection in the case of full match nucleic acid hybridization by measuring the oxida tion signal of guanine approximately at +1.00 V [27, 32-36].

Same experimental procedure was followed in the presence of DNA probe selective to mtDNA target as well as control experiments performed by spermine alone. The hybridization of 3’bio-ZNA probe with mtDNA target and other oligonucleotides containing mismatch (such as; wtDNA, C-mtDNA, T-mtDNA) as well as noncomplementary oligonucleotides (NC-1 and NC-2). Additionally, a batch of experiments on the hybridization of 3’bio-ZNA probe with PCR products was performed in the medium of PCR Template. 10

ACCEPTED MANUSCRIPT 2.5. Voltammetric measurements The measurements were performed by differential pulse voltammetry (DPV) in ABS (pH 4.80)

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by scanning between +0.50 V and +1.40 V at the pulse amplitude, 50 mV and scan rate, 50 mV/ s.

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ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1. Voltammetric detection of Factor V Leiden mutation using magnetic beads assay based on ZNA in combination with CNF-SPEs: Firstly, the experimental conditions of magnetic beads assay in combination with ZNA probe was optimized by using voltammetric method based on the changes at guanine signal masured by

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CNF-SPE in order to perform efficient ZNA:DNA hybridization; such as, th e concentration of probe,

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mtDNA target and Mg+2 , pH and hybridization temperature, etc. The results were presented in Fig.

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S1-S6 as well as discussed (see supporting information). Further experiments related the detection of Factor V Leiden mutation by CNF-SPEs were done under the optimized conditions.

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The nucleic acid hybridization between 50 μg/mL 3’bio-ZNA probe and mtDNA target in its different concentrations from 10 to 60 µg/mL (respectively, equals 1.4 µM to 8.4 µM) at MB -STR

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surface was carried out at 25 °C for 60 min as described previously. The representative voltammograms and the line graph presenting the average guanine signals were given in Fig. 2.

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There was a gradual increase at the guanine signal in the presence of full match hybridization of ZNA:DNA until 50 µg/mL of mtDNA target and then it levelled off (shown in Fig . 2A and Fig. S7 ).

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Thus, 50 µg/mL of mtDNA concentration was chosen as the optimum concentration of target DNA for our further studies by CNF-SPEs.

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The detection limit (DL) was calculated according to the Miller and Miller method [ 37] and found to be 3.79 µg/mL (7.53 pmol in 20 µL sample, 376 nM) in the linear concentration range of mtDNA target from 10 to 50 µg/mL (respectively, equals 1.4 µM to 7 µM) with the equation y=12.16x + 1209.30 and R 2=0.97 (shown Fig. 2B as inset). Figure 2.

3.2. Selectivity of ZNA probe on magnetic beads assay on voltammetric detection of Factor V Leiden mutation by CNF-SPEs:

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ACCEPTED MANUSCRIPT The selectivity of ZNA probe to Factor V Leiden mutation was then tested against to oligonucleotides having different mutation, where the mutant base was located at mtDNA target (Fig. S8-I), or noncomplementary oligonucleotides (Fig. S8-II). The average guanine signal was obtained as 1786 ± 177.40 nA with the RSD % as 9.93 % (n=3) by CNF-SPEs, after the full match hybridization of ZNA probe with mtDNA target (Fig. S8-I,II-d). On the otherhand, it was measured

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as 1295 ± 63.60 nA (RSD %, 4.90 %, n=2) in the presence of hybridization of ZNA probe with wtDNA

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target (not shown). On the other hand, the average guanine signal was measured as 1485.70 ± 522.70nA, 1593.80 ± 91.30 nA, 1290 ± 254.60 nA and 999 ± 355 nA with the RSDs % as 35.2 %, 5.70 %,

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19.7 % and 35.5 % after the hybridization of ZNA probe with C-mtDNA, T-mtDNA (Fig. S8-I), or

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NC-1 and NC-2 (Fig. S8-II), respectively. Hence, it could be concluded that ZNA probe exhibited a selective behavior even in the presence of oligonucleotides having a different single-base mutation,

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or non-complementary sequences.

3.3. Voltammetric detection of Factor V Leiden mutation using mag netic beads assay based on Z NA in

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combination with CNF-MULTI SPEx8:

Similar to the optimization studies that were performed by CNF-SPE, the effect of the changes

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at the concentration of probe, mtDNA target and Mg+2, pH and hybridization temperature upon to the response measured by CNF-MULTI SPEx8 was examined. Accordingly, these results were

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presented in Fig. S9-S14 as well as discussed (see supporting information). Further experiments related the detection of Factor V Leiden mutation by CNF-MULTI SPEx8 were done under the optimized conditions.

After the full match hybridization occurred at MB-STR surface between 3’bio-ZNA probe and mtDNA target varying from 10 and 60 µg/mL, a gradual increase at the oxidation signal of guanine was obtained until 50 µg/mL mt DNA target (equals to 7 µM) and then it levelled off (shown in Fig. 3A and Fig. S15). Thus, the optimum mtDNA concentration was chosen as 50 µg/mL for further studies using CNF-MULTI SPEx8.

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ACCEPTED MANUSCRIPT The detection limit (DL) was calculated according to the Miller and Miller method [37] and found to be 11.63 µg/mL (16.24 pmol in 10 µL sample, 1.624 µM) in the linear concentration range of mtDNA target from 10 to 50 µg/mL (respectively, equals 1.4 µM to 7 µM) with the equation y=22.89x+1025.60 and R 2=0.93 (shown Fig. 3-B as inset).

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Figure 3.

3.4. Selectivity of ZNA probe on magnetic beads assay on voltammetric detection of Factor V Leiden

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mutation by CNF-MULTI SPEx8:

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The selectivity of ZNA probe to Factor V Leiden mutation was tested against to oligonucleotides having different mutation, where the mutant base was located at mtDNA target, or

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noncomplementary oligonucleotides; such as, C-mtDNA / T-mtDNA / NC-1 / NC-2. The average

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guanine signal was obtained as 2087.75 ± 266 nA with the RSD % as 12.74 % (n=3) by MULTIx8 CNF-SPE, after the full match hybridization of ZNA probe with mtDNA target (Fig.S16-I,II-b). On the contrary, it was measured as 1801.83 ± 270.60 nA (RSD %, 15.02 %, n=3) in the presence of

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hybridization of ZNA probe with wtDNA target (not shown). On the other hand, the average

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guanine signal was measured as 1747 ± 72.12nA, 1640.50 ± 57.28 nA, 882.50 ± 51.62 nA and 942.50 ± 38.89 nA with the RSDs % as 4.13%, 3.49%, 5.85 % and 4.13 % after the hybridization of ZNA probe with C-mtDNA, T-mtDNA (Fig. S16-I), or NC-1 and NC-2 (Fig. S16-II), respectively by using

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CNF-MULTI SPEx8. It could be concluded that ZNA probe exhibited a selective behavior even in the presence of oligonucleotides having a different single-base mutation, or non-complementary sequences.

3.5. Voltammetric detection of Factor V Leiden mutation in the medium of PCR Template using ZNA probe on magnetic beads assay in combination with CNF-SPE and CNF-MULTI: The electrochemical detection of Factor V Leiden mutation was also studied in the medium of PCR template was also tested in the presence of 3’bio-ZNA probe and the PCR products with the length of 143 nt and 220 nt by using CNF-SPE (Fig. 4) and CNF-MULTI SPEx8 (Fig. 5). After the hybridization of 3 ’bio ZNA probe with mtPCR-1/ wtPCR-1, the average guanine signals were 14

ACCEPTED MANUSCRIPT measured by CNF-SPE as 1226 ± 101.82 nA and 913 ± 66.47 nA with the RSDs % (n=3) as 8.31 % and 7.28 %, respectively (shown in Fig. 4-I). According to the experiments related to the hybridization of 3’bio ZNA probe and mtPCR-2/ wtPCR-2, the average guanine signals were measured using CNF-SPEs as 1414 ± 499.22 nA and 412±42.43 nA with the RSDs % (n=3) as 35.31 % and 10.3 0 %, respectively (Fig. 4-II).

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Figure 4.

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After the hybridization of 3’bio ZNA probe and mtPCR-1/wtPCR-1, the average guanine signals

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were measured using CNF-MULTI SPEx8 as 1622.50 ± 41.72 nA and 1446.50 ± 210.01 nA with the RSDs % (n=3) as 2.57 % and 14.52 %, respectively (Fig. 5-I). Moreover, the average guanine signals

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were measured using CNF-MULTI SPEx8 as 1672 ± 97.58 nA and 1327 ± 53.74 nA with the RSDs %

probe and mtPCR-2 or wtPCR-2.

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(n=3) as 5.84 % and 4.05 %, respectively (Fig . 5-II) in the presence of the hybridization of 3’bio ZNA

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Figure 5.

Additionally, the “Discrimination percentage (D %)" was calculated in order to define the

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difference between the responses measured in a hybridization of ZNA probe with mutant type-PCR-1 and the one of ZNA-wild type-PCR-1 (single base mutant type; A>G). Similarly, the

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same experimental scheme was followed for the PCR-2 samples in the PCR Template medium and given in Table S1 and S2 for both electrode CNF-SPE and CNF-MULTI SPEx8. ZNA probe could selectively recognize the single-base mutated target, mtPCR-1 in comparison to wtPCR-1, or mtPCR-2 in comparison to wtPCR-2.

4. Conclusions A genomagnetic assay in combination with zip nucleic acid (ZNA) was introduced for the first time herein for electrochemical detection of SNP related to Factor V Leiden mutation. A new generation nucleic acid; ZNA was applied for electrochemical monitoring of nucleic ac id 15

ACCEPTED MANUSCRIPT hybridization related to Factor V Leiden mutation based on magnetic beads assay. No report related to ZNA based electrochemical biosensing assay has been available in the literature yet. Under the principle of magnetic beads assay in combination with ZNA probes, a novel, simple, sensitive and low-cost method for highly selective detection of SNP related to Factor V Leiden mutation was reported. SNP analysis was also studied in the medium of PCR template in the presence of

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3’bio-ZNA probe and the single stranded PCR products with the length of 143 nt and 220 nt. It can

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be concluded that our assay can be applied in the future by using assymetric PCR. Therefore, SNP analysis can be improved as reported before [38,39].

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In addition, to the best of our knowledge, the single-use electrodes; CNF-SPEs and CNF-MULTI

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SPEx8 were used for the first time herein for voltammetric detection of FV Leiden mutation. By the advantage of this novel genomagnetic assay based on ZNA probe, the analysis of a single point

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mutation of G>A; G>C and G>T was carried out for diagnosis of Factor V Leiden (FV Leiden) mutation.

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Under optimum experimental parameters, the DLs were calculated and found to be 3.79 µg/mL (7.53 pmol in 20 µL sample, 376 nM) and 11.63 µg/mL (16.24 pmol in 10 µL sample, 1.624 µM), for

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CNF-SPEs and CNF-MULTI SPEx8, respectively. ZNA probe based magnetic beads assay was implemented successfully into the label-free electrochemical detection of Factor V Leiden mutation (G>A) since ZNA probe exhibited a selective behavior even in the

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presence of oligonucleotides having a different single-base mutation (G>C and G>T) related to not only Factor V Leiden mutation, but also other types of SNPs. Morover, ZNA probe based assay presents a good selectivity in the presence of any non-complementary DNA sequences. Another objective of the present work has been to develop a novel assay which enables to carry out selective and sensitive analysis of a disease, a bioma rker, or any of environmental/food contaminants; such as pathogen and particularly single point mutation in nucleic acids. A very sensitive and selective electrochemical analysis of Factor V Leiden mutation was performed in our study using ZNA probe resulted in a relatively shorter time, in a practical way, selectively and by

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ACCEPTED MANUSCRIPT time-saving method in comparison to other biosensor studies in the literature [17-21], ELISA and spectroscopic techniques [40-50].

Ackno wledgments: A.E acknowle dges the financial support from Turkish Scie ntific and Technological Research Council (TUBITAK; Proje ct no. 114Z400) as a project inve stigator, and she also would like to e xpress her gratitude to the Turkish Academy of Scie nces (TUBA) as a Principal me mber for its partial support. E.E

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acknowle dge s a project scholarship through by proje ct (TUBITAK Proje ct no. 114Z400).

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ACCEPTED MANUSCRIPT Figure Captions Figure 1. The representative experimental scheme of ZNA:DNA hybridization occured at the surface of MB-STR and voltammetric detection of voltammetric detection of Factor V Leiden mutation by CNF-SPE.

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Figure 2. (A) Voltammograms representing the guanine oxidation signals measured by (a) 50 μg/mL 3’bio-ZNA probe alone, and after hybridization between 50 μg/mL 3 ’bio-ZNA

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probe and mtDNA target in its different concentrations (b) 10, (c) 20, (d) 30, (e) 40, (f) 50

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and (g) 60 µg/mL by CNF-SPE (B)The calibration graph based on the average guanine signal (n=3) obtained after nucleic acid hybridization in the presence of 50 µg/mL

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3’bio-ZNA probe and 10-50 µg/mL mtDNA target sequence.

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Figure 3. (A) Voltammograms representing the guanine oxidation signals measured by (a) 50 µg/mL 3’bio-ZNA probe alone, and after hybridization between 50 μg/mL 3 ’bio-ZNA

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probe and mtDNA target in its different concentrations (b) 10, (c) 20, (d) 30, (e) 40, (f) 50 µg/mL mt DNA by CNF-MULTI SPEx8. (B) The calibration graph based on the average

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guanine signal (n=3) obtained after nucleic acid hybridization in the presence of 50 µg/mL 3’bio-ZNA probe and 10-50 µg/mL mtDNA target sequence.

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Figure 4. (I) (A) Voltammograms (B) histograms representing the signals measured (a) CNF-SPE in ABS, (b) MB-STR control in ABS, (c) 50 μg/mL 3’bio-ZNA probe control in PCR template, and the guanine signals measured by DPV using CNF-MULTI SPEx8 after hybridization of 50 μg/mL 3’bio-ZNA probe with 50 µg/mL (d) mtPCR-1, (e) wtPCR-1 in PCR template (n=3). (II) (A) Voltammograms (B) histograms representing the signals measured (a) CNF-SPE in ABS, (b) MB-STR control in ABS, (c) 50 μg/mL 3’bio-ZNA probe control in PCR template, and the guanine signals measured by DPV using CNF-MULTI SPEx8 after hybridization of 50 μg/mL 3 ’bio-ZNA probe with 50 µg/mL (d) mtPCR-2, (e) wtPCR-2 in PCR template (n=3). 23

ACCEPTED MANUSCRIPT Figure 5. (I) (A) Voltammograms (B) histograms representing the signals measured (a) CNF-MULTI SPEx8 in ABS, (b) MB-STR control in ABS, (c) 50 μg/mL 3’bio-ZNA probe control in PCR template, and the guanine signals measured by DPV using CNF-MULTI SPEx8 after hybridization of 50 μg/mL 3 ’bio-ZNA probe with 50 µg/mL (d) mtPCR-1, (e) wtPCR-1 in PCR template (n=3). (II) (A) Voltammograms (B) histograms representing the

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signals measured (a) CNF-MULTI SPEx8 in ABS, (b) MB-STR control in ABS, (c) 50 µg/mL

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3’bio-ZNA probe control in PCR template, and the guanine signals measured by DPV

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(d) mtPCR-2, (e) wtPCR-2 in PCR template (n=3).

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using CNF-MULTI SPEx8 after hybridization of 50 μg/mL 3’bio-ZNA probe with 50 µg/mL

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ACCEPTED MANUSCRIPT Highlights  Genomagnetic assay in combination with zip nucleic acid (ZNA) was developed for electrochemical detection of SNP related to Factor V Leiden (FV Leiden) mutation.  Magnetic beads were used for preparation of samples containing ZNA-DNA hybrid.  The voltammetric detection of single nucleotide mutation ‘FV Leiden’ was

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

performed by using single-use multi-channel array of electrodes. The applicability of ZNA based assay in comparison to DNA based one was shown using synthetic PCR samples.

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Figure 1

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Figure 5