DNA-based Electrochemical Biosensor for Imipramine Detection

DNA-based Electrochemical Biosensor for Imipramine Detection

Available online at www.sciencedirect.com ScienceDirect Procedia Engineering 120 (2015) 574 – 577 EUROSENSORS 2015 DNA-based electrochemical biosen...

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Available online at www.sciencedirect.com

ScienceDirect Procedia Engineering 120 (2015) 574 – 577

EUROSENSORS 2015

DNA-based electrochemical biosensor for imipramine detection Joanna Jankowska-Śliwińskaa*, Marek Dawgula, Dorota G. Pijanowskaa* a

Nałęcz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland

Abstract The main aim of our research was development of a novel selective electrochemical method for imipramine (IMI) detection. This substance enable to interact with DNA and it is also electrochemically active. Two major groups of electrochemical sensors were used in the experiments with bare gold electrodes and DNA-modified. Determination of imipramine by means of non-modified electrodes was not selective and limit of detection was shifted towards higher concentrations of IMI. The sensitivity of mixed and mono-GC sequence DNA-modified electrodes was much higher than for the bare gold ones. © by Elsevier Ltd. This an open Ltd. access article under the CC BY-NC-ND license ©2015 2015Published The Authors. Published by isElsevier (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015. Peer-review under responsibility of the organizing committee of EUROSENSORS 2015

Keywords: : electrochemical detection; imipramine detection; DNA biosensor; intercalation into DNA;

1. Introduction The DNA biosensors provide fast, simple, sensitive and selective detection of target substance through its interaction with double-stranded DNA immobilized on the active sensors surface. Imipramine (IMI) is a tricyclic antidepressant (TCA), which belongs to the dibenzazepine group. Due to side-effects for depressive patients treated using these drugs, which may be toxicity and/or inability to drive mechanical vehicles [1], their determination is important in clinical diagnostics. There are a few reports related to electrochemical IMI detection including utilization of highly boron-doped diamond electrodes [1], functionalized carbon paste electrodes [2] and indium-tin oxide (ITO) electrodes modified with molecularly imprinted polymer (MIP) and Au-nanoparticles [3]. Nomenclature AA CPZ

ascorbic acid chlorpromazine

* Corresponding author. Tel.: +48 22 6599143; fax: +48 22 6597030. E-mail address: [email protected]

1877-7058 © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015

doi:10.1016/j.proeng.2015.08.727

Joanna Jankowska-Śliwińska et al. / Procedia Engineering 120 (2015) 574 – 577

DPV IMI LOD LSV PCT SD

differential pulse voltammetry imipramine limit of detection linear sweep voltammetry acetaminophen (in latine: paracetamolum) standard deviation

2. Experimental 2.1. Chemicals The 21-mer modified (at 3’-end) oligonucleotide HS-(CH2)3-3’-AAG GCC GAC TCA GTA GTT CGC-5’ and complementary strand 5’-TTC CGG CTG AGT CAT CAA GCG-3’ were synthesized by Oligo.pl (Warsaw, Poland). The 21-mer modified (at 3’-end) oligonucleotides HS-(CH2)3-3’-(A)21-5’ and HS-(CH2)3-3’-(G)21-5’ and respectively complementary strands 5’-(T)21-3’, 5’-(C)21-3’ were synthesized by Genomed. Acetaminophen, L-ascorbic acid, imipramine and 6-mercapto-1-hexanol were obtained from Sigma-Aldrich. Chlorpromazine was obtained from MP Biomedicals, LLC. Analytical grade reagents and purified water from Millipore Milli-Q system were used for the preparation of phosphate buffer solution. 2.2. Sensors fabrication and measurements In experiments, gold paste electrochemical sensors manufactured by microdosing robot, fabricated in our laboratory [4], were modified with the use of short thiol-labeled oligonucleotides HS-(CH2)3-3’-AAG GCC GAC TCA GTA GTT CGC-5’ and complementary strands 5’-TTC CGG CTG AGT CAT CAA GCG-3’ forming mixed sequence DNA. After immobilization of thiol–labeled strands, extant free surface was blocked by means of 6-mercapto-1-hexanol. Then hybridization with complementary strand was performed at room temperature for 1 h. For elimination of non-specific binding of DNA strands, after each step the electrodes were vigorously rinsed with MQ water. The biosensors prepared according to the described procedure were incubated in solutions of imipramine at concentration range from 0.0005 to 50.0 μM for 2 min, afterwards electrodes were rinsed. In the next stage of experiments sensors were modified (according to above-described procedure) with use one of the short thiol-labeled oligonucleotides HS-(CH2)3-3’-(A)21-5’ or HS-(CH2)3-3’-(G)21-5’ then hybridized with complementary strands 5’-(T)21-3’ and 5’-(C)21-3’, to form mono-AT, and mono-GC sequence DNA, respectively. Prepared biosensors were incubated in solutions of imipramine at concentration range from 0.005 to 0.1 μM for 2 min, next electrodes were rinsed. Afterwards electrochemical measurements were performed in 0.17 M phosphate buffer solution (pH 7.5), by differential pulse voltammetry (DPV) with the use of PalmSens potentiostat (Palm Instruments BV, The Netherlands). For comparison the same procedure was applied for measurements of imipramine with use of bare gold electrodes and DNA modified ones. The last stage of experiments were measurements in sample containing 400 nM IMI, 50 μM CPZ, 200 μM PCT and 2000 μM AA with the use of two measuring methods DPV and linear sweep voltammetry (LSV). 3. Results Comparison of the performances of the electrochemical sensors for imipramine with bare gold electrodes and DNA modified ones are shown in Fig. 1a and Fig. 1b. Linear concentration range of imipramine determination by means of DNA-modified electrodes is significantly shifted towards lower concentrations. In this case of DNAmodified biosensors low detection limit is c.a. 0.0005 μM, while for sensors with bare gold electrodes – c.a. 1 μM.

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Fig. 1. (a) Exemplary differential pulse voltammograms with corrected baseline obtained with bare gold electrodes incubated in imipramine solutions of concentration from 1 to 30 μM. Measurements performed in 0.17 M phosphate buffer (pH 7.5), (b) Exemplary differential pulse voltammograms with corrected baseline obtained on DNA-modified gold electrodes incubated in imipramine solutions of concentration from 0.0005 to 0.1 μM. Measurements performed in 0.17 M phosphate buffer (pH 7.5). DPV method with the following parameters Estep: 7 mV, Epulse:0.80 mV, scan rate: 30 mV/s.

As it can be seen in Fig. 2. the best results (the highest oxidation peaks) and lower standard deviation values were obtained for Au electrodes modified with mixed sequence DNA, while Au modification with mono-GC sequence DNA effects in lower imipramine oxidation peaks and higher standard deviation values. For Au modification with mono-AT sequence DNA imipramine oxidation peaks do not occur. 0.4

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Fig. 2. Calibration curves corresponding with imipramine oxidation peaks height (n=4, ±SD) obtained for electrodes modified with mixed sequence DNA (blue series), mono-GC DNA (red series) and mono-AT DNA (green series). DPV method with the following parameters Estep: 7 mV, Epulse: 0.80 mV, scan rate: 30 mV/s.

In the case of non-modified electrodes, determination of imipramine was not selective and shifted towards high concentration range (Fig. 3). The resulting sensitivity of DNA-modified electrodes was much higher than for the bare gold electrodes. For the above mentioned three group of sensors – 1) bare gold electrodes measured in imipramine solutions, 2) bare gold electrodes measured in phosphate buffer solution after 2-minute incubation and 3) mixed sequence DNA-modified electrodes, imipramine linear concentration ranges are as follows: 10.0 – 50.0 μM, 2.0 – 30.0μM, 0.005 – 0.05 μM, respectively. As it can be seen in Fig. 4, in the case of DPV method both peaks corresponding with peak potentials chlorpromazine (c.a. +0.55V) and imipramine (c.a. +0.74 V) are visible. Using LSV method only one, chlorpromazine oxidation peak (c.a. +0.66 V) is visible, what corresponds with results obtained previously described in paper [5].

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Fig. 3. Calibration curves for imipramine based on differential pulse voltammograms with corrected baseline obtained in IMI solutions on bare gold electrodes (red series), in phosphate buffer of pH 7.5 after 2 min incubation in IMI solutions on bare gold electrodes (series green) and on DNA-modified gold electrodes in phosphate buffer of pH 7.5 (blue series), (n=4, ±SD). Bare Au DNA-modified Au (mixed sequence) DNA-modified Au (mono-G-C)

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Fig. 4. (a) Exemplary DPV with corrected baseline obtained for bare gold electrode – red line, for DNA-modified gold electrode (mixed sequence) – blue line, for DNA - modified gold electrode mono-GC – green line. DPV method: Estep: 7 mV, Epulse: 0.80 mV, scan rate: 30 mV/s. (b) Exemplary LSV with corrected baseline obtained for bare gold electrode – red line, for DNA-modified gold electrode (mixed sequence) – blue line, for DNA - modified gold electrode mono-GC – green line. All measurements performed in 0.17 M phosphate buffer (pH 7.5) after 2-min incubation in samples containing mixture of IMI – 400 nM, CPZ – 50 μM, PCT – 200 μM, AA – 2000 μM. LSV method Estep: 5 mV, scan rate: 100 mV/s.

Summarizing, determination of imipramine by means of non-modified electrodes was not selective and limit of detection was shifted towards higher concentrations of imipramine. The sensitivity of mixed and mono-GC sequence DNA-modified electrodes was much higher than for the bare gold ones. Acknowledgements This research was financed by National Centre of Science (Poland), project no. UMO-2012/07/N/ST4/01841. References [1] T.A. Ivandini, B.V. Sarada, C. Terashima, T.N. Rao, D.A. Tryk, et al., Electrochemical detection of tricyclic antidepressant drugs by HPLC using highly boron-doped diamond electrodes, J. Electroanal. Chem 521 (2002) 117–126. [2] A. Ferancova, E. Korgova, T. Buzinkaiova, W. Kutner, I. Stepanek, J. Labuda, Electrochemical sensors using screen-printed carbon electrode assemblies modified with the β-cyclodextrin or carboxymethylated β-cyclodextrin polymer films for determination of tricyclic antidepressive drugs, Anal. Chim. Acta, 447 (2001) 47-54. [3] X. Xu, G. Zhou, H. Li, Q. Liu, S. Zhang, J. Kong, A novel molecularly imprinted sensor for selectively probing imipramine created on ITO electrodes modified by Au nanoparticles, Talanta 78 (2009) 26-32. [4] M. Dawgul, B. Rozum, J. Jankowska-Śliwińska, J. Kruk, W. Torbicz, D. G. Pijanowska, An innovative method for complete microsensors fabrication, 26TH EUROPEAN CONFERENCE ON SOLID-STATE TRANSDUCERS, EUROSENSOR 2012 Book Series: Procedia Engineering 47 (2012) 1430-1433. [5] J. Jankowska-Śliwińska, M. Dawgul, D. G. Pijanowska, DNA intercalation-based amperometric biosensor for chlorpromazine detection, 28TH EUROPEAN CONFERENCE ON SOLID-STATE TRANSDUCERS, EUROSENSORS 2014 Book Series: Procedia Engineering 87 (2014) 747-750.