- Email: [email protected]
ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation
Accepted Manuscript ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation Arzum Erdem, Ece Eksin PII:
S0003-2670(19)30470-2
DOI:
https://doi.org/10.1016/j.aca.2019.04.036
Reference:
ACA 236725
To appear in:
Analytica Chimica Acta
Received Date: 16 December 2018 Revised Date:
14 April 2019
Accepted Date: 16 April 2019
Please cite this article as: A. Erdem, E. Eksin, ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.04.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Graphical Abstract
ACCEPTED MANUSCRIPT 1
ZNA probe immobilized single-use electrodes for impedimetric detection of
2
nucleic acid hybridization related to single nucleotide mutation
3 4
Arzum Erdem1,2* and Ece Eksin1,2
6 7
1 2
RI PT
5 Faculty of Pharmacy, Analytical Chemistry Department,
Biotechnology Department, Graduate School of Natural and Applied Sciences,
8
Ege University, Bornova, Izmir, 35100, TURKEY
SC
9 *Correspondence: (A. Erdem):
11
[email protected] and [email protected]; Tel: +90-232-311 5131
M AN U
10
12 13 14
Special issue: New Directions in Electroanalytical Chemistry
15 Abstract
17
The development of a low-cost and disposable biosensing technologies has received a great interest
18
of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single
19
nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based
20
on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden mutation.
21
After optimization of experimental parameters, the sequence selective hybridization between ZNA
22
probe and target related to FV Leiden mutation was evaluated via electrochemical impedance
23
spectroscopy technique (EIS) by measuring changes at the value of the charge transfer resistance, Rct.
24
Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified
25
screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-
26
SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of
27
ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA
28
biosensor was also tested in synthetic PCR samples containing a single base mutation.
AC C
EP
TE D
16
29 30
Keywords: Zip nucleic acids; ZNA; Factor V Leiden mutation; Electrochemical impedance
31
spectroscopy; Electrochemical nucleic acid biosensors; multi-channel screen printed array of
32
electrodes. 1
ACCEPTED MANUSCRIPT 1.Introduction
34
Zip nucleic acids (ZNA) exhibit a high affinity against to target oligonucleotide sequence and have
35
ability to discriminate between a perfect match and a single base-pair-mismatched complementary
36
sequence [1-4]. The Factor V Leiden (FV Leiden) with the most common inherited prothrombotic
37
conditions has been received a great attention and studied by the researchers [5]. The diagnosis of
38
FV Leiden requires the activated Protein C (APC) resistance assay, a coagulation screening test, or
39
DNA analysis of F5 the gene encoding Factor V, to identify the Leiden mutation, a specific G-to-A
40
substitution at nucleotide 1691 that predicts a single-amino acid replacement (R506Q) [5,6].
41
The diagnosis of FV Leiden thrombophilia is established in a proband by identification of a 1691G>A
42
variant coupled with coagulation tests such as the APC resistance assay. It was reported that there
43
was considerable overlap in APC ratios with a normal Factor V genotype and heterozygotes for FV
44
Leiden. The authors concluded that the APC resistance assay in its present form is not a useful
45
screening test for FV Leiden heterozygotes [7]. Until the performance of this assay is improved,
46
patients should have molecular diagnostic testing performed to determine their FV Leiden status.
47
Under this aim, different techniques for SNP genotyping have been reported in recent years [8]
48
including, DNA sequencing [9], mass spectroscopy [10] polymerase chain reaction (PCR) [11,12]
49
techniques. These approaches could specifically detect SNP, but the intrinsic drawbacks (e.g, low
50
throughput and specificity) limit their applications. Alternatively, DNA microarray and denaturing
51
high performance liquid chromatography have been considered as fast and efficient techniques for
52
SNP analysis [13,14]. However, these methods require expensive facilities and radioactive/
53
fluorescent tags as well as require complex procedures; because of this reason, they are not suitable
54
techniques for the development of simple and low-cost analysis methods for point-of-care (PoC)
55
devices. Thus, there is an urgent need to develop selective, sensitive, fast and easy to use platforms
56
for detection of FV Leiden mutation. There are several biosensor studies including immuno-reaction
57
based optic biosensors [15,16] voltammetric biosensors [17,18] for FV Leiden mutation analysis.
58
No report on the development of single-use biosensor has been available yet in the literature
59
impedimetric detection of FV Leiden mutation via electrochemical impedance spectroscopy (EIS)
60
technique. EIS is a powerful method of analysing the complex electrical resistance of the system. It is
61
sensitive to surface phenomena and a valuable method in electrochemical research. In the field of
62
biosensors, it is well-suited to the detection of binding events on the transducer surface [19]. Thus,
63
the impedimetric techniques have been developed in order to characterize the fabricated biosensors
64
and to monitor the catalytic reactions of biomolecules such as enzymes, proteins, nucleic acids,
65
whole cells, antibodies etc. [20,21].
66
Nowadays, electrochemical DNA sensing strategies based on nanotechnology have become one of
67
the most exciting forefronts area due to the challenging advances of nanomaterials, e.g., magnetic
AC C
EP
TE D
M AN U
SC
RI PT
33
2
ACCEPTED MANUSCRIPT particles [22-24] carbon nanomaterials [25-28] by using different electrochemical transducers
69
[29,30].
70
Carbon nanofibers are also easily massproduced at a lower cost, better mechanical stability, and a
71
larger surface-active groups-to-volume ratio than carbon nanotubes. Due to their physical and
72
chemical properties, carbon nanofibers possess a lot of edge sites on the outer wall, leading to high
73
tensile strength, elasticity, high thermal and electric conductivity, which facilitate electron-transfer
74
reactions of analytes [31]. Therefore, there is numerous study which implements carbon nanofibers
75
modification onto the electrode surface. There is a great attention recently to use of CNFs in the
76
development of biosensors due to their unique properties. For example, Baker et al. [32] reported
77
that DNA molecules have been covalently immobilized on vertically aligned carbon nanofibers
78
(VACNFs). According to the results, it was reported that carbon nanofiber samples were efficiently
79
functionalized with DNA molecules with excellent biomolecular recognition properties, according to
80
the hybridization measurements. Thus, it was concluded that VACNFs have shown a higher binding
81
specificity for complementary DNA in contrast to the one of unmodified electrode.
82
To our best knowledge, we introduce a new generation nucleic acid “ZNA” probe based biosensor for
83
the first time in this present work. It was developed for the detection of single nucleotide mutation
84
related to FV Leiden in combination with multi-channel electrochemical array system. This novel
85
assay involves ZNA probe immobilization onto the surface of carbon nanofiber modified screen
86
printed electrodes (CNF-SPE) or multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE).
87
After hybridization occured between ZNA probe and mtDNA target onto the surface of electrode, the
88
impedimetric measurement was performed and accordingly, the Rct values were recorded as a
89
response of hybridization related to FV Leiden detection. The selectivity of ZNA probe was tested in
90
the presence of mutation-free DNA sequences (G>C or G>T) as well as synthetic PCR samples (143 nt
91
and 220 nt) containing a single base mutation (G>A).
93 94 95 96
SC
M AN U
TE D
EP
AC C
92
RI PT
68
97 98 99 100 101 102 3
ACCEPTED MANUSCRIPT 2.Experimental
104
2.1.Apparatus
105
Electrochemical measurements were performed by AUTOLAB-30 and AUTOLAB-302 PGSTAT
106
electrochemical analysis system with NOVA (version 1.1.2 EcoChemie, The Netherlands) or GPES
107
4.9.007 software package (Eco Chemie, The Netherlands) using electrochemical impedance (EIS)
108
technique. The impedimetric measurements with CNF-SPEs was done in a Faraday cage (EcoChemie,
109
The Netherlands).
110
2.2.Chemicals
111
The amino linked from 5' end of ZNA probe and the othe oligonucleotides; complementary mutant
112
type DNA target, wild type DNA oligonucleotide, the oligonucleotides having different types of single
113
base mutation, noncomplementary DNA sequences and PCR products were purchased from (as
114
lyophilized powder) TIB Molbiol (Germany). The sequences of oligonucleotides and PCR products are
115
given in Supporting information.
116
ZNA probe stock solution as 472 µg mL-1 was prepared in Dulbecco’s modified Phosphate Buffer
117
Solution (pH 7.40) and kept frozen. The stock solutions of other oligonucleotides were prepared in
118
ultrapure water (i.e, RNase/DNase free). The diluted solutions of ZNA probe, DNA probe, wtODN, C-
119
mtDNA ODN, T-mtDNA ODN, NC-1, NC-2 and PCR products were prepared in 50 mM phosphate
120
buffer solution containing 20 mM NaCl (PBS, pH 7.40). The diluted solutions of mtDNA target was
121
prepared in PBS (pH 7.40), acetate buffer solution (ABS, pH 4.80), or carbonate buffer (CBS, pH 9.50),
122
that was preferentially used according to the protocol followed herein.
123
Other chemicals were in analytical reagent grade and they were supplied from Sigma-Aldrich and
124
Merck.
125
The detailed information about carbon nanofibers enriched screen printed electrodes (CNF-SPEs) and
126
multi-channel screen printed array of electrodes (MULTIx8 CNF-SPEs) is given in Supporting
127
information.
128
2.3.Procedure
129
The procedure included (i) immobilization of ZNA probe onto the surface of CNF-SPE or MULTIx8
130
CNF-SPE (ii) hybridization of ZNA probe with complementary mtDNA target, or other
131
oligonucleotides; wtDNA oligonucleotide, noncomplementary ODNs (C-mtDNA ODN, T-mtDNA ODN,
132
NC-1, NC-2) as well as complementary mutant type PCR-1,2 and wild type PCR-1,2 onto the electrode
133
surface, (iii) impedimetric measurement.
134
2.3.1.Immobilization of ZNA probe onto the surface of electrodes
135
ZNA probe was prepared in PBS (pH, 7.40) and immobilized onto the surface of working electrode;
136
CNF-SPE / MULTIx8 CNF-SPE via passive adsorption during 30 min as reported in our previous work
137
[33]. Same experimental procedure was followed in the presence of DNA probe selective to mtDNA target
AC C
EP
TE D
M AN U
SC
RI PT
103
4
ACCEPTED MANUSCRIPT as well as control experiments performed spermine alone. Then, the electrode was washed with PBS
139
(pH 7.40) in order to eliminate unspecific binding.
140
2.3.2.Hybridization between ZNA probe/DNA probe and its complementary mtDNA target, or other
141
ODNs
142
The required amount of mtDNA target or other ODNs prepared in PBS (pH 7.40) was dropped onto
143
the probe immobilized working electrode of CNF-SPE / MULTIx8 CNF-SPE during 20 min. Then, the
144
electrode was washed with PBS (pH 7.40) in order to eliminate unspecific binding.
145
2.3.3.Impedimetric measurements
146
Impedimetric measurements were performed in the presence of 2.5 mM K3[Fe(CN)6] and 2.5 mM
147
K4[Fe(CN)6] (1:1). The impedance was measured in the frequency range between 100 kHz and 0.1 Hz
148
at a potential of +0.23 V with an amplitude of 10 mV. The frequency interval was divided into 98
149
logarithmically equidistant measure points. All measurements were performed by AUTOLAB-30
150
system and NOVA software package (version 1.1.2 Eco Chemie, The Netherlands).
151
The real and imaginary impedance (Z’ and -Z’’) are the components of the complex impedance (Z). An
152
equivalent circuit model (Randles circuit) was utilized for fitting of impedimetric results. The electron
153
transfer was limited at higher frequencies and the linear section seen at lower frequencies may be
154
attributed to the diffusion as explained earlier studies [19] According to the Randles circuit, Rs is the
155
solution resistance. Q is the constant phase element. The charge transfer resistance (Rct) is defined as
156
the resistance related to the dielectric and insulating characteristics at the electrode/electrolyte
157
interface. W is the Warburg impedance due to mass transfer to the electrode surface and observed
158
at higher frequencies.
161 162 163 164 165 166
SC
M AN U
TE D
EP
160
AC C
159
RI PT
138
167 168 169 170 171 172 5
ACCEPTED MANUSCRIPT 3.Results and Discussion
174
Impedimetric detection of FV Leiden mutation using ZNA probe in combination with CNF-SPEs:
175
In order to perform efficient ZNA-DNA hybridization, the experimental conditions, such as, the
176
hybridization temperature, the concentration of ZNA probe, mtDNA target and the effect of Mg+2, pH
177
and hybridization time were optimized. Additionally the interference effect of spermine is tested
178
and all results were presented in Fig. S1-S7 as well as discussed in the supporting information.
179
Further experiments related to the detection of FV Leiden mutation (G>A) were done under the
180
optimized conditions.
RI PT
173
181
The effect of mtDNA target concentration upon the hybridization process
183
The nucleic acid hybridization between 0.5 µg mL-1 5’ZNA probe and mtDNA target in different
184
concentrations varying from 2 to 14 µg mL-1 (0.3 µM to 2 µM) was carried out under optimum
185
conditions. The changes at the Rct value was evaluated and shown in Fig. 1. There was a gradual
186
increase at the Rct value after the full match hybridization of ZNA probe with its target, mtDNA up to
187
12 µg mL-1 (1.7 µM) concentration level of mtDNA, and then it levelled off (Fig. 1 and Fig. S8). The
188
highest Rct was measured as 1081.7 ± 45.3 Ohm (RSD%, 4.2%, n=3). Thus, 12 µg mL-1 mtDNA target
189
was used in our further studies performed by CNF-SPEs.
190
The limit of detection (LOD) was calculated according to the Miller and Miller method [34] and found
191
to be 1.48 µg mL-1 (207 nM) in the linear concentration range of mtDNA target with the equation
192
y=47.75x + 144.90 and R2=0.98 (Figure S8 inset).
195
M AN U
TE D
194
Figure 1.
EP
193
SC
182
Selectivity of ZNA probe in contrast to DNA probe
197
The selectivity of the ZNA probe based biosensor was tested againist to wild type DNA target (wtDNA
198
ODN). The average Rct was recorded in the case of full match hybridization between 5’ZNA probe and
199
mtDNA target as 1081.7 ± 45.3 Ohm (RSD %, 4.2 %, n=3), whereas it was obtained as 644.5 ± 68.6
200
Ohm (RSD %, 10.6 %, n=3) in the case of hybridization between 5’ZNA probe and wtDNA target. On
201
the otherhand, the Rct values were measured as 1061 Ohm and 1187 Ohm after the hybridization of
202
DNA probe respectively with mtDNA, or wtDNA ODN (Figure 2). It was concluded that the 5’ ZNA
203
probe presented more selective behavior to detect SNP in contrast to DNA probe.
AC C
196
204 205
Figure 2.
206
6
ACCEPTED MANUSCRIPT 207
The hybridization efficiency
208
effectiveness [35]. It was calculated for each hybridization occured between DNA probe/ZNA probe
209
with each type of target; mutant type or wild type according to the equation 1.
210
(HE %) is used as the signature of the full match hybridization
The hybridization efficiency (HE %) = [∆ ∆ Rct / Rct hybrid] x 100
Eq 1.
( ∆ Rct = Rct hybrid – Rct probe )
212
HE % is accepted as 100 % in the case of hybridization between ZNA probe with its complementray
213
mutant type target. The lower HE % value is expected in the case of hybridization between ZNA
214
probe and wild type target. The differentiation between the values of HE % that is calculated in the
215
presence of ZNA probe should be higher in comparison to the differentiation between HE %s
216
obtained in the presence of DNA probe. Therefore, we can consider that ZNA probe can recognize
217
SNP more selectively than DNA probe. After the possible interaction between spermine and mtDNA,
218
the value of HE % was calculated and found to be 60 %, whereas HE % was acccepted as 100 % in
219
the case of full hybridization between ZNA probe and mtDNA (Fig. S7). According to the data
220
obtained in control experiment, it was concluded that the spermine did not show any interference
221
effect on hybridization process.
222
The selectivity of ZNA probe to FV Leiden mutation was then tested against to other oligonucleotides
223
having different mutations (Fig. S9). In the presence of hybridization of ZNA/DNA probe with its
224
complementary strand, the highest Rct value is expected in contrast to the ones measured in the
225
presence of other ODNs. In order to examine the selectivity of the ZNA based platform, a batch of
226
experiment was performed in the presence of other oligonucleotides containing single-base
227
mutation located at mtDNA target sequence, or non-complementary sequences. As expected, the
228
highest Rct value was obtained as 1081.7 ± 45.3 Ohm (RSD%, 4.2%, n=3), after the full match
229
hybridization of ZNA probe with mtDNA target. The average Rct and HE % values that obtained after
230
hybridization of ZNA probe with mtDNA target / C-mtDNA/ T-mtDNA/ NC-1/NC-2 presented in Table
231
S1. According to HE % values, it can be concluded that ZNA probe exhibited a selective behaviour
232
even in the presence of oligonucleotides having a different single-base mutation (G>C or G>T), or
233
non-complementary sequences.
SC
M AN U
TE D
EP
AC C
234
RI PT
211
235
Detection of FV Leiden mutation in synthetic PCR products using ZNA based biosensor in contrast
236
to DNA based one:
237
FV Leiden mutation in 143 nt lenght PCR products was detected using ZNA probe / DNA probe
238
immobilized CNF-SPEs, and the results were given in Figure 3 and Table S2. The HE % was calculated
239
related to each experiment on possible hybridization between ZNA probe/DNA probe with
240
mtPCR/wtPCR (Eq. 1). After the hybridization of ZNA probe with wtPCR-1, the value of HE % was 7
ACCEPTED MANUSCRIPT 241
calculated as 34 %, whereas HE % was acccepted as 100 % in the case of hybridization of ZNA probe
242
with mtPCR-1. However, the values of HE % were found to be 79 % and 58 %, respectively in the case
243
of hybridization of DNA probe with mtPCR-1, or wtPCR-1. According to the HE % values (shown in
244
table S2), it is obvious that a selective detection of SNP can be performed by ZNA probe, even this
245
target sequence is a part of PCR product with the length of 143 nt.
247
Figure 3.
248
RI PT
246
Similarly, a batch of experiments was performed using synthetic PCR products in the length of 220 nt
250
(named as mtPCR-2 and wtPCR-2), and the results were shown in Figure 4 and Table S3. After the
251
hybridization of ZNA probe with mtPCR-2, or wtPCR-2, the average Rct values were measured as
252
895.5±48.8 Ohm and 612.5 ± 36.1 Ohm with the RSDs % (n=3) as 5.4 % and 5.9 %, respectively. After
253
hybridization of DNA probe with mtPCR-2, or wtPCR-2, the average Rct values were measured as
254
921.0 ±241.8 Ohm and 1153.0 ± 287.1 Ohm with the RSDs % (n=3) as 26.3 % and 24.9 %, respectively
255
(Fig. 4).
256
According to HE % values that calculated for each synthetic PCR products in length of 220 nt, (shown
257
in Table S3), it was concluded that DNA probe could not present any selective behavior in
258
hybridization process with mtPCR-2, or wtPCR-2. On the otherhand, ZNA probe selectively detect the
259
single-base mutated target, mtPCR-2 over to wtPCR-2, even the target sequence is a part of
260
synthetic PCR product in length of 220 nt.
TE D
M AN U
SC
249
261
Figure 4.
262
Impedimetric detection of FV Leiden mutation by multi-channel array of electrodes (MULTIx8 CNF-
264
SPE):
265
It is aimed to implement our assay to multi-channel array of electrodes which are specially designed
266
for the development of multiple simultaneous analysis. Under this aim, the effect of the changes at
267
the concentration of probe, mtDNA target, hybridization temperature upon to the response
268
measured by MULTIx8 CNF-SPE was examined. In view of that these results were presented in Figure
269
S10-S12 as well as discussed in the supporting information. Further experiments related the
270
detection of FV Leiden mutation by MULTIx8 CNF-SPE were done under the optimized conditions.
271
After the full match hybridization between 5’ZNA probe and mtDNA target in different
272
concentrations varying between 2 and 12 µg mL-1, a gradual increase at the Rct value was obtained
273
until 10 µg mL-1 mtDNA target, and then it levelled off (shown in Fig. 5 and Fig. S13). The average Rct
274
value was measured as 2419 ± 216.0 Ohm with the RSD% as 8.9% (n=3) after the hybridization of 1
AC C
EP
263
8
ACCEPTED MANUSCRIPT 275
µg mL-1 5’ZNA probe with 10 mtDNA target. Thus, the optimum mtDNA concentration was chosen as
276
10 µg mL-1 (equals to 1.4 µM) for further studies with MULTIx8 CNF-SPE.
277 278
Figure 5.
279 The LOD was calculated according to the Miller and Miller method [34] and found to be 0.95 µg mL-1
281
(133 nM) in the linear concentration range of mtDNA target with the equation y=225.69x+150.35 and
282
R2 = 0.99 (shown Fig. S13 inset).
RI PT
280
283
Selectivity of ZNA probe in contrast to DNA probe by MULTIx8 CNF-SPE:
285
The selectivity of ZNA probe to FV Leiden mutation was tested against to oligonucleotides having
286
different mutation (G>C or G>T), where the mutant base was located at mtDNA target,
287
noncomplementary oligonucleotides; such as, C-mtDNA / T-mtDNA / NC-1 / NC-2 (Fig. S14). The
288
values of average Rct with the calculated HE % were given in Table S4. According to the HE % values,
289
ZNA probe exhibited a selective behaviour to its complementary target in contrast to other DNA
290
oligonucleotides containing different single-base mutation, or noncomplementary DNA sequences.
291
or
M AN U
SC
284
Detection of FV Leiden mutation in synthetic PCR products using ZNA based multi-channel array of
293
electrodes in contrast to DNA based one:
294
The impedimetric detection of FV Leiden mutation in the synthetic PCR products with the length of
295
143 nt was carried out using MULTIx8 CNF-SPE and the results were given in Figure 6 and Table S5.
296
After the hybridization of ZNA probe with wtPCR-1, the value of HE % was calculated as 59 %,
297
whereas HE % was acccepted as 100 % in the case of hybridization of ZNA probe with mtPCR-1. On
298
the other hand, the values of HE % were found to be 57 % and 93 %, respectively after hybridization
299
of DNA probe with mtPCR-1, or wtPCR-1. It can be concluded that a more selective detection was
300
performed by ZNA probe to a single-base mutated target in PCR product with 143 nt in contrast to
301
the result obtained by DNA probe.
303
EP
AC C
302
TE D
292
Figure 6.
304 305
Similarly, the selectivity of ZNA based biosensor was tested when the FV Leiden mutation was a part
306
of PCR products with 220 nt; mtPCR-2 and wtPCR-2, and the results were shown in Figure 7 and Table
307
S6. After the hybridization of ZNA probe mtPCR-2, the average Rct value was recorded as 987.0 ±
308
130.1 Ohm (RSD%, 13.2%, n=3) and HE % is accepted as 100% for this case. After the hybridization of
309
DNA probe with mtPCR-2 or wtPCR-2, the Rct values were measured as 1015.5 ± 40.3 Ohm (RSD%, 9
ACCEPTED MANUSCRIPT 310
4.0%, n=3) and 875.0 ± 224.9 Ohm (RSD%, 25.7%, n=3), respectively (Fig. 7). According to HE %
311
values, it can be concluded that ZNA probe selectively detected the single-base mutated target of
312
mtPCR-2 comparison to wtPCR-2, even the target sequence was a part of PCR product in length of
313
220 nt.
314
Figure 7.
RI PT
315 4.Conclusion
317
In the present study, the impedimetric detection of a single nucleotide mutation related to FV Leiden
318
mutation was performed by using the new generation nucleic acids (ZNAs) in combination with
319
carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed
320
array of electrodes (MULTIx8 CNF-SPE). The DLs were found to be 1.48 µg mL-1 (207 nM) and, 0.95 µg
321
mL-1 (133 nM) for CNF-SPEs and MULTIx8 CNF-SPEs, respectively. The DLs were lower that our earlier
322
ZNA based biosensor studies related to FV Leiden mutation [36,37]. The selectivity of ZNA probe to
323
mutation-free DNA sequences, or synthetic PCR samples in different length; 143 and 220 nt
324
containing a single base mutation, or without any mutant base was also examined using
325
impedimetric ZNA biosensor.
326
Herein, the impedimetric analysis of FV Leiden mutation was performed in relatively shorter time (i.e
327
30 min) and selectively in comparison to earlier studies [17,18]. As an another advantage, detection
328
is performed without using any metallic nanoparticles or inosine base substituted probe (metallic
329
nanoparticles or inosine base substituted probe) which makes our assay more practical and cost
330
effective earlier studies [16, 38-40].
331
The remarkable hybridization properties of ZNA, with respect to both affinity and specificity, make it
332
a technology-improving, molecule for molecular biology research and biotechnology innovation. An
333
advanced disposable biosensing platforms were successfully used herein by incorporation of ZNAs
334
with the advantages of their unique chemical and structural properties. Moreover, CNF modified
335
electrodes presented some important advantages such as, being disposable, cost-effective, easy-to
336
use, and not requiring any time consuming modification step, or any sophisticated instruments with
337
a special training in comparison to other biosensor studies in the literature [16-18,38-40].
M AN U
TE D
EP
AC C
338
SC
316
339
Acknowledgements:
340
A.E acknowledges the financial support from Turkish Scientific and Technological Research Council
341
(TÜBİTAK; Project no. 114Z400) as a project investigator, and she also would like to express her
342
gratitude to the Turkish Academy of Sciences (TÜBA) as a Principal member for its partial support.
343
E.E acknowledges a project scholarship through by project (TÜBİTAK Project no. 114Z400).
344 10
ACCEPTED MANUSCRIPT References
346 347
[1] F. Pellestor, P. Paulasova, The peptide nucleic acids, efficient tools for molecular diagnosis (Review) Int. J. Mol. Med. 13 (2004) 521–525.
348 349
[2] B. Pons, M. Kotera, G. Zuber, J.P. Behr, Online synthesis of diblock cationic oligonucleotides for enhanced hybridization to their complementary sequence, Chembiochem, 7 (2006) 1173–1176.
350 351 352
[3] V. Moreau, E. Voirin, C. Paris, M. Kotera, M. Nothisen, J.S., Remy, J.P. Behr, P.; Erbacher, N. LenneSamuel, Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription, Nucleic Acids Res. 37 (2009) e130.
353 354 355
[4] R. Noir, M. Kotera, B. Pons, J.S. J.P. Remy Behr, Oligonucleotide- Oligospermine Conjugates (Zip Nucleic Acids): A Convenient Means of Finely Tuning Hybridization Temperatures, J. Am. Chem. Soc. 9 (2008) 13500-13505.
356
[5] J.L. Kujovich, Factor V Leiden thrombophilia, Genet. Med. (Nature) 13 (2011) 1-16.
357 358
[6] F.R. Rosendaal, P.H. Reitsma, Genetics of venous thrombosis, J. Thromb. Haemost. 7 (2009) 301304.
359 360
[7] J.L. Zehnder, R.C. Benson, Sensitivity and Specificity ofthe APC Resistance Assay in Detection of Individuals with Factor V Leiden, Am J Clin Pathol. 106 (1996) 107-111.
361 362
[8] K. Chang, S. Deng, M. Chen, Novel biosensing methodologies for improving the detection of single nucleotide polymorphism, Biosens. Bioelectron. 66 (2015) 297-307.
363 364
[9] A.C. Syvanen, Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2 (2001) 930–942.
365 366
[10] M.P. Millis, Medium-throughput SNP genotyping using mass spectrometry: multiplex SNP genotyping using the iPLEX® Gold assay. Methods Mol. Biol. 700 (2011) 61–76.
367 368 369 370
[11] K. Fujii, Y. Matsubara, J. Akanuma, K. Takahashi, S. Kure, Y. Suzuki, M. Imaizumi, K. Iinuma, O. Sakatsume, P. Rinaldo, K. Narisawa, Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency. Hum. Mutat. 15 (2000), 189–196.
371 372
[12] L. Beaudet, J. Bedard, B. Breton, R.J. Mercuri, M.L. Budarf, Homogeneous Assays for SingleNucleotide Polymorphism Typing Using AlphaScreen. Genome Res. 11 (2011) 600–608.
373 374
[13] C. Ding, S. Jin, High-throughput methods for SNP genotyping. Methods Mol. Biol. 578 (2009) 245–254.
375 376 377
[14] C. Deulvot, H. Charrel, A. Marty, F. Jacquin, C. Donnadieu, I. Lejeune-Henaut, J. Burstin, G. Aubert. Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea. BMC Genomics 11 (2010) 468.
378 379
[15] Y. Ren, S. Rezania, K.A. Kang, Biosensor for diagnosing Factor V Leiden, a single amino acid mutated abnormality of Factor V, Adv. Exp. Med. Biol. 614 (2008) 245-252.
AC C
EP
TE D
M AN U
SC
RI PT
345
11
ACCEPTED MANUSCRIPT [16] K.A. Kang, Y. Ren, V.R. Sharma, S.C. Peiper, Near real-time immuno-optical sensor for diagnosing single point mutation: A model system: Sensor for factor V leiden diagnosis, Biosens. Bioelectron. 24 (2009) 2785-2790.
383 384 385
[17] M. Ozsoz, A. Erdem, K. Kerman, D. Ozkan, B. Tugrul, N. Topcuoglu, H. Ekren, M. Taylan, Electrochemical Genosensor Based on Colloidal Gold Nanoparticles for the Detection of Factor V Leiden Mutation Using Disposable Pencil Graphite Electrodes, Anal. Chem. 75 (2003) 2181-2187.
386 387 388
[18] D. Ozkan, A. Erdem, P. Kara, K. Kerman, B. Meric, J. Hassmann, M. Ozsoz, Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor, Anal. Chem. 74 (2002) 5931-5936.
389 390
[19] F. Lisdat, and D. Schafer, The use of electrochemical impedance spectroscopy for biosensing, Anal. Bioanal. Chem. 391 (2008) 1555–1567.
391 392
[20] A. Erdem and G. Congur, Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C, Sens. Act. B: Chem. 196 (2014) 168-174.
393 394
[21] M. Steichen, C.B. Herman, Electrochemical detection of the immobilization and hybridization of unlabeled linear and hairpin DNA on gold, Electrochem. Commun. 7 (2005) 416–420.
395 396
[22] I. Giouroudi, G. Kokkinis, Recent Advances in Magnetic Microfluidic Biosensors, Nanomaterials 7 (2017) 171.
397 398
[23] Y. Xianyua, Q. Wang, Y. Chen, Magnetic particles-enabled biosensors for point-of-care testing, TrAC Trends in Anal. Chem. 106 (2018) 213-224.
399 400
[24] M. Pohanka, Magnetic Particles in Electrochemical Analyses, Int. J. Electrochem. Sci. 13 (2018) 12000 – 12009.
401 402
[25] T. Pasinszki, M. Krebsz, T. T.Tung, D. Losic, Carbon Nanomaterial Based Biosensors for NonInvasive Detection of Cancer and Disease Biomarkers for Clinical Diagnosis, Sensors 17 (2017) 1919.
403 404
[26] N.L. Teradal, R. Jelinek, Carbon Nanomaterials in Biological Studies and Biomedicine, Adv. Healthcare Mater. 6 (2017) 1700574.
405 406
[27] S. Gupta, C.N. Murthy, C. Ratna Prabha, Recent advances in carbon nanotube based electrochemical biosensors, Int. J. Biol.l Macromol. 108 (2018) 687-703.
407 408
[28] Z. Zhu, An Overview of Carbon Nanotubes and Graphene for Biosensing Applications, NanoMiicro Letters 9 (2017) 25.
409 410
[29] A. Erdem, Nanomaterial-based electrochemical DNA sensing strategies, Talanta 74 (2007) 318– 325.
411 412
[30] S.H. Lee, J.H. Sung, T.H. Park, Nanomaterial-Based Biosensor as an Emerging Tool for Biomedical Applications, Annals of Biomed. Eng. 40 (2012) 1384-1397.
413 414
[31] J. Wang, Y. Lin, Functionalised carbon nanotubes and nanofibers for biosensing applications. TrAC Trends Anal. Chem. 27 (2008) 619–626.
AC C
EP
TE D
M AN U
SC
RI PT
380 381 382
12
ACCEPTED MANUSCRIPT [32] S.E. Baker, K.Y. Tse, E. Hindin, B.M. Nichols, T.L. Clare, R.J. Hamers, Covalent functionalization for biomolecular recognition on vertically aligned carbon nanofibers. Chem. Mater., 17 (2005) 4971– 4978.
418 419 420
[33] E. Eksin, A. Erdem, Chitosan-carbon Nanofiber Modified Single-use Graphite Electrodes Developed for Electrochemical Detection of DNA Hybridization Related to Hepatitis B Virus, Electroanalysis, 28 (2016) 2514-2521.
421 422
[34] J. N. Miller, J. C. Miller in Statistics and Chemometrics for Analytical Chemistry, 5th ed., Pearson Education, Essex, UK 2005, pp. 121.
423 424 425
[35] A. Ganguly, J. Benson, P. Papakonstantinou, Sensitive Chronocoulometric Detection of miRNA at Screen-Printed Electrodes Modified by Gold-Decorated MoS2 Nanosheets, ACS Appl. Bio .Mater. 1 (2018) 1184−1194.
426 427
[36] A. Erdem, E. Eksin, Zip nucleic acid based single-use biosensor for electrochemical detection of Factor V Leiden mutation, Sens. Act. B. 288 (2019) 634–640.
428 429
[37] A. Erdem, E. Eksin, Magnetic beads assay based on Zip nucleic acid for electrochemical detection of Factor V Leiden mutation, Int. J. Biol. Macromol. 125 (2019) 839–846.
430 431 432
[38] H. Orum, M.H. Jakobsen, T. Koch, J. Vuust, M.B. Borre, Detection of the Factor V Leiden Mutation by Direct Allele-specific Hybridization of PCR Amplicons to Photoimmobilized Locked Nucleic Acids, Clin. Chem. 45 (1999) 1898–1905.
433 434 435
[39] E. Palecek, M. Masarik, R. Kizek, D. Kuhlmeier, J. Hassmann, J. Schülein, Sensitive Electrochemical Determination of Unlabeled MutS Protein and Detection of Point Mutations in DNA, Anal. Chem. 76 (2004) 5930-5936.
436 437
[40] M. Schimid, G.A. Schalasta, A Rapid and Reliable PCR Based Method for Detecting the Blood Coagulation Factor V Leiden Mutation, Biochemica 3 (1997) 12-15.
440 441 442 443
SC
M AN U
TE D
EP
439
AC C
438
RI PT
415 416 417
444 445 446 447 13
ACCEPTED MANUSCRIPT 448 Figure Captions
450
Figure 1. Nyquist diagrams obtained by (a) CNF-SPE, (b) 0.5 µg mL-1 5’ZNA probe immobilized CNF-
451
SPE, after the hybridization between 0.5 µg mL-1 5’ ZNA probe and mtDNA target in its different
452
concentrations (c) 2, (d) 4, (e) 6, (f) 8, (g) 10, (h) 12, (i) 14 µg mL-1 by CNF-SPE. Inset was the
453
equivalent circuit model used for fitting of the impedance data.
454
Figure 2. The Nyquist diagrams of (a) CNF-SPE, (b) 0.5 µg mL-1 (A) 5’ ZNA probe, (B) DNA probe
455
immobilized CNF-SPE, hybridization between (A) 5’ZNA probe or (B) DNA probe and 12 µg mL-1 (c)
456
mtDNA target or (d) wtDNA ODN. Inset was the equivalent circuit model used for fitting of the
457
impedance data.
458
Figure 3. Histograms representing the average Rct values measured by DPV using CNF-SPEs after
459
hybridization of 0.5 µg mL-1 ZNA probe or DNA probe with 12 µg mL-1 mtDNA target, mtPCR-1 and
460
wtPCR-1 using CNF-SPEs (n=3).
461
Figure 4. Histograms representing the average Rct values measured by DPV using CNF-SPEs after
462
hybridization of 0.5 µg mL-1 ZNA probe or DNA probe with 12 µg mL-1 mtDNA target, mtPCR-2 and
463
wtPCR-2 using CNF-SPEs (n=3).
464
Figure 5. Nyquist diagrams obtained by (a) MULTIx8 CNF-SPE, (b) 1 µg mL-1 5’ZNA probe immobilized
465
MULTIx8 CNF-SPE, after the hybridization between 1 µg mL-1 5’ ZNA probe and mtDNA target in its
466
different concentrations (c) 2, (d) 4, (e) 6, (f) 8, (g) 10, (h) 12 µg mL-1 by MULTIx8 CNF-SPE. Inset was
467
the equivalent circuit model used for fitting of the impedance data.
468
Figure 6. Histograms representing the average Rct values measured by DPV using MULTIx8 CNF-SPEs
469
after hybridization of 1 µg mL-1 ZNA probe or DNA probe with 10 µg mL-1 mtDNA target, mtPCR-1 and
470
wtPCR-1 (n=2).
471
Figure 7. Histograms representing the average Rct values measured by DPV using MULTIx8 CNF-SPEs
472
after hybridization of 1 µg mL-1 ZNA probe or DNA probe with 10 µg mL-1 mtDNA target, mtPCR-2 and
473
wtPCR-2 (n=3).
475
SC
M AN U
TE D
EP
AC C
474
RI PT
449
476 477 478 479 480 481 14
ACCEPTED MANUSCRIPT 482 483
Figures
M AN U
SC
RI PT
484
485 486
Figure 1.
490 491 492 493 494 495 496 497
EP
489
AC C
488
TE D
487
498 499 500 501 502 503 15
ACCEPTED MANUSCRIPT
506
Figure 2.
507
M AN U
508 509 510 511 512
517 518 519 520 521 522 523 524
EP
516
AC C
515
TE D
513 514
SC
505
RI PT
504
525 526 527 528 529 530 16
ACCEPTED MANUSCRIPT 531
M AN U
SC
RI PT
532
533 534
Figure 3.
535 536
540 541 542 543 544 545 546 547
EP
539
AC C
538
TE D
537
548 549 550 551 552 553 17
ACCEPTED MANUSCRIPT 554
M AN U
SC
RI PT
555
556 557 558
Figure 4.
559
563 564 565 566 567 568 569 570 571
EP
562
AC C
561
TE D
560
572 573 574 575 576 18
ACCEPTED MANUSCRIPT 577
SC
RI PT
578
M AN U
579 580
Figure 5.
581 582 583 584
588 589 590 591 592 593 594 595
EP
587
AC C
586
TE D
585
596 597 598 599 600 601 19
ACCEPTED MANUSCRIPT 602
M AN U
SC
RI PT
603
604 605
Figure 6.
606 607
611 612 613 614 615 616 617 618
EP
610
AC C
609
TE D
608
619 620 621 622 623 624 20
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
625
626 627
Figure 7.
628
AC C
EP
TE D
629
21
ACCEPTED MANUSCRIPT
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
AC C
EP
TE D
M AN U
SC
RI PT
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
ACCEPTED MANUSCRIPT Credit Author Statement
AC C
EP
TE D
M AN U
SC
RI PT
A.E designed, assisted and analyzed the experiments and wrote the manuscript. E.E performed the experiments and wrote the draft of manuscript.
S0003-2670(19)30470-2
DOI:
https://doi.org/10.1016/j.aca.2019.04.036
Reference:
ACA 236725
To appear in:
Analytica Chimica Acta
Received Date: 16 December 2018 Revised Date:
14 April 2019
Accepted Date: 16 April 2019
Please cite this article as: A. Erdem, E. Eksin, ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.04.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Graphical Abstract
ACCEPTED MANUSCRIPT 1
ZNA probe immobilized single-use electrodes for impedimetric detection of
2
nucleic acid hybridization related to single nucleotide mutation
3 4
Arzum Erdem1,2* and Ece Eksin1,2
6 7
1 2
RI PT
5 Faculty of Pharmacy, Analytical Chemistry Department,
Biotechnology Department, Graduate School of Natural and Applied Sciences,
8
Ege University, Bornova, Izmir, 35100, TURKEY
SC
9 *Correspondence: (A. Erdem):
11
[email protected] and [email protected]; Tel: +90-232-311 5131
M AN U
10
12 13 14
Special issue: New Directions in Electroanalytical Chemistry
15 Abstract
17
The development of a low-cost and disposable biosensing technologies has received a great interest
18
of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single
19
nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based
20
on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden mutation.
21
After optimization of experimental parameters, the sequence selective hybridization between ZNA
22
probe and target related to FV Leiden mutation was evaluated via electrochemical impedance
23
spectroscopy technique (EIS) by measuring changes at the value of the charge transfer resistance, Rct.
24
Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified
25
screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-
26
SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of
27
ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA
28
biosensor was also tested in synthetic PCR samples containing a single base mutation.
AC C
EP
TE D
16
29 30
Keywords: Zip nucleic acids; ZNA; Factor V Leiden mutation; Electrochemical impedance
31
spectroscopy; Electrochemical nucleic acid biosensors; multi-channel screen printed array of
32
electrodes. 1
ACCEPTED MANUSCRIPT 1.Introduction
34
Zip nucleic acids (ZNA) exhibit a high affinity against to target oligonucleotide sequence and have
35
ability to discriminate between a perfect match and a single base-pair-mismatched complementary
36
sequence [1-4]. The Factor V Leiden (FV Leiden) with the most common inherited prothrombotic
37
conditions has been received a great attention and studied by the researchers [5]. The diagnosis of
38
FV Leiden requires the activated Protein C (APC) resistance assay, a coagulation screening test, or
39
DNA analysis of F5 the gene encoding Factor V, to identify the Leiden mutation, a specific G-to-A
40
substitution at nucleotide 1691 that predicts a single-amino acid replacement (R506Q) [5,6].
41
The diagnosis of FV Leiden thrombophilia is established in a proband by identification of a 1691G>A
42
variant coupled with coagulation tests such as the APC resistance assay. It was reported that there
43
was considerable overlap in APC ratios with a normal Factor V genotype and heterozygotes for FV
44
Leiden. The authors concluded that the APC resistance assay in its present form is not a useful
45
screening test for FV Leiden heterozygotes [7]. Until the performance of this assay is improved,
46
patients should have molecular diagnostic testing performed to determine their FV Leiden status.
47
Under this aim, different techniques for SNP genotyping have been reported in recent years [8]
48
including, DNA sequencing [9], mass spectroscopy [10] polymerase chain reaction (PCR) [11,12]
49
techniques. These approaches could specifically detect SNP, but the intrinsic drawbacks (e.g, low
50
throughput and specificity) limit their applications. Alternatively, DNA microarray and denaturing
51
high performance liquid chromatography have been considered as fast and efficient techniques for
52
SNP analysis [13,14]. However, these methods require expensive facilities and radioactive/
53
fluorescent tags as well as require complex procedures; because of this reason, they are not suitable
54
techniques for the development of simple and low-cost analysis methods for point-of-care (PoC)
55
devices. Thus, there is an urgent need to develop selective, sensitive, fast and easy to use platforms
56
for detection of FV Leiden mutation. There are several biosensor studies including immuno-reaction
57
based optic biosensors [15,16] voltammetric biosensors [17,18] for FV Leiden mutation analysis.
58
No report on the development of single-use biosensor has been available yet in the literature
59
impedimetric detection of FV Leiden mutation via electrochemical impedance spectroscopy (EIS)
60
technique. EIS is a powerful method of analysing the complex electrical resistance of the system. It is
61
sensitive to surface phenomena and a valuable method in electrochemical research. In the field of
62
biosensors, it is well-suited to the detection of binding events on the transducer surface [19]. Thus,
63
the impedimetric techniques have been developed in order to characterize the fabricated biosensors
64
and to monitor the catalytic reactions of biomolecules such as enzymes, proteins, nucleic acids,
65
whole cells, antibodies etc. [20,21].
66
Nowadays, electrochemical DNA sensing strategies based on nanotechnology have become one of
67
the most exciting forefronts area due to the challenging advances of nanomaterials, e.g., magnetic
AC C
EP
TE D
M AN U
SC
RI PT
33
2
ACCEPTED MANUSCRIPT particles [22-24] carbon nanomaterials [25-28] by using different electrochemical transducers
69
[29,30].
70
Carbon nanofibers are also easily massproduced at a lower cost, better mechanical stability, and a
71
larger surface-active groups-to-volume ratio than carbon nanotubes. Due to their physical and
72
chemical properties, carbon nanofibers possess a lot of edge sites on the outer wall, leading to high
73
tensile strength, elasticity, high thermal and electric conductivity, which facilitate electron-transfer
74
reactions of analytes [31]. Therefore, there is numerous study which implements carbon nanofibers
75
modification onto the electrode surface. There is a great attention recently to use of CNFs in the
76
development of biosensors due to their unique properties. For example, Baker et al. [32] reported
77
that DNA molecules have been covalently immobilized on vertically aligned carbon nanofibers
78
(VACNFs). According to the results, it was reported that carbon nanofiber samples were efficiently
79
functionalized with DNA molecules with excellent biomolecular recognition properties, according to
80
the hybridization measurements. Thus, it was concluded that VACNFs have shown a higher binding
81
specificity for complementary DNA in contrast to the one of unmodified electrode.
82
To our best knowledge, we introduce a new generation nucleic acid “ZNA” probe based biosensor for
83
the first time in this present work. It was developed for the detection of single nucleotide mutation
84
related to FV Leiden in combination with multi-channel electrochemical array system. This novel
85
assay involves ZNA probe immobilization onto the surface of carbon nanofiber modified screen
86
printed electrodes (CNF-SPE) or multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE).
87
After hybridization occured between ZNA probe and mtDNA target onto the surface of electrode, the
88
impedimetric measurement was performed and accordingly, the Rct values were recorded as a
89
response of hybridization related to FV Leiden detection. The selectivity of ZNA probe was tested in
90
the presence of mutation-free DNA sequences (G>C or G>T) as well as synthetic PCR samples (143 nt
91
and 220 nt) containing a single base mutation (G>A).
93 94 95 96
SC
M AN U
TE D
EP
AC C
92
RI PT
68
97 98 99 100 101 102 3
ACCEPTED MANUSCRIPT 2.Experimental
104
2.1.Apparatus
105
Electrochemical measurements were performed by AUTOLAB-30 and AUTOLAB-302 PGSTAT
106
electrochemical analysis system with NOVA (version 1.1.2 EcoChemie, The Netherlands) or GPES
107
4.9.007 software package (Eco Chemie, The Netherlands) using electrochemical impedance (EIS)
108
technique. The impedimetric measurements with CNF-SPEs was done in a Faraday cage (EcoChemie,
109
The Netherlands).
110
2.2.Chemicals
111
The amino linked from 5' end of ZNA probe and the othe oligonucleotides; complementary mutant
112
type DNA target, wild type DNA oligonucleotide, the oligonucleotides having different types of single
113
base mutation, noncomplementary DNA sequences and PCR products were purchased from (as
114
lyophilized powder) TIB Molbiol (Germany). The sequences of oligonucleotides and PCR products are
115
given in Supporting information.
116
ZNA probe stock solution as 472 µg mL-1 was prepared in Dulbecco’s modified Phosphate Buffer
117
Solution (pH 7.40) and kept frozen. The stock solutions of other oligonucleotides were prepared in
118
ultrapure water (i.e, RNase/DNase free). The diluted solutions of ZNA probe, DNA probe, wtODN, C-
119
mtDNA ODN, T-mtDNA ODN, NC-1, NC-2 and PCR products were prepared in 50 mM phosphate
120
buffer solution containing 20 mM NaCl (PBS, pH 7.40). The diluted solutions of mtDNA target was
121
prepared in PBS (pH 7.40), acetate buffer solution (ABS, pH 4.80), or carbonate buffer (CBS, pH 9.50),
122
that was preferentially used according to the protocol followed herein.
123
Other chemicals were in analytical reagent grade and they were supplied from Sigma-Aldrich and
124
Merck.
125
The detailed information about carbon nanofibers enriched screen printed electrodes (CNF-SPEs) and
126
multi-channel screen printed array of electrodes (MULTIx8 CNF-SPEs) is given in Supporting
127
information.
128
2.3.Procedure
129
The procedure included (i) immobilization of ZNA probe onto the surface of CNF-SPE or MULTIx8
130
CNF-SPE (ii) hybridization of ZNA probe with complementary mtDNA target, or other
131
oligonucleotides; wtDNA oligonucleotide, noncomplementary ODNs (C-mtDNA ODN, T-mtDNA ODN,
132
NC-1, NC-2) as well as complementary mutant type PCR-1,2 and wild type PCR-1,2 onto the electrode
133
surface, (iii) impedimetric measurement.
134
2.3.1.Immobilization of ZNA probe onto the surface of electrodes
135
ZNA probe was prepared in PBS (pH, 7.40) and immobilized onto the surface of working electrode;
136
CNF-SPE / MULTIx8 CNF-SPE via passive adsorption during 30 min as reported in our previous work
137
[33]. Same experimental procedure was followed in the presence of DNA probe selective to mtDNA target
AC C
EP
TE D
M AN U
SC
RI PT
103
4
ACCEPTED MANUSCRIPT as well as control experiments performed spermine alone. Then, the electrode was washed with PBS
139
(pH 7.40) in order to eliminate unspecific binding.
140
2.3.2.Hybridization between ZNA probe/DNA probe and its complementary mtDNA target, or other
141
ODNs
142
The required amount of mtDNA target or other ODNs prepared in PBS (pH 7.40) was dropped onto
143
the probe immobilized working electrode of CNF-SPE / MULTIx8 CNF-SPE during 20 min. Then, the
144
electrode was washed with PBS (pH 7.40) in order to eliminate unspecific binding.
145
2.3.3.Impedimetric measurements
146
Impedimetric measurements were performed in the presence of 2.5 mM K3[Fe(CN)6] and 2.5 mM
147
K4[Fe(CN)6] (1:1). The impedance was measured in the frequency range between 100 kHz and 0.1 Hz
148
at a potential of +0.23 V with an amplitude of 10 mV. The frequency interval was divided into 98
149
logarithmically equidistant measure points. All measurements were performed by AUTOLAB-30
150
system and NOVA software package (version 1.1.2 Eco Chemie, The Netherlands).
151
The real and imaginary impedance (Z’ and -Z’’) are the components of the complex impedance (Z). An
152
equivalent circuit model (Randles circuit) was utilized for fitting of impedimetric results. The electron
153
transfer was limited at higher frequencies and the linear section seen at lower frequencies may be
154
attributed to the diffusion as explained earlier studies [19] According to the Randles circuit, Rs is the
155
solution resistance. Q is the constant phase element. The charge transfer resistance (Rct) is defined as
156
the resistance related to the dielectric and insulating characteristics at the electrode/electrolyte
157
interface. W is the Warburg impedance due to mass transfer to the electrode surface and observed
158
at higher frequencies.
161 162 163 164 165 166
SC
M AN U
TE D
EP
160
AC C
159
RI PT
138
167 168 169 170 171 172 5
ACCEPTED MANUSCRIPT 3.Results and Discussion
174
Impedimetric detection of FV Leiden mutation using ZNA probe in combination with CNF-SPEs:
175
In order to perform efficient ZNA-DNA hybridization, the experimental conditions, such as, the
176
hybridization temperature, the concentration of ZNA probe, mtDNA target and the effect of Mg+2, pH
177
and hybridization time were optimized. Additionally the interference effect of spermine is tested
178
and all results were presented in Fig. S1-S7 as well as discussed in the supporting information.
179
Further experiments related to the detection of FV Leiden mutation (G>A) were done under the
180
optimized conditions.
RI PT
173
181
The effect of mtDNA target concentration upon the hybridization process
183
The nucleic acid hybridization between 0.5 µg mL-1 5’ZNA probe and mtDNA target in different
184
concentrations varying from 2 to 14 µg mL-1 (0.3 µM to 2 µM) was carried out under optimum
185
conditions. The changes at the Rct value was evaluated and shown in Fig. 1. There was a gradual
186
increase at the Rct value after the full match hybridization of ZNA probe with its target, mtDNA up to
187
12 µg mL-1 (1.7 µM) concentration level of mtDNA, and then it levelled off (Fig. 1 and Fig. S8). The
188
highest Rct was measured as 1081.7 ± 45.3 Ohm (RSD%, 4.2%, n=3). Thus, 12 µg mL-1 mtDNA target
189
was used in our further studies performed by CNF-SPEs.
190
The limit of detection (LOD) was calculated according to the Miller and Miller method [34] and found
191
to be 1.48 µg mL-1 (207 nM) in the linear concentration range of mtDNA target with the equation
192
y=47.75x + 144.90 and R2=0.98 (Figure S8 inset).
195
M AN U
TE D
194
Figure 1.
EP
193
SC
182
Selectivity of ZNA probe in contrast to DNA probe
197
The selectivity of the ZNA probe based biosensor was tested againist to wild type DNA target (wtDNA
198
ODN). The average Rct was recorded in the case of full match hybridization between 5’ZNA probe and
199
mtDNA target as 1081.7 ± 45.3 Ohm (RSD %, 4.2 %, n=3), whereas it was obtained as 644.5 ± 68.6
200
Ohm (RSD %, 10.6 %, n=3) in the case of hybridization between 5’ZNA probe and wtDNA target. On
201
the otherhand, the Rct values were measured as 1061 Ohm and 1187 Ohm after the hybridization of
202
DNA probe respectively with mtDNA, or wtDNA ODN (Figure 2). It was concluded that the 5’ ZNA
203
probe presented more selective behavior to detect SNP in contrast to DNA probe.
AC C
196
204 205
Figure 2.
206
6
ACCEPTED MANUSCRIPT 207
The hybridization efficiency
208
effectiveness [35]. It was calculated for each hybridization occured between DNA probe/ZNA probe
209
with each type of target; mutant type or wild type according to the equation 1.
210
(HE %) is used as the signature of the full match hybridization
The hybridization efficiency (HE %) = [∆ ∆ Rct / Rct hybrid] x 100
Eq 1.
( ∆ Rct = Rct hybrid – Rct probe )
212
HE % is accepted as 100 % in the case of hybridization between ZNA probe with its complementray
213
mutant type target. The lower HE % value is expected in the case of hybridization between ZNA
214
probe and wild type target. The differentiation between the values of HE % that is calculated in the
215
presence of ZNA probe should be higher in comparison to the differentiation between HE %s
216
obtained in the presence of DNA probe. Therefore, we can consider that ZNA probe can recognize
217
SNP more selectively than DNA probe. After the possible interaction between spermine and mtDNA,
218
the value of HE % was calculated and found to be 60 %, whereas HE % was acccepted as 100 % in
219
the case of full hybridization between ZNA probe and mtDNA (Fig. S7). According to the data
220
obtained in control experiment, it was concluded that the spermine did not show any interference
221
effect on hybridization process.
222
The selectivity of ZNA probe to FV Leiden mutation was then tested against to other oligonucleotides
223
having different mutations (Fig. S9). In the presence of hybridization of ZNA/DNA probe with its
224
complementary strand, the highest Rct value is expected in contrast to the ones measured in the
225
presence of other ODNs. In order to examine the selectivity of the ZNA based platform, a batch of
226
experiment was performed in the presence of other oligonucleotides containing single-base
227
mutation located at mtDNA target sequence, or non-complementary sequences. As expected, the
228
highest Rct value was obtained as 1081.7 ± 45.3 Ohm (RSD%, 4.2%, n=3), after the full match
229
hybridization of ZNA probe with mtDNA target. The average Rct and HE % values that obtained after
230
hybridization of ZNA probe with mtDNA target / C-mtDNA/ T-mtDNA/ NC-1/NC-2 presented in Table
231
S1. According to HE % values, it can be concluded that ZNA probe exhibited a selective behaviour
232
even in the presence of oligonucleotides having a different single-base mutation (G>C or G>T), or
233
non-complementary sequences.
SC
M AN U
TE D
EP
AC C
234
RI PT
211
235
Detection of FV Leiden mutation in synthetic PCR products using ZNA based biosensor in contrast
236
to DNA based one:
237
FV Leiden mutation in 143 nt lenght PCR products was detected using ZNA probe / DNA probe
238
immobilized CNF-SPEs, and the results were given in Figure 3 and Table S2. The HE % was calculated
239
related to each experiment on possible hybridization between ZNA probe/DNA probe with
240
mtPCR/wtPCR (Eq. 1). After the hybridization of ZNA probe with wtPCR-1, the value of HE % was 7
ACCEPTED MANUSCRIPT 241
calculated as 34 %, whereas HE % was acccepted as 100 % in the case of hybridization of ZNA probe
242
with mtPCR-1. However, the values of HE % were found to be 79 % and 58 %, respectively in the case
243
of hybridization of DNA probe with mtPCR-1, or wtPCR-1. According to the HE % values (shown in
244
table S2), it is obvious that a selective detection of SNP can be performed by ZNA probe, even this
245
target sequence is a part of PCR product with the length of 143 nt.
247
Figure 3.
248
RI PT
246
Similarly, a batch of experiments was performed using synthetic PCR products in the length of 220 nt
250
(named as mtPCR-2 and wtPCR-2), and the results were shown in Figure 4 and Table S3. After the
251
hybridization of ZNA probe with mtPCR-2, or wtPCR-2, the average Rct values were measured as
252
895.5±48.8 Ohm and 612.5 ± 36.1 Ohm with the RSDs % (n=3) as 5.4 % and 5.9 %, respectively. After
253
hybridization of DNA probe with mtPCR-2, or wtPCR-2, the average Rct values were measured as
254
921.0 ±241.8 Ohm and 1153.0 ± 287.1 Ohm with the RSDs % (n=3) as 26.3 % and 24.9 %, respectively
255
(Fig. 4).
256
According to HE % values that calculated for each synthetic PCR products in length of 220 nt, (shown
257
in Table S3), it was concluded that DNA probe could not present any selective behavior in
258
hybridization process with mtPCR-2, or wtPCR-2. On the otherhand, ZNA probe selectively detect the
259
single-base mutated target, mtPCR-2 over to wtPCR-2, even the target sequence is a part of
260
synthetic PCR product in length of 220 nt.
TE D
M AN U
SC
249
261
Figure 4.
262
Impedimetric detection of FV Leiden mutation by multi-channel array of electrodes (MULTIx8 CNF-
264
SPE):
265
It is aimed to implement our assay to multi-channel array of electrodes which are specially designed
266
for the development of multiple simultaneous analysis. Under this aim, the effect of the changes at
267
the concentration of probe, mtDNA target, hybridization temperature upon to the response
268
measured by MULTIx8 CNF-SPE was examined. In view of that these results were presented in Figure
269
S10-S12 as well as discussed in the supporting information. Further experiments related the
270
detection of FV Leiden mutation by MULTIx8 CNF-SPE were done under the optimized conditions.
271
After the full match hybridization between 5’ZNA probe and mtDNA target in different
272
concentrations varying between 2 and 12 µg mL-1, a gradual increase at the Rct value was obtained
273
until 10 µg mL-1 mtDNA target, and then it levelled off (shown in Fig. 5 and Fig. S13). The average Rct
274
value was measured as 2419 ± 216.0 Ohm with the RSD% as 8.9% (n=3) after the hybridization of 1
AC C
EP
263
8
ACCEPTED MANUSCRIPT 275
µg mL-1 5’ZNA probe with 10 mtDNA target. Thus, the optimum mtDNA concentration was chosen as
276
10 µg mL-1 (equals to 1.4 µM) for further studies with MULTIx8 CNF-SPE.
277 278
Figure 5.
279 The LOD was calculated according to the Miller and Miller method [34] and found to be 0.95 µg mL-1
281
(133 nM) in the linear concentration range of mtDNA target with the equation y=225.69x+150.35 and
282
R2 = 0.99 (shown Fig. S13 inset).
RI PT
280
283
Selectivity of ZNA probe in contrast to DNA probe by MULTIx8 CNF-SPE:
285
The selectivity of ZNA probe to FV Leiden mutation was tested against to oligonucleotides having
286
different mutation (G>C or G>T), where the mutant base was located at mtDNA target,
287
noncomplementary oligonucleotides; such as, C-mtDNA / T-mtDNA / NC-1 / NC-2 (Fig. S14). The
288
values of average Rct with the calculated HE % were given in Table S4. According to the HE % values,
289
ZNA probe exhibited a selective behaviour to its complementary target in contrast to other DNA
290
oligonucleotides containing different single-base mutation, or noncomplementary DNA sequences.
291
or
M AN U
SC
284
Detection of FV Leiden mutation in synthetic PCR products using ZNA based multi-channel array of
293
electrodes in contrast to DNA based one:
294
The impedimetric detection of FV Leiden mutation in the synthetic PCR products with the length of
295
143 nt was carried out using MULTIx8 CNF-SPE and the results were given in Figure 6 and Table S5.
296
After the hybridization of ZNA probe with wtPCR-1, the value of HE % was calculated as 59 %,
297
whereas HE % was acccepted as 100 % in the case of hybridization of ZNA probe with mtPCR-1. On
298
the other hand, the values of HE % were found to be 57 % and 93 %, respectively after hybridization
299
of DNA probe with mtPCR-1, or wtPCR-1. It can be concluded that a more selective detection was
300
performed by ZNA probe to a single-base mutated target in PCR product with 143 nt in contrast to
301
the result obtained by DNA probe.
303
EP
AC C
302
TE D
292
Figure 6.
304 305
Similarly, the selectivity of ZNA based biosensor was tested when the FV Leiden mutation was a part
306
of PCR products with 220 nt; mtPCR-2 and wtPCR-2, and the results were shown in Figure 7 and Table
307
S6. After the hybridization of ZNA probe mtPCR-2, the average Rct value was recorded as 987.0 ±
308
130.1 Ohm (RSD%, 13.2%, n=3) and HE % is accepted as 100% for this case. After the hybridization of
309
DNA probe with mtPCR-2 or wtPCR-2, the Rct values were measured as 1015.5 ± 40.3 Ohm (RSD%, 9
ACCEPTED MANUSCRIPT 310
4.0%, n=3) and 875.0 ± 224.9 Ohm (RSD%, 25.7%, n=3), respectively (Fig. 7). According to HE %
311
values, it can be concluded that ZNA probe selectively detected the single-base mutated target of
312
mtPCR-2 comparison to wtPCR-2, even the target sequence was a part of PCR product in length of
313
220 nt.
314
Figure 7.
RI PT
315 4.Conclusion
317
In the present study, the impedimetric detection of a single nucleotide mutation related to FV Leiden
318
mutation was performed by using the new generation nucleic acids (ZNAs) in combination with
319
carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed
320
array of electrodes (MULTIx8 CNF-SPE). The DLs were found to be 1.48 µg mL-1 (207 nM) and, 0.95 µg
321
mL-1 (133 nM) for CNF-SPEs and MULTIx8 CNF-SPEs, respectively. The DLs were lower that our earlier
322
ZNA based biosensor studies related to FV Leiden mutation [36,37]. The selectivity of ZNA probe to
323
mutation-free DNA sequences, or synthetic PCR samples in different length; 143 and 220 nt
324
containing a single base mutation, or without any mutant base was also examined using
325
impedimetric ZNA biosensor.
326
Herein, the impedimetric analysis of FV Leiden mutation was performed in relatively shorter time (i.e
327
30 min) and selectively in comparison to earlier studies [17,18]. As an another advantage, detection
328
is performed without using any metallic nanoparticles or inosine base substituted probe (metallic
329
nanoparticles or inosine base substituted probe) which makes our assay more practical and cost
330
effective earlier studies [16, 38-40].
331
The remarkable hybridization properties of ZNA, with respect to both affinity and specificity, make it
332
a technology-improving, molecule for molecular biology research and biotechnology innovation. An
333
advanced disposable biosensing platforms were successfully used herein by incorporation of ZNAs
334
with the advantages of their unique chemical and structural properties. Moreover, CNF modified
335
electrodes presented some important advantages such as, being disposable, cost-effective, easy-to
336
use, and not requiring any time consuming modification step, or any sophisticated instruments with
337
a special training in comparison to other biosensor studies in the literature [16-18,38-40].
M AN U
TE D
EP
AC C
338
SC
316
339
Acknowledgements:
340
A.E acknowledges the financial support from Turkish Scientific and Technological Research Council
341
(TÜBİTAK; Project no. 114Z400) as a project investigator, and she also would like to express her
342
gratitude to the Turkish Academy of Sciences (TÜBA) as a Principal member for its partial support.
343
E.E acknowledges a project scholarship through by project (TÜBİTAK Project no. 114Z400).
344 10
ACCEPTED MANUSCRIPT References
346 347
[1] F. Pellestor, P. Paulasova, The peptide nucleic acids, efficient tools for molecular diagnosis (Review) Int. J. Mol. Med. 13 (2004) 521–525.
348 349
[2] B. Pons, M. Kotera, G. Zuber, J.P. Behr, Online synthesis of diblock cationic oligonucleotides for enhanced hybridization to their complementary sequence, Chembiochem, 7 (2006) 1173–1176.
350 351 352
[3] V. Moreau, E. Voirin, C. Paris, M. Kotera, M. Nothisen, J.S., Remy, J.P. Behr, P.; Erbacher, N. LenneSamuel, Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription, Nucleic Acids Res. 37 (2009) e130.
353 354 355
[4] R. Noir, M. Kotera, B. Pons, J.S. J.P. Remy Behr, Oligonucleotide- Oligospermine Conjugates (Zip Nucleic Acids): A Convenient Means of Finely Tuning Hybridization Temperatures, J. Am. Chem. Soc. 9 (2008) 13500-13505.
356
[5] J.L. Kujovich, Factor V Leiden thrombophilia, Genet. Med. (Nature) 13 (2011) 1-16.
357 358
[6] F.R. Rosendaal, P.H. Reitsma, Genetics of venous thrombosis, J. Thromb. Haemost. 7 (2009) 301304.
359 360
[7] J.L. Zehnder, R.C. Benson, Sensitivity and Specificity ofthe APC Resistance Assay in Detection of Individuals with Factor V Leiden, Am J Clin Pathol. 106 (1996) 107-111.
361 362
[8] K. Chang, S. Deng, M. Chen, Novel biosensing methodologies for improving the detection of single nucleotide polymorphism, Biosens. Bioelectron. 66 (2015) 297-307.
363 364
[9] A.C. Syvanen, Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2 (2001) 930–942.
365 366
[10] M.P. Millis, Medium-throughput SNP genotyping using mass spectrometry: multiplex SNP genotyping using the iPLEX® Gold assay. Methods Mol. Biol. 700 (2011) 61–76.
367 368 369 370
[11] K. Fujii, Y. Matsubara, J. Akanuma, K. Takahashi, S. Kure, Y. Suzuki, M. Imaizumi, K. Iinuma, O. Sakatsume, P. Rinaldo, K. Narisawa, Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency. Hum. Mutat. 15 (2000), 189–196.
371 372
[12] L. Beaudet, J. Bedard, B. Breton, R.J. Mercuri, M.L. Budarf, Homogeneous Assays for SingleNucleotide Polymorphism Typing Using AlphaScreen. Genome Res. 11 (2011) 600–608.
373 374
[13] C. Ding, S. Jin, High-throughput methods for SNP genotyping. Methods Mol. Biol. 578 (2009) 245–254.
375 376 377
[14] C. Deulvot, H. Charrel, A. Marty, F. Jacquin, C. Donnadieu, I. Lejeune-Henaut, J. Burstin, G. Aubert. Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea. BMC Genomics 11 (2010) 468.
378 379
[15] Y. Ren, S. Rezania, K.A. Kang, Biosensor for diagnosing Factor V Leiden, a single amino acid mutated abnormality of Factor V, Adv. Exp. Med. Biol. 614 (2008) 245-252.
AC C
EP
TE D
M AN U
SC
RI PT
345
11
ACCEPTED MANUSCRIPT [16] K.A. Kang, Y. Ren, V.R. Sharma, S.C. Peiper, Near real-time immuno-optical sensor for diagnosing single point mutation: A model system: Sensor for factor V leiden diagnosis, Biosens. Bioelectron. 24 (2009) 2785-2790.
383 384 385
[17] M. Ozsoz, A. Erdem, K. Kerman, D. Ozkan, B. Tugrul, N. Topcuoglu, H. Ekren, M. Taylan, Electrochemical Genosensor Based on Colloidal Gold Nanoparticles for the Detection of Factor V Leiden Mutation Using Disposable Pencil Graphite Electrodes, Anal. Chem. 75 (2003) 2181-2187.
386 387 388
[18] D. Ozkan, A. Erdem, P. Kara, K. Kerman, B. Meric, J. Hassmann, M. Ozsoz, Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor, Anal. Chem. 74 (2002) 5931-5936.
389 390
[19] F. Lisdat, and D. Schafer, The use of electrochemical impedance spectroscopy for biosensing, Anal. Bioanal. Chem. 391 (2008) 1555–1567.
391 392
[20] A. Erdem and G. Congur, Dendrimer modified 8-channel screen-printed electrochemical array system for impedimetric detection of activated protein C, Sens. Act. B: Chem. 196 (2014) 168-174.
393 394
[21] M. Steichen, C.B. Herman, Electrochemical detection of the immobilization and hybridization of unlabeled linear and hairpin DNA on gold, Electrochem. Commun. 7 (2005) 416–420.
395 396
[22] I. Giouroudi, G. Kokkinis, Recent Advances in Magnetic Microfluidic Biosensors, Nanomaterials 7 (2017) 171.
397 398
[23] Y. Xianyua, Q. Wang, Y. Chen, Magnetic particles-enabled biosensors for point-of-care testing, TrAC Trends in Anal. Chem. 106 (2018) 213-224.
399 400
[24] M. Pohanka, Magnetic Particles in Electrochemical Analyses, Int. J. Electrochem. Sci. 13 (2018) 12000 – 12009.
401 402
[25] T. Pasinszki, M. Krebsz, T. T.Tung, D. Losic, Carbon Nanomaterial Based Biosensors for NonInvasive Detection of Cancer and Disease Biomarkers for Clinical Diagnosis, Sensors 17 (2017) 1919.
403 404
[26] N.L. Teradal, R. Jelinek, Carbon Nanomaterials in Biological Studies and Biomedicine, Adv. Healthcare Mater. 6 (2017) 1700574.
405 406
[27] S. Gupta, C.N. Murthy, C. Ratna Prabha, Recent advances in carbon nanotube based electrochemical biosensors, Int. J. Biol.l Macromol. 108 (2018) 687-703.
407 408
[28] Z. Zhu, An Overview of Carbon Nanotubes and Graphene for Biosensing Applications, NanoMiicro Letters 9 (2017) 25.
409 410
[29] A. Erdem, Nanomaterial-based electrochemical DNA sensing strategies, Talanta 74 (2007) 318– 325.
411 412
[30] S.H. Lee, J.H. Sung, T.H. Park, Nanomaterial-Based Biosensor as an Emerging Tool for Biomedical Applications, Annals of Biomed. Eng. 40 (2012) 1384-1397.
413 414
[31] J. Wang, Y. Lin, Functionalised carbon nanotubes and nanofibers for biosensing applications. TrAC Trends Anal. Chem. 27 (2008) 619–626.
AC C
EP
TE D
M AN U
SC
RI PT
380 381 382
12
ACCEPTED MANUSCRIPT [32] S.E. Baker, K.Y. Tse, E. Hindin, B.M. Nichols, T.L. Clare, R.J. Hamers, Covalent functionalization for biomolecular recognition on vertically aligned carbon nanofibers. Chem. Mater., 17 (2005) 4971– 4978.
418 419 420
[33] E. Eksin, A. Erdem, Chitosan-carbon Nanofiber Modified Single-use Graphite Electrodes Developed for Electrochemical Detection of DNA Hybridization Related to Hepatitis B Virus, Electroanalysis, 28 (2016) 2514-2521.
421 422
[34] J. N. Miller, J. C. Miller in Statistics and Chemometrics for Analytical Chemistry, 5th ed., Pearson Education, Essex, UK 2005, pp. 121.
423 424 425
[35] A. Ganguly, J. Benson, P. Papakonstantinou, Sensitive Chronocoulometric Detection of miRNA at Screen-Printed Electrodes Modified by Gold-Decorated MoS2 Nanosheets, ACS Appl. Bio .Mater. 1 (2018) 1184−1194.
426 427
[36] A. Erdem, E. Eksin, Zip nucleic acid based single-use biosensor for electrochemical detection of Factor V Leiden mutation, Sens. Act. B. 288 (2019) 634–640.
428 429
[37] A. Erdem, E. Eksin, Magnetic beads assay based on Zip nucleic acid for electrochemical detection of Factor V Leiden mutation, Int. J. Biol. Macromol. 125 (2019) 839–846.
430 431 432
[38] H. Orum, M.H. Jakobsen, T. Koch, J. Vuust, M.B. Borre, Detection of the Factor V Leiden Mutation by Direct Allele-specific Hybridization of PCR Amplicons to Photoimmobilized Locked Nucleic Acids, Clin. Chem. 45 (1999) 1898–1905.
433 434 435
[39] E. Palecek, M. Masarik, R. Kizek, D. Kuhlmeier, J. Hassmann, J. Schülein, Sensitive Electrochemical Determination of Unlabeled MutS Protein and Detection of Point Mutations in DNA, Anal. Chem. 76 (2004) 5930-5936.
436 437
[40] M. Schimid, G.A. Schalasta, A Rapid and Reliable PCR Based Method for Detecting the Blood Coagulation Factor V Leiden Mutation, Biochemica 3 (1997) 12-15.
440 441 442 443
SC
M AN U
TE D
EP
439
AC C
438
RI PT
415 416 417
444 445 446 447 13
ACCEPTED MANUSCRIPT 448 Figure Captions
450
Figure 1. Nyquist diagrams obtained by (a) CNF-SPE, (b) 0.5 µg mL-1 5’ZNA probe immobilized CNF-
451
SPE, after the hybridization between 0.5 µg mL-1 5’ ZNA probe and mtDNA target in its different
452
concentrations (c) 2, (d) 4, (e) 6, (f) 8, (g) 10, (h) 12, (i) 14 µg mL-1 by CNF-SPE. Inset was the
453
equivalent circuit model used for fitting of the impedance data.
454
Figure 2. The Nyquist diagrams of (a) CNF-SPE, (b) 0.5 µg mL-1 (A) 5’ ZNA probe, (B) DNA probe
455
immobilized CNF-SPE, hybridization between (A) 5’ZNA probe or (B) DNA probe and 12 µg mL-1 (c)
456
mtDNA target or (d) wtDNA ODN. Inset was the equivalent circuit model used for fitting of the
457
impedance data.
458
Figure 3. Histograms representing the average Rct values measured by DPV using CNF-SPEs after
459
hybridization of 0.5 µg mL-1 ZNA probe or DNA probe with 12 µg mL-1 mtDNA target, mtPCR-1 and
460
wtPCR-1 using CNF-SPEs (n=3).
461
Figure 4. Histograms representing the average Rct values measured by DPV using CNF-SPEs after
462
hybridization of 0.5 µg mL-1 ZNA probe or DNA probe with 12 µg mL-1 mtDNA target, mtPCR-2 and
463
wtPCR-2 using CNF-SPEs (n=3).
464
Figure 5. Nyquist diagrams obtained by (a) MULTIx8 CNF-SPE, (b) 1 µg mL-1 5’ZNA probe immobilized
465
MULTIx8 CNF-SPE, after the hybridization between 1 µg mL-1 5’ ZNA probe and mtDNA target in its
466
different concentrations (c) 2, (d) 4, (e) 6, (f) 8, (g) 10, (h) 12 µg mL-1 by MULTIx8 CNF-SPE. Inset was
467
the equivalent circuit model used for fitting of the impedance data.
468
Figure 6. Histograms representing the average Rct values measured by DPV using MULTIx8 CNF-SPEs
469
after hybridization of 1 µg mL-1 ZNA probe or DNA probe with 10 µg mL-1 mtDNA target, mtPCR-1 and
470
wtPCR-1 (n=2).
471
Figure 7. Histograms representing the average Rct values measured by DPV using MULTIx8 CNF-SPEs
472
after hybridization of 1 µg mL-1 ZNA probe or DNA probe with 10 µg mL-1 mtDNA target, mtPCR-2 and
473
wtPCR-2 (n=3).
475
SC
M AN U
TE D
EP
AC C
474
RI PT
449
476 477 478 479 480 481 14
ACCEPTED MANUSCRIPT 482 483
Figures
M AN U
SC
RI PT
484
485 486
Figure 1.
490 491 492 493 494 495 496 497
EP
489
AC C
488
TE D
487
498 499 500 501 502 503 15
ACCEPTED MANUSCRIPT
506
Figure 2.
507
M AN U
508 509 510 511 512
517 518 519 520 521 522 523 524
EP
516
AC C
515
TE D
513 514
SC
505
RI PT
504
525 526 527 528 529 530 16
ACCEPTED MANUSCRIPT 531
M AN U
SC
RI PT
532
533 534
Figure 3.
535 536
540 541 542 543 544 545 546 547
EP
539
AC C
538
TE D
537
548 549 550 551 552 553 17
ACCEPTED MANUSCRIPT 554
M AN U
SC
RI PT
555
556 557 558
Figure 4.
559
563 564 565 566 567 568 569 570 571
EP
562
AC C
561
TE D
560
572 573 574 575 576 18
ACCEPTED MANUSCRIPT 577
SC
RI PT
578
M AN U
579 580
Figure 5.
581 582 583 584
588 589 590 591 592 593 594 595
EP
587
AC C
586
TE D
585
596 597 598 599 600 601 19
ACCEPTED MANUSCRIPT 602
M AN U
SC
RI PT
603
604 605
Figure 6.
606 607
611 612 613 614 615 616 617 618
EP
610
AC C
609
TE D
608
619 620 621 622 623 624 20
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
625
626 627
Figure 7.
628
AC C
EP
TE D
629
21
ACCEPTED MANUSCRIPT
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
AC C
EP
TE D
M AN U
SC
RI PT
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
ACCEPTED MANUSCRIPT Credit Author Statement
AC C
EP
TE D
M AN U
SC
RI PT
A.E designed, assisted and analyzed the experiments and wrote the manuscript. E.E performed the experiments and wrote the draft of manuscript.