Label-free DNA sensor based on diazonium immobilisation for detection of DNA damage in breast cancer 1 gene

Accepted Manuscript Title: Label-free DNA sensor based on diazonium immobilisation for detection of DNA damage in breast cancer 1 gene Authors: Seyedeh Zeinab Mousavisani, Jahan-Bakhsh Raoof, Anthony P.F. Turner, Reza Ojani, Wing Cheung Mak PII: DOI: Reference:

S0925-4005(18)30431-3 https://doi.org/10.1016/j.snb.2018.02.152 SNB 24247

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4-12-2017 12-2-2018 20-2-2018

Please cite this article as: Seyedeh Zeinab Mousavisani, Jahan-Bakhsh Raoof, Anthony P.F.Turner, Reza Ojani, Wing Cheung Mak, Label-free DNA sensor based on diazonium immobilisation for detection of DNA damage in breast cancer 1 gene, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.02.152 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.

Label-free DNA sensor based on diazonium immobilisation for detection of DNA damage

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in breast cancer 1 gene

Seyedeh Zeinab Mousavisania, b, Jahan-Bakhsh Raoofb, Anthony P.F. Turnera, Reza Ojanib, Wing Cheung Maka*

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Biosensors and Bioelectronics Centre, Department of Physics, Chemistry and Biology (IFM),

Eletroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty

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b

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Linkoping University, 58183, Linkoping (Sweden)

Corresponding Author:[email protected]

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*

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of Chemistry, University of Mazandaran, Babolsar, Iran

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Research highlights:  Label free DNA biosensor for detection of DNA damage in breast cancer 1 gene. Diazonium chemistry for stable immobilisation of DNA onto screen-printed carbon electrode.

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The DNA sensor could detect the protective effect of glutathione against DNA damage.

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The DNA sensor showed good reproducibility and high stability.

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

Abstract

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Electrochemical DNA biosensors offer simple and rapid tools for detection of DNA sequences or

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damaged genes associated with human disease. The performance of electrochemical DNA sensors

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is critically dependent on the quality of the DNA immobilisation. Many DNA biosensors were

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focused on studying DNA hybridisation that usually preformed in relatively mild assay conditions, while the development of stable DNA biosensors to study DNA damage under a much harsher

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condition typically in the presence of reactive oxygen species is more challenging. In this article,

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we developed an electrochemical DNA biosensor based on a stable diazonium-modified screenprinted carbon electrode (SPCE) for the detection of damage in DNA sequences related to the

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BRCA1 gene by using electrochemical impedance spectroscopy (EIS). The successful preparation

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of the DNA sensor was confirmed by FTIR-ATR, contact angle and electrochemical measurements. The DNA sensor exhibited good reproducibility and high stability and could also

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have potential for investigation of the glutathione antioxidant effect.

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Keywords: Screen-printed carbon electrode, DNA damage, Electrochemical impedance spectroscopy, Diazonium immobilisation

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Introduction Deoxyribonucleic acid (DNA) is a fundamentally important biomolecule that can be attacked via reactive oxygen species (ROS) resulting in oxidative damage. If DNA damage is not repaired in a timely fashion, it can induce gene mutation and consequently lead to carcinogenesis and ageing [1-3].

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Breast cancer is one of the most prevalent and significant diseases occurring in women. Breast

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cancer therapy at advanced stages is often in vain and defacing, making early detection a high

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priority in clinical medicine. Approximately 40% of hereditary breast cancers, and more than 80%

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of both inherited breast and ovarian cancers are due to breast cancer 1 (BRCA1) gene mutations [4]. Hence, the development of a fast and reliable method for detection of oxidative damage in

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DNA sequences related to BRCA1 gene is of great importance for genetic research and clinical

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diagnosis, since the accumulation of damaged DNA, and failures in the repair system (including

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mutation prone repair and hyperactive repair systems) can lead the breast cancer [5]. Various techniques have been used to detect DNA damage including fluorescence [6], single-

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cell gel electrophoresis [7], capillary-zone electrophoresis [8], high-performance liquid chromatography with electrochemical detection (HPLC-ECD) [9], 32P-postlabeling [10] and gas

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chromatography with detection by mass spectrometry (GC/MS) [11]. All these techniques are arduous, time-consuming or involve expensive equipment. Electrochemical DNA biosensors have been suggested as powerful alternative tools for the detection of DNA damage due to their high sensitivity, quick response time, simplicity and relatively low cost [12].

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The performance of electrochemical DNA sensors is critically dependent on the quality of the DNA immobilisation [13]. Hence, stable and controllable immobilisation of DNA onto electrode surfaces is the one of the most important factors in DNA sensor design. Carbon electrodes are

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favoured as a transducers for electrochemical DNA sensors due to their high conductivity, low background current and wide operational voltage windows [13].

There are three major routes for immobilisation of DNA: physical adsorption; streptavidinbiotin assisted immobilisation and covalent immobilisation. Physical adsorption provides a simple method for DNA immobilisation without the need for chemical modification. However, it suffers

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several limitations such as random orientation, relatively weak and unstable attachment of DNA

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onto electrode surfaces and desorption of DNA, which may occur on change of ionic strength, pH

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or temperature [14]. The streptavidin-biotin assisted immobilisation approach provides a more

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stable and controllable method for DNA immobilisation. However, there are still drawbacks with this method such as the relatively high cost of streptavidin, crowding effects and limits to the

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density of DNA probe that can be immobilised onto the electrode surface [15,16]. In contrast,

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covalent immobilisation allows stable attachment of high density of DNA probes onto electrode

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surfaces [16,17]. Covalent immobilisation of DNA on the surface of gold electrodes can be easily performed using thiol-metal interactions, which are due to the strong affinity between thiol groups

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and gold surfaces, via the creation of self-assembled monolayers (SAM) on gold surfaces, followed by grafting of DNA probes onto the SAM [18]. SAMs are widely used for the fabrication of

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electrochemical DNA sensors, but they have some substantial limitations such as low thermal stability [19,20], tedious and time consuming procedures for SAM surface modification and the instability of the resulting SAM in the presence of electrical potentials [21] and UV irradiation [22].

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Covalent immobilisation based on diazonium chemistry is an attractive alternative offering advantages such as fast and easy preparation, and high stability [23,24]. Long-term stability of diazonium grafted layers under atmospheric conditions has been well documented [25] and

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diazonium-grafted surfaces are very stable to exaggerated sonication [26,27], high temperatures [26] and electric potentials [28,29]. Diazonium-grafted surfaces have been applied for the immobilisation of various biomolecules, such as peptides [28], antibodies and proteins [30-33]. However, there are only a few examples of the use of diazonium chemistry for DNA immobilisation and these are focused on DNA hybridisation assays [34-37]. The assay conditions

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for DNA hybridisation are relatively mild in terms of ionic strength, temperature and pH, and they

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are performed in buffers such as phosphate (pH 7.0) or citrate (2 × SSC, pH 7.4) [38]. In contrast,

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the experimental conditions for DNA biosensors used to study DNA damage are much harsher,

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typically being performed at non-neutral pH [39,40], while the damaging reactions will create reactive oxygen species (ROS) [39,40]. Thus, the development of stable DNA biosensors to study

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DNA damage is more challenging. To the best of our knowledge, this is the first report of using

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diazonium salt as a platform for the fabrication of a DNA biosensor for the detection of DNA

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damage. In this study, we developed an electrochemical DNA biosensor based on a diazoniummodified screen-printed carbon electrode for the detection of damage in DNA sequences related

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to the BRCA1 gene. In addition, the protective effect of glutathione as an antioxidant in reducing

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DNA damage was investigated using the screen-printed DNA biosensors developed.

2. Experimental 2.1 Chemicals

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All chemicals were of analytical grade and used as received. 4-aminobenzoic acid, tetrafluoroboric acid solution, sodium nitrite 99.5%, N-hydroxysuccinimide (NHS), N-ethyl-N’(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), potassium ferricyanide (III),

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potassium ferrocyanide (II), sulphuric acid (H2SO4), copper (II) sulfate pentahydrate (CuSO4.5H2O), hydrogen peroxide (H2O2, 30% w/v), methylene blue (MB), L-ascorbic acid (LAA), L-glutathione- reduced were obtained from Sigma–Aldrich (Sweden).

The amine terminated DNA probes designed based on E908X WT breast cancer 1 and its complementary target, were acquired from biomers.net (Germany) and they had the following

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sequences, respectively.

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5'-NH2-AGG-GTG-TCT-GAA-GGA-GGG-GG-3'

2.2 Electrochemical measurements

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5'- CCC-CCT-CCT-TCA-GAC-ACC-CT-3'

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All electrochemical experiments were performed at room temperature using an Ivium Stat.

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XR electrochemical analyser equipped with appropriate software (Ivium, Eindhoven,

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Netherlands). Electrochemical impedance spectroscopy (EIS) was carried out in 0.10 M Tris buffer solution (TBS) (pH 7.0) containing 0.1 M KCl and 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture

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within the frequency range of 0.05 Hz - 100 kHz at a bias potential of 0 mV vs. open circuit potential (OCP) and the amplitude of 5 mV. All of the experiments were repeated 3 times.

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The different stages of DNA sensor preparation were characterised using cyclic voltammetry

(CV) over a potential range + 0.5 to -0.3 V, with a scan rate of 50 mV s−1 in the same solution that was used for EIS measurements. Differential pulse voltammetry (DPV) was performed from − 0.5 V to + 0.1 V, with a pulse amplitude of 10 mV at a scan rate 20 mVs-1 in 0.1 M PBS (pH 7.4).

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Screen-printed carbon electrodes (SPCEs) (DRP-110) consisted of a carbon working electrode (4 mm in diameter), a carbon counter electrode and a silver (to form a Ag/AgCl)reference

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electrode were purchased from Dropsens (Spain).

2.3 Characterisations

Fourier transform infrared (FTIR) was performed with VERTEX 70 (Bruker, USA) equipped with a germanium attenuated total reflectance (ATR) sample cell. Samples were dropped onto the surface of the ATR cell and FTIR spectra were recorded in the frequency region of 800-4000 cmwith a resolution of 4 cm-1 and run for 32 scan cycles at room temperature under continuous

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purging of N2.

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Contact angle measurements were performed using a CAM200 Optical Contact Angle Meter

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(KVS Instrument, Finland). For this purpose, 3 μL of fresh Milli Q water (18.2 MΩ) was dropped onto bare SPCE, ACOOH/SPCE and DNA/ACOOH/SPCE and three images were recorded for

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each of them.

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2.4 Synthesis of 4-aminobenzoic acid tetrafluoroborate (ACOOH) The synthesis of 4-aminobenzoic acid tetrafluoroborate was performed according to a

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procedure described in the literature [41]. In brief, 1.16 gr of 4-aminobenzoic acid was dissolved by heating it in 9 ml of aqueous tetrafluoroboric acid solution (50% w/w). After cooling in an ice

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bath, 0.73 gr of sodium nitrite dissolved in 2 ml cold MilliQ water was added gently to the reaction mixture with stirring. The slurry was cooled in an ice bath, filtered, and rinsed with ice water and cold ether and finally dried under vacuum. The white solid obtained was kept at – 4 °C in the dark.

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The IR spectrum obtained established the presence of the diazonium functional group in the synthesised product.

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2.5. Modification of SPCE with ACOOH salt Modification of the SPCE by electrochemical reduction of diazonium was performed in 0.5 M cold sulphuric acid containing 5 mM of ACOOH. For this purpose, 100 µL of diazonium solution was dropped onto the SPCE and 10 sequential scans from 0 to -1 V at a scan rate of 0.2 Vs-1 were performed [42]. Fig. S1 shows cyclic voltammograms for the electroreduction of

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diazonium salt.

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2.6 Preparation of the DNA biosensor

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Following electrochemical grafting of diazonium, the electrode was sonicated for 1 min in water to eliminate weakly bounded diazonium molecules, and slowly dried in a stream of nitrogen.

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Then a 10µL droplet of aqueous solution containing of EDC (200mM) and NHS (50 mM), was

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placed onto the electrode surface for 30 min to activate the existing carboxyl groups in the grafted

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diazonium. After washing with water and drying under nitrogen, the electrode surface was covered with10µL of 1 μM aminated DNA (in 0.1 M TBS buffer pH 7.0, containing 0.1 M NaCl) for 60

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min. After that, 1 mM ethanolamine (pH 8) was drop cast onto the surface of electrode for 30 min, in order to deactivate unreacted carboxylate groups. Eventually, the modified electrode was rinsed

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with water to eliminate unspecifically chemisorbed DNA. The ability of the proposed DNA sensor to detect DNA damage was assessed using EIS. Scheme 1 depicts the whole process of sensor fabrication and DNA damage detection.

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2.7 DNA biosensors to study DNA damage and the protective effect of glutathione In order to cause DNA damage by Fenton’s reagent, the DNA sensor was immersed in 0.1 M of TBS (pH 7.0) containing damaging agent (1.0 × 10-3 M CuSO4, 1.0 × 10-3 M H2O2 and 1× 10-6

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M AA), under stirring for 60 min. For investigating of induced DNA damage, EIS measurements were performed before and after incubation of the DNA/ACOOH/SPCE in the damaging solution. In addition, glutathione was used as a model to investigate the potential application of the DNA/ACOOH/SPCE for the evaluation of antioxidant capacity. For this purpose, the degree of DNA damage was investigated in the presence of glutathione and the Nyquist plots were recorded.

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In order to correct for electrode-to-electrode or film-to-film variation, the normalised value of Rct

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(Eq. (1)) was employed instead of the absolute Rct as the measurement signal for replicate

∆R

= (R ct,t − R ct,0 )/R ct,o (1)

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R

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experiments:

Where Rct,t and Rct,0 are resistance charge transfer value after and before incubation of

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DNA/ACOOH/SPCE in damaging solution, respectively. Control experiments were conducted by dipping the DNA sensor in a solution containing either CuSO4 (1.0 × 10-3 M), H2O2 (1.0 × 10-3 M)

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or buffer (0.1 M of TBS (pH 7.0). In addition, the measurement signal, ∆R/R, for the proposed

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sensor after incubation in different solutions was obtained and compared in section 3.4.

2.8 Monitoring DNA damage by DPV signals of MB In order to investigate DNA damage using DPV, the DNA/ACOOH/SPCEs that had been treated or untreated by damaging solution, were further modified with complementary target DNA.

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For this purpose, 10 µL of 0.1 M TBS (PH 7.0) containing 0.1 M NaCl and 1.0 µM of complementary target DNA was dropped on the surface of the DNA/ACOOH/SPCE and incubated for 1 h to carry out hybridisation between complementary target DNA and the probe. During this

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time, the electrodes were kept in Petri dishes in a wet chamber to protect the drops from evaporation. After rinsing with water, the electrodes were immersed in 0.1 M PBS (PH 7.4) containing 20 µM MB for 5 min under stirring (100 rpm). After accumulation of MB, the electrodes were rinsed with water for 10 s. Then, the oxidation peak currents of MB were recorded by dropping 100 µL of 0.1 M PBS (pH 7.4) onto the electrode and recording the DPV response of

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MB.

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3.1. Characterisation of the diazonium salt

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3. Results and discussion

In order to characterise the chemical structure of the synthesised diazonium salt, the FTIR-

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ATR spectrum was recorded (Fig. 1A). Spectra a and b corresponded to the 4-aminobenzoic acid

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and 4-aminobenzoic acid tetrafluoroborate, respectively. As shown in spectrum b, new bands

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appeared at about 2304 cm-1and 1040 cm−1 can be ascribed to the presence of (C-N=N) groups [43] and BF4– ions [44], respectively in the 4-aminobenzoic acid tetrafluoroborate. The observed

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peak shift in the carboxylic acid region from 1656 cm-1 to 1718 cm-1 was due to the electron

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acceptor properties of the newly formed diazo group.

3.2. Electrochemical characterisation of the DNA sensor CV and EIS techniques were applied for the electrochemical characterisation of the different steps of sensor fabrication. Fig. 1B represents the cyclic voltammograms of SPCE (a),

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ACOOH/SPCE (b) and DNA/ACOOH/SPE (c) in 0.1 M TBS (pH 7.0) containing 0.1 M KCl and 5.0 mM [Fe(CN)6]3-/4- in equimolar ratio as redox couple (1:1), respectively. As seen in Fig. 1B, a couple of redox peaks could be observed at bare SPCE (a) with a cathodic

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peak current (Ipc) of - 128.07 μA, an anodic peak current (Ipa) of 131.34 μA, and peak-to-peak potential separation (ΔEp= Epa–Epc) of 0.14 V (curve a). Following electrografting of ACOOH onto the SPCE surface, the anodic and cathodic peaks were barely visible. This observation was attributed to the presence of insulating organic diazonium salts on the SPCE surface, as well as electrostatic repulsion between negatively charged COOH group from the diazonium salt that

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hinders the diffusion of [Fe(CN)6] 3−/4− to the electrode surface [45]. After covalent immobilisation

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of amine-functionalised DNA probes onto the ACOOH/SPCE surface, the peaks for the redox

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couple appeared again (curve c), which may be due to the less negative charge density of DNA

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compared to ACOOH, and neutralisation of the excess ACOOH by ethanolamine, which further reduces the negative surface charge density [41,46].

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The CV results were confirmed by the EIS measurements. EIS is a direct and non-destructive

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technique for investigating the interfacial properties, such as charge transfer resistance, after each

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modification step in the sensor fabrication process. The charge-transfer resistance (Rct) values were extracted by fitting Nyquist plots to an equivalent circuit shown in the inset of Fig. 1(C), which

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comprised Warburg impedance (W), solution resistance (Rs), constant phase element (CPE) and charge transfer resistance (Rct). Fig. 1C shows the Nyquist plots of bare SPCE (a), ACOOH/SPCE

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(b) and DNA/ACOOH/SPCE (c). The charge transfer resistance (Rct) significantly increased from 154 Ω for the bare SPCE to 6159 Ω following the ACOOH electrografting process. This increase can be ascribed to formation of a highly packed negatively charged ACOOH film on the electrode surface that was effectively hindering, via electrostatic repulsion, the diffusion of the [Fe(CN)6]3-

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/4-

redox couple to the sensor surface [41,46], and is in agreement with the CV results. After

immobilisation of DNA onto the surface of ACOOH/SPCE and treatment with ethanol amine (c), the Rct value dramatically decreased to 1179 Ω. During the DNA immobilisation step, the ACOOH

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groups were activated by EDC and NHS forming a neutrally charged active NHS ester, followed by reaction of amine functionalised DNA. DNA molecules, due to their relatively high molecular weight and intrinsic negative charge will affect the immobilisation efficiency and result in the formation of a relatively less dense DNA surface, while the electrode surface without immobilised DNA remains as neutrally charged active NHS ester. Thus, the negative charge density of

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DNA/ACOOH/SPCE is less than that of ACOOH/SPCE and facilitated the diffusion of the

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negative [Fe(CN)6]3−/4− redox couple to the electrode surface causing a decrease in the Rct value

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after DNA immobilisation. After treatment of DNA/ACOOH/SPCE with ethanolamine, the Rct

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value was further decreased to 1179 Ω (c), and this decrease can be related to the blocking of

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group [41,46,47] (Fig. S2).

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unreacted COOH which further reduced the surface charge because of the introduction of OH

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3.3. Contact angle measurements

The surface contact angles of the modified SPCEs following each modification step were

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measured. Fig. 2 shows the average values of contact angle and the corresponding images. The measured contact angles of the bare SPCE, ACOOH/SPCE and DNA/ACOOH/SPCE were 110.03º

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± 0.90º, 36.43º ± 0.85º and 26.53º ± 0.82º, respectively. As can be observed, the contact angle of the SPCE decreased consecutively after modification with diazonium salt and DNA, displaying surface hydrophilicity enhancement due to the introduction of COOH groups and subsequently, DNA molecules [41].

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3.4 Detection of DNA damage by EIS Fig.3A shows Nyquist plots of the DNA biosensor before (a) and after incubation with damaging solution (b) for 60 min under stirring. It can be seen that following incubation of the DNA

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biosensor with damaging solution, the charge transfer resistance was significantly increased from 1179 Ω (curve a) to 3350 Ω (curve b), which was reflected in the apparent increase in the semicircular part of the spectrum. Many different types of DNA modifications are produced due to reactive oxygen species including base modification, such as oxidation of nitrogen bases, deamination, depurination, while another type of DNA damage is due to strand breakage [48]. 8-

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oxoguanine is a major product of oxidation damage and has been widely used as an indicator for

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cancer risk. Beside 8-oxoguanine, more than 20 different oxidative modifications of DNA bases

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have been identified [49,50]. The oxidative forms of DNA bases comprise electron rich carbonyl

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group and hydroxyl group that result in an increase in the electro-negativity of the DNA structure. Similarly, deamination will introduce an electron rich carbonyl group into the DNA bases and

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increase the electro-negativity of the DNA. The increase in electro-negativity of the DNA structure

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will affect the diffusion of the negatively charge [Fe(CN)6]3−/4− redox probe and could cause an

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increase in Rct. While, DNA damage resulting from depurination (loss of purine bases), will make the negatively charged phosphate backbone of DNA more available (less shielded by the purine

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bases). Therefore, the negatively charged backbone of DNA becomes more exposed and this affects the diffusion of the negatively charged [Fe(CN)6]3−/4− redox probe and could cause an

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increase in the Rct. In the case of strand break, this will reduce the negative charge of DNA and could cause a decrease in the Rct. Regarding impedimetric biosensors for detection of DNA damage, some articles report an increase in charge transfer (Rct) [51-54] while other articles report a decrease in Rct [55,56] after DNA damage. This could be explained depending on which DNA

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modifications dominated during the particular experimental conditions. In our study, we observed an increase in the Rct after DNA damage that could have been due to the domination of oxidative base modification, deamination and depurination, while the contribution of strand breakage was

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less. Further experiments were performed in order to investigate the effects of various reagents on the degree of DNA damage (Fig. 3B). The ∆R/R values of the biosensors in the presence of buffer (a), Cu2+ (b), H2O2 (c), Cu2+/AA (d), Cu2+/H2O2 (e) and Cu2+/AA/H2O2 (f) were 0.054 ± 0.01, 0.727 ± 0.03, 0.633 ± 0.04, 0.843 ± 0.03, 1.14 ± 0.05 and 1.83 ± 0.06, respectively. As can be seen,

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no obvious change in the Rct value was observed after incubation of the sensor with buffer solution

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(a). In the presence of 1.0 × 10-3 M CuSO4 (b), the R/R value was increased due to the auto

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oxidation of Cu (II) by oxygen and the production of ROS, which creates oxidative damage in the

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DNA [51,57]. Similarly, in the presence of 1.0 × 10-3 H2O2, the R/R value increased indicating the damage to the DNA (c). The enhancement of R/R in the presence of a mixture of CuSO4 +

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AA (d) can be ascribed to induced DNA damage by •OH produced following auto oxidation of ascorbic acid in the presence of metal ions under aerobic condition [58-60]. In addition, the

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reaction between Cu2+ ions with H2O2 produces •OH, which cause DNA damage (e) [61]. As can be observed, the Cu2+/H2O2/AA system (f) showed the highest degree of DNA damage compared

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with the above reactions (b-f), via the following mechanism. (Eqs. (2)-(4)) [62]. Cu2+ + AH− → Cu+ + A

(2)

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Cu+ + 𝐻2 𝑂2 ↔ Cu2+ + • OH + OH − (3) Cu2+ + AH− → Cu+ + A

(4)

During the Fenton reaction (3), the reduced Cu1+, reacts with H2O2 to produce Cu2+and hydroxyl radical. In the presence of ascorbate, the generated Cu2+ can be reactivated (4), thus

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allowing additional •OH to be generated [62].Therefore, we adopted the Cu2+/H2O2/AA system for further studies, since this created the highest degree of DNA damage and had the harshest

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conditions.

3.5. Optimisation of experimental parameters

The effect of incubation time was studied by placing the biosensors into a damaging solution composed of Cu2+/AA/H2O2 under stirred conditions between 0 and 120 minutes, followed by EIS analysis. As illustrated in Fig. 4A, ∆R/R increased with increasing incubation time, and reached a

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maximum value after 60 min. Thus, 60 min was selected as an optimum time for the study.

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In addition to incubation time, the H2O2 concentration dependency of Fenton reagent (ranging

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from 1.0 × 10-3 to 1.0 × 10-6 M) towards the extent of DNA damage was investigated. As shown

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in Fig. S3, the highest degree of DNA damage was observed with Fenton reagent composed of 1.0 × 10-3 M H2O2 and the degree of DNA damage decreased as the H2O2 concentration decreased.

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Hence, Fenton reagent composed of 1.0 × 10-3 M of H2O2 was chosen for further experimental

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study.

3.6 Confirmation of detection of DNA damage by a hybridisation assay

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Although EIS provides a simple and label-free technique to detect DNA damage, it is necessary to confirm that the responses obtained from EIS are truly representative of DNA damage. In this

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context, we used a complementary target DNA to characterise the damaged DNA via hybridisation assay on the sensor surface, followed by DPV analysis with MB as an electroactive indicator. The differential pulse voltammograms in the presence of MB probe of the control DNA/ACOOH/SPCE without hybridisation reaction (a), and DNA/ACOOH/SPCE that incubated

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with (b) and without (c) the damaging solution followed by hybridisation reaction were recorded. As shown in Fig. 4B, the oxidation peak current of MB at the surface of DNA/ACOOH/SPCE appeared at - 0.25 V with a peak current of 4.64 μA (curve a, Fig 4B). After hybridisation of the

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untreated DNA/ACOOH/SPCE with damaging solution, the peak current was obviously increased to 11.10 μA (curve c, Fig 4B). After hybridisation of the DNA/ACOOH/SPCE which had been treated by damaging solution, the peak current obtained was 5.95 μA (curve b, Fig 4B), which is less than curve c. This is thought to be due to the DNA/ACOOH/SPCE treated by damaging solution not forming as much ds DNA following hybridisation compared to the untreated

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DNA/ACOOH/SPCE, because the induced damage by •OH produces some modifications at the

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DNA level, including strand breaks, base and sugar lesions, DNA-protein cross-linking and base-

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free sites. These results indicate that MB as an intercalative indicator has more affinity to double

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stranded DNA (ds-DNA) compared to single stranded DNA (ss-DNA) [39]. Thus, the results

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obtained from this section confirm that the EIS detected DNA damage (Fig. 3A).

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3.7 Potential application for antioxidant capacity evaluation

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Using the optimal experiment conditions determined for DNA damage detection, the antioxidant effect of glutathione was studied using EIS techniques. Fig. 5 shows the Nyquist plots

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of DNA/ACOOH/SPCE before (a) and after incubation with the Fenton reagent in the absence (b), and the presence (c) of glutathione (5.0 × 10-4 M). The concentration of glutathione was within

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physiological concentration [63]. As seen in this figure, the charge transfer resistance of the DNA/ACOOH/SPCE electrodes incubated with Fenton reagent was significantly increased to3350 Ω (curve b), while the electrodes incubated with Fenton reagent in the presence of glutathione only showed a smaller increased in the charge transfer resistance to 1812 Ω (curve c). This demonstrates

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that glutathione can decrease DNA damage and that it has a good antioxidant effect in this system as detected by the DNA sensor. This observation is consistent with glutathione preventing radical formation in the Cu2+/H2O2/AA system, as reported elsewhere [64]. This is maybe due to the fact

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that glutathione can stabilise copper in the oxidation state (+1), and thereby decrease the ability of Cu+1to participate in •OH production [64].

3.8 Reproducibility and stability studies

The reproducibility of the developed DNA sensor was studied by measuring the Rct value for

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five different sensors which had been prepared in the same way. The relative standard deviation

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was 3.8%. The stability of the DNA sensor was examined by recording the variation in Rct value

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of DNA sensors stored at 4 ◦C for 3 weeks. The response of the sensors showed no significant

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change and retained about 83% of the initial value after 3 weeks. The good stability of the sensor

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4. Conclusions

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demonstrated the advantages of diazonium salt for covalent immobilisation of DNA.

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A new and fast impedimetric DNA sensor for the direct detection of damage to DNA sequences related to the BRCA1 gene has been developed. Diazonium salt was employed for

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modification of SPCEs for covalent immobilisation of DNA. The DNA damage was monitored by following the changes in charge transfer resistance after incubation of the DNA sensor in damaging

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solutions. Moreover, this sensor was successfully applied to study the antioxidant capacity of glutathione in reducing DNA damage. This is the first report that uses a diazonium salt-modified SPCE to rapidly and accurately detect DNA damage. The sensor showed excellent stability retains

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up to 83% of the signal response in 3 weeks storage. This fabrication method for DNA sensors provides a good platform for a range of applications in DNA biosensing.

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Acknowledgement Seyedeh Zeinab Mousavisani acknowledges the Ministry of Science Research and Technology of Iran (www.msrt.ir) to support her study visit at Linköping University.

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Authors Biographies

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Seyedeh Zeinab Mousavisani is a Ph.D student of analytical chemistry, University of Mazandaran, Babolsar, Iran. She received her B.S. and M.S. degrees in applied chemistry and Analytical chemistry from University of Mazandaran, Babolsar, Iran, in 2009 and 2012,

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respectively; Her research interests are DNA biosensors and fuel cells.

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Jahan-Bakhsh Raoof is a Professor in the Department of Chemistry at the University of

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Mazandaran, Babolsar, Iran. He received the B.S. and M.S. degrees in Analytical chemistry from

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Tabriz University in 1988 and 1990, respectively, and received the Ph.D in Analytical chemistry (Electrochemistry) from the Tabriz University, Tabriz, Iran in 1996. His research investigations

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have dealt with various aspects of electrochemistry, with an emphasis on sensors, biosensors and

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nanotechnology. He has published over 90 research articles in this area, He has supervised 7

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doctoral students in Electrochemistry, and 20 Master’s students.

Anthony Turner is Emeritus Professor of Biosensors and Bioelectronics at Linköping University.

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He was awarded Higher Doctorates from the University of Kent and the University of Bedfordshire and has >750 publications and patents (>350 refereed journal papers and reviews) in the field of biosensors and biomimetic sensors with an h-index of 76. He is probably best known for his role in the development of commercial glucose sensors, publishing the first textbook on Biosensors, as

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Editor-In-Chief of Biosensors & Bioelectronics (Elsevier) and for chairing the World Congress on

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Biosensors.

Reza Ojani is a full professor of analytical chemistry, University of Mazandaran, Babolsar, Iran. BS from Gilan University, 1988; MS from Tabriz University in analytical chemistry, 1991; Ph.D from Tabriz University in analytical chemistry, 1996. His research interests cover electrochemical

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sensors, biosensors, electrochemical behavior of nanoparticles and fuel cell.

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Wing Cheung Mak is currently an Associate Professor and Head of the Unit of the Biosensors and Bioelectronics of the Department of Physics, Chemistry and Biology (IFM) with research

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focus on bridging technologies between biomaterials, biosensors, regenerative medicine, and

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distributed healthcare with particularly interest on in vitro theranostics (therapy + diagnostics),

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printable bioelectronics and theranostics devices. He has considerable experience on industrial R&D as technical manager and entrepreneur (shareholder of a university spin-off diagnostic

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company - Supernova Diagnostics Inc., and recently being merged with Jupiter Diagnostics Ltd.). He is also the lead investigator IF Sensing Ltd. based in Manchester – UK focus on the

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development of innovative diagnostic solutions for kidney care. He is the author of >50 publications with a h-index of 20 with publications covering the fields in biomaterials, biosensors, materials science, bio-colloids and surface engineering. He is the inventor of four patent families and more than fifteen patents in the field of healthcare and diagnostics.

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Figure Captions

Scheme 1 Schematic representation of DNA sensor fabrication and DNA damage detection

Fig. 1(A) ATR-FTIR spectra of (a) 4-Aminobenzoic acid and (b) 4-Aminobenzoic acid tetrafluoroborate. (B) CVs of bare SPCE (a), ACOOH/SPCE (b) and DNA/ACOOH/SPCE (c) at

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a scan rate 50 mV s-1. (C) Nyquist plots of bare SPCE (a), ACOOH/SPCE (b) and

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DNA/ACOOH/SPCE in 0.1 M TBS (pH 7.0) containing 0.1 M KCl and 5.0 mM

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K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) redox couple.

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Fig. 2 Contact angle measurements for SPCE, ACOOH/SPCE and DNA/ACOOH/SPCE. Fig. 3(A) Nyquist plots of DNA/ACOOH/SPCE (a) before and (b) after incubation with damaging

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solution for 60 min with stirring. (B) Bar chart of ∆R/R for DNA sensor after and before incubation

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with just buffer, TBS (pH 7.0) (a), or TBS (pH 7.0) consist of (b) 1.0 × 10-3 M CuSO4, (c) 1× 10-3 M H2O2 (d) 1.0 × 10-3 M CuSO4 + 1×10-6 M AA, (e) 1.0 × 10-3 M CuSO4 + 1× 10-3 M H2O2 and

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(f) 1.0 × 10-3 M CuSO4 + 1× 10-3 M H2O2 + 1×10-6 M AA.

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Fig. 4(A) Effect of incubation time on DNA damage degree. (B) DPV curves of MB in 0.1 M PBS (PH 7.4) before hybridisation of DNA/ACOOH/SPCE (a), after hybridisation of treated (b) and untreated DNA/ACOOH/SPCE (c) with damaging solution. Fig. 5 Nyquist plots of DNA/ACOOH/SPCE in 0.1 M TBS (pH 7) containing 0.1 M KCl and 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) before (a) and after incubation with damaging solution (1.0 28

× 10-3 M CuSO4 +1.0 × 10-3 M H2O2+1× 10-6 M AA) for 60 min under stirring in the absence (b) and presence of Glutathione (5× 10-4M) (c).

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