Nanopore-Penetration Sensing Effects for Target DNA Sequencing via Impedance Difference Between Organometallic-Complex-Decorated Carbon Nanotubes with Twisted Single-Stranded or Double-Stranded DNA

We offer a highly sensitive and reproducible dielectric-spectroscopy 11 assay of deoxyribonucleic acid (DNA) sequence on a platform of quantum 12 graphene-like structures arranged on nanoporous alumina to correctly identifying 13 an infectious agent in a native double-stranded (ds) DNA. The hybridization 14 of complementary target DNA with probe DNA in the sensor sensitive layer 15 leads to penetration of the formed single-stranded (ss) target DNA into the 16 underlayer nanoporous anodic alumina through the nanocavities of LB-film from 17 organometallic complexes. This results in linking of MWCNT ends, shielding of 18 Helmholtz double layer and following decrease of electrical capacitance of the 19 sensor. The novel electrochemical impedimetric DNA sensor with self-organized 20 multi-walled carbon nanotube (MWCNT) bundles decorated by organometallic 21 complexes as transducer has been utilized to detect the viral DNA in the biological 22 samples of patients with virus infection at DNA concentration as low as 1.0– 23 1.3 ng/μL. 24 A. S. Babenko Bioorganics Department, Belarusian State Medical University, Minsk, Belarus H. V. Grushevskaya ( ) · N. G. Krylova · I. V. Lipnevich · R. F. Chakukov Physics Department, Belarusian State University, Minsk, Belarus e-mail: grushevskaja@bsu.by; nina-kr@tut.by; lipnevich@bsu.by V. P. Egorova Belarusian State Pedagogical University, Minsk, Belarus © Springer Nature B.V. 2020 J. Bonča, S. Kruchinin (eds.), Advanced Nanomaterials for Detection of CBRN, NATO Science for Peace and Security Series A: Chemistry and Biology, https://doi.org/10.1007/978-94-024-2030-2_17 UN CO RR EC TE D PR OO F A. S. Babenko et al.

Keywords Multi-walled carbon nanotube (MWCNT) · Nanopore-penetration 25 sensing effect · Double-stranded DNA · Single-stranded DNA 26 27 Advances in molecular biology in recent decades are connected with utilizing third-28 generation DNA-nanosequencing label-free methods [1]. But, the sensitivity of 29 these methods appears to be not enough to recognize a viral infection at the stage 30 of fewness lesions of body cells (so called the window period, or the serologic 31 window). In addition the modern DNA-sequencing methods take quite a lot of 32 time for target viral genome identification. Human parvovirus infection leads to the 33 serious complications, including transient aplastic crisis, chronic anemia, and fetal 34 death. In such cases effectiveness of the infectious diseases treatment often depends 35 on the correct identifying an infectious agent, namely on the performance of medical 36 diagnostic methods. High-sensitive methods to detect viral infection is challenge. 37 To reveal human parvovirus infection for medical practice we offer a dielec-38 tric spectroscopy method based on highly selective hybridization interactions of 39 noncovalent complementary single-stranded ss-DNA molecules. Interacting probe 40 ss-DNA and target genomic linear ss-DNA of samples under investigation form 41 a ds-DNA helix of homoduplex on sensor surface for detection of parvovirus 42 infection. The novel high-sensitive method reliably detects the presence or absence 43 of parvovirus in a sample and does not demand expensive consumable materials. 44 The goal of the paper is to study effects of penetration of DNA in nanopores 45 at electrochemical DNA sensing on organometallic-complex-decorated MWCNTs 46 deposited on a nanoporous surface. We will utilize the novel label-free electrochem-47 ical DNA-nanosensor based on carbon nanotubes (CNTs) to identify viral status 48 of native genomic DNA via impedance difference between the metal-decorated 49 MWCNTs with twisted ss-or ds-DNA.

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We utilized two types of ss-DNA probes to recognize parvovirus sequences: direct 53 primer sequence to 3 → 5 -ss-DNA-chain and revers one to 5 → 3 -ss-DNA-54 chain. The direct and revers primers are denoted through B19V F 4 and B19V R4, 55 respectively. The ds-DNA samples have been obtained from blood serum of patients 56 with parvovirus infection (DNA pvi , i = 1, 2, 3) and of practically healthy donors 57 (DNA hi , i = 1, 2, 3) as negative control. Spectrophotometric data for the infection 58 DNA samples are presented in Table 17.1. The spectrofluorimetric method was used 59 also to measure the concentration of DNA hi , i = 1, 2, 3. The concentration of DNA 60 was estimated about 4.5-5.0 ng/μL.

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To calibrate a label-free sequencing an oncogene KRAS, native DNA isolated 62 from colon-cancer tumor, and placental DNA were utilized as a marker gene. 63 The tumor tissue of patients with established diagnosis of colon cancer carrying 64 a mutation single nucleotide polymorphism (SNP) in the second KRAS-exon, 65 codon 12, GGT>GAT were used. Probe DNA KRAS m is a label-free probe 66 oligonucleotide sequence for the KRAS-gene with SNP. RNA and proteins contents 67 in high-purity ds-DNA (1.03 mg/ml in 10 −5 M Na 2 CO 3 buffer medium) isolated 68 from placenta tissue of healthy donors were less that 0.1% (optical density ratio 69 D 260 /D 230 = 2.378 and D 260 /D 280 = 1.866, respectively).

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All DNA probes were purchased in "Primetech ALC" (Minsk, Belarus). Length 71 of the oligonucleotides does not exceed 20 nucleotides.

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All used materials belong to class of analytical pure reagents. (charging-discharging processes in the capacitors), the sensor was connected as 94 the capacitance C into the relaxation resistance (R) -capacitor (C) oscillator 95 (self-excited RC-oscillator) [1,4]. Operating of such RC-generator is based on 96 the principle of self-excitation of an amplifier with a positive feedback on the 97 quasi-resonance frequency. The capacitance C of the sensor entered in measuring 98 RC-oscillating circuit has been calculated by the formula C = 1/(2πRf ), where R 99 is the measuring resistance, f is the frequency of quasi-resonance. 100 Biosensitive nanostructured layers which transduce hybridization signals have 101 been fabricated by Langmuir-Blodgett (LB) technique. The biosensitive coating 102 consists of five monomolecular LB-layers (LB-monolayers) fabricated from 103 nanocyclic complexes of high-spin octahedral iron with dithionylpyrrole (DTP) 104 ligands [5]. Complexes of carboxylated hydrophilic MWCNT with different 105 DNA-probes have been deposited on the LB-film of metal-containing conducting 106 dithionylpyrrole polymer [4]. The synthesized LB-nanoheterostructures were 107 suspended on the interdigital electrode system. The fabricated capacitive DNA-108 nanosensors are sensors of non-Faraday type.

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The response to the interactions between the DNA samples and the viral DNA 110 probes has been detected on nanosensors F14, F24, F32 with the direct primers as 111 DNA probes and on the nanosensors R0, R4, R18 with the revers primers as DNA 112 probes. A DNA-nanosensor K11 with the mutant DNA probe KRAS m recognized 113 SNP in the DNA samples of colon cancer tissue. All electrochemical measurements 114 are performed in deionized water.

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All results were confirmed by the method of sequencing by Sanger. We have 116 discriminated mutation and wild type in 100% of 20 samples.

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Langmuir-Blodgett technique. A fabrication of the LB-monolayers was carried 118 out on an automated hand-made Langmuir trough with controlled deposition on 119 a substrate, and with computer user interface working under Microsoft Windows 120 operational system. Control of the surface tension has been performed by a highly 121 sensitive resonant inductive sensor. The Y-type transposition of monolayers on 122 supports was performed by their vertical dipping. The complexes Fe(II)DTP 3 of 123 high-spin Fe(II) with DTP ligands were synthesized by LB-method at compression 124 of H-dithionilepyrrole molecules on the surface of subphase with salts of three-125 valence Fe [5]. Horizontally and vertically arranged LB-MWCNT-bundles can be 126 fabricated from the carboxylated multi-walled CNTs [6][7][8][9][10][11]. 127 We use the LB-technique to deposit two LB-monolayers of stearic acid 128 micelles with DNA/MWCNT complexes inside on five-monolayer LB-film of 129 the organometallic Ce-containing Fe(II)DTP-complexes. 130 Fabrication of micellar DNA/MWCNT complexes. The micellar complexes ds-131 DNA/MWCNT and oligonucleotide/MWCNT were obtained by means of ultrasonic 132 treatment of alcoholic solution of ds-DNA or oligonucleotide with MWCNT [6]. 133 Then, the complexes were mixed with a solution of stearic acid in deionized water 134 or in hexane. The resulting mixtures were homogenized by ultrasonic treatment to 135 form hydrophilic or hydrophobic (reverse) micelles of stearic acid with complexes 136 ds-DNA/MWCNT or oligonucleotide/MWCNT inside them [12].
Raman spectroscopy studies. Spectral studies in visible range were carried out 138 using a confocal micro-Raman spectrometer Nanofinder HE ("LOTIS-TII", Tokyo, 139 Japan-Belarus) by laser excitation at wavelengths 355, 473 and 532 nm with power 140 in range from 0.0001 to 20 mW at room (RT) and low temperatures. Fe(II) with the dithionilepyrrole ligands. Pyrrole rings of conducting polymer are 147 able to reversible oxidation and reduction [13]. Cation-active (cationic) oxidized 148 pyrrole rings holds dsDNA molecules fixed [14]. This self-redox activity provides 149 DNA fixation during enough time for complementary hybridization, electrostatic 150 repulsion of non-specific-bounded target DNA molecules and leaving from sensor 151 surface in the phase of pyrrole reduction.  Table 17.2 demonstrates that the sensitive layer contains CNTs with some number 157 of walls (about two walls). MWCNTs, which has been selected for transducer, 158 practically do not contain impurities, as characteristic vibration mode D are absent 159 in Raman spectrum of micellar MWCNTs. Since a part of MWCNT charge carriers 160 is localized on the support defects the peak D appears in the Raman spectrum of 161 LB-MWCNT-film at laser excitation with wavelength λ = 532 nm (see Table 17.2). 162 However, decreasing in amplitude the peak D shifts to low frequencies at laser 163 excitation with wavelength λ = 355 nm (Table 17.2). It testifies that the charge 164 carriers confined on the MWCNT surface a can not participate in charge transport 165 onto high-excited impurity levels. Presence of them on the lower excited impurity 166 DNA-levels due π − π -interactions quenches light scattering with wave length 167 λ = 532 nm in both ds-DNA and oligonucleotides (Tables 17.3

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Quinone can direct interact with DNA by forming covalently bonds with DNA 169 bases as menadione, p-benzoquinone and mitomycin C [23][24][25] or by intercalating 170 into DNA helix as anthracyclines [26]. Quinones can also interact with CNTs due to 171 π − π stacking onto the polyaromatic surface of nanotubes [27]. We utilized such a 172 quinone as thymoquinone to prove the presence of recognized complementary target 173 ss-DNA in the senor covering. According data in Fig. 17

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The DNA-nanosensors were placed into deionized water. An electrical double 182 (Helmholtz) layer is formed on the interface. Typical frequency dependencies of 183 the sensor capacity are presented in Fig. 17.1b, c. The principe of target DNA 184 sequence detection is based on shielding near-electrode Helmholtz layer that leads 185 to decrease electric capacitance of double layer in a case of complementary target 186  Since a diameter of this ds-DNA is larger than the nanocavities, the shielding effect 200 is absent and a capacitance of sensor increases (Fig. 17.1c).

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The dielectric loss is measured as the inverse capacity C −1 of sensor. Spectra 202 of Cole-Cole plots are dependencies of the dielectric losses on signal power W . 203 These dependencies correspond to the dependencies of dielectric loss constant 204 17 Nanopore-Penetration Sensing Effects on the real part of complex dielectric permeability (dielectric dispersion). As one 205 can see in Fig. 17.2, the Cole-Cole plots of pure sensors without sensitive coating 206 are characterized by the presence of three Cole-Cole plots with characteristic 207 frequencies λ 0 , λ 1 , λ 2 of dipole relaxation in the range in signal power value W 208 from 2 to 25 V*V. In addition to a capacitance of electrically charged Helmholtz 209 double layer, there is a Warburg impedance element of diffusion layer at signal 210 power more than 40 V*V.

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Deposition of sensor coating leads to appearing of additional frequencies λ p and 212 λ ON of relaxation oscillations of dipoles in the LB-DTP-film and oligonucleotide-213 MWCNT-LB-film, as Fig. 17.2 demonstrates. In Fig. 17.2c, d the data of electro-214 chemical response of the sensors on hybridization of probe oligonucleotides with 215 DNA isolated from the blood of patient with parvovirus infection are presented. 216 As one can see, hybridization of oligonucleotide with complementary viral DNA 217 results in appearing additional frequency λ DNA of dipole relaxation at signal power 218 17-19 V*V. For revers primer B19V R4 along with the appearing the Cole-Cole 219 plots λ DNA dielectric losses increase (screening effect).

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The characteristic Cole-Cole plots λ DNA are absent at non-complementary 221 hybridization between target DNA of all samples obtained from healthy donors and 222 both the DNA probes B19V R4 and B19V F 4 (see Fig. 17.2a, b). Since dielectric 223 losses decrease and accordingly the screening effect is absent an increase of capacity 224 emerges after the noncomplementary hybridization.

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The Raman and impedance spectroscopic assays demonstrate that charge CNT-227 carriers are confined on CNT-surface. Quinone, intercalating into ds-DNA helix, 228 keeps sterically its nucleosides away from CNT-surface. Since the CNT charge 229 carriers can not be transported (localized) on the remote impurity defects, a number 230 of free CNT charge carriers increases by the number of charge carriers localized 231 before. Meanwhile π − π -bonds between nucleosides and CNT-surface break. Due 232 to an attenuation of π − π -interactions conformational mobility of DNA increases 233 and a conformation of DNA molecules attaching to CNT-ends that MWCNTs linked 234 with DNA form a network is an energy-efficient DNA configuration. Hopping 235 conduction of DNA appears after doping in the sites of contact between the DNA 236 molecules and the end groups. The number of contacts increases with thymoquinone 237 concentration (see Fig. 17.1a). Now, a transport of electrical charge occurs along 238 two systems. Since both of systems are high-conductivity ones, screening of near-239 electrode Helmholtz layer by the DNA-CNT network is more effective than by 240 the MWCNT LB-bundles. The native-DNA sequencing performed and presented 241 in the paper is based on this double shielding of external electrical fields.