Electrochemical oligonucleotide – based biosensor for the determination of lead ion Marta Jarczewska, Ewa Kierzkowska, Robert Zi´ołkowski, Łukasz G´orski, El˙zbieta Malinowska PII: DOI: Reference:

S1567-5394(14)00095-4 doi: 10.1016/j.bioelechem.2014.06.013 BIOJEC 6760

To appear in:

Bioelectrochemistry

Received date: Revised date: Accepted date:

11 April 2014 30 June 2014 30 June 2014

Please cite this article as: Marta Jarczewska, Ewa Kierzkowska, Robert Zi´ olkowski, L  ukasz G´ orski, El˙zbieta Malinowska, Electrochemical oligonucleotide – based biosensor for the determination of lead ion, Bioelectrochemistry (2014), doi: 10.1016/j.bioelechem.2014.06.013

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ACCEPTED MANUSCRIPT Electrochemical oligonucleotide – based biosensor for the determination of lead ion

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Marta Jarczewska, Ewa Kierzkowska, Robert Ziółkowski, Łukasz Górski*, Elżbieta Malinowska

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Institute of Biotechnology, Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

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*Corresponding author. Tel.: +48 222 347573, Fax: +48 226 282741. E-mail address: [email protected] (Ł. Górski)

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Abstract

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The possibility of utilization of gold electrodes modified with short guanine-rich ssDNA probes for determination of Pb2+ was examined. Interaction between guanine residues and lead ion followed by formation of G-quadruplex structures was confirmed by electrochemical impedance spectroscopy investigations. An external cationic redox label, methylene blue, was employed in voltammetric measurements for analytical signal generation. It was shown that due to the G-quadruplexes formation, the oligonucleotides in the recognition layer fold, which enhances the electron transfer between methylene blue and the electrode surface. The MB current signal rises proportionally to the lead ion concentration in the range from 0.05 to 1 µmol /L. The developed biosensor demonstrated high selectivity towards Pb2+ ion, with only minor response towards interfering metal cations. The calculated limit of detection was of 34.7 nmol /L. The utilization of the biosensor for Pb2+ determination in real samples of water was also tested.

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Keywords: Electrochemical sensors, DNA-based biosensors, Metal ion determination

ACCEPTED MANUSCRIPT 1. Introduction

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Lead ion is one of the most toxic pollutants, which can severely affect human health and the environment. The Pb2+ ions accumulation can cause the substantial damage to the brain, central nervous system, muscles and renal systems [1,2]. Therefore, the routine monitoring of lead ion in the environmental and biological samples is of a crucial significance and has become an important subject of chemical analysis. There are several methods which have been used for Pb2+ ion detection, and most common techniques are inductively coupled plasma mass spectrometry, atomic absorbance spectrometry and capillary electrophoresis [2,3]. However, the apparatus complexity and the necessity of sample pretreatment are important drawbacks of above mentioned techniques. On the contrary, electrochemical methods have overcome those limitations as they show several important advantages such as: low-cost, simplicity, selectivity and sensitivity [4]. One of frequently used electrochemical techniques for lead ion determination is potentiometry with the application of Ion Selective Electrodes (ISE). As the Pb2+ ions show affinity towards sulfur atoms, one of the best Pb2+ ionophores is the thioamide derivative of ptert-butyl-calix[4]arene, which is also commercially available as lead ionophore IV [5]. Determination of lead ions can be also performed with the utilization of stripping voltammetry [6,7]. In the case of adsorptive stripping voltammetry, the detection limit of 1.67•10-10 mol /L has been achieved [8]. To further improve the selectivity and lower detection limit, complexing agents such as thiolated calixarenes were employed [9] and the utilization of bismuth modified electrodes was also proposed [10,11]. In the last few years, much work was dedicated to nucleic acid based sensors, as they represent a new research area with the possibility of application in environmental, clinical and food analysis [12]. DNA-based biosensors employ the group of “functional nucleic acids”, which refer to naturally occurring double and single stranded DNA and RNAs, including ribozymes, microRNA and riboswitches [13]. Moreover, there is also a wide range of artificially synthesized oligonucleotides such as aptamers, DNAzymes, ribozymes and allosteric nucleic acid enzymes [14]. Depending on the interaction manner, DNA-based biosensors were employed for the detection of hybridization or DNA damage [12,15], but also for determination of organic and inorganic compounds (Hg2+, K+, Cd2+, adenosine) [16], proteins (PDGF, thrombin) and whole cells (E. coli) [17, 18]. In the case of Pb2+ ion determination, a number of DNAzyme - based sensors were introduced [3,19] and the detection limit of 6.2 pmol /L was demonstrated [20]. Moreover, significant attention was paid to the utilization of oligonucleotides and aptamers of defined sequence, as lead ion has a strong affinity towards adenine [21] and guanine residues, with the latter formation of G-quadruplex structure [2,22 - 24]. Most of Pb2+ DNA sensors utilize fluorescence as the detection method because of its sensitivity and possibility of combining DNA molecules with gold nanoparticles [3], porphirins [24] and fluorophore - quencher systems [19]. Herein, we report on the use of DNA oligonucleotides as a receptor layer for electrochemical determination of lead ions. The action of proposed sensor is based on the stabilization of G-quadruplex structure in the presence of lead ion. As a result, the oligonucleotide strands undergo a conformational change leading to the enhancement of electron transfer between MB molecules and electrode surface. This process can be quantified by MB redox current which is proportional to the Pb2+ concentration. To our knowledge, this is the first biosensor, where the specific interaction between external redox - MB and G quadruplex structure was employed for the detection of lead ions. Moreover, the developed system can be used for analysis of real samples.

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ACCEPTED MANUSCRIPT 2. Experimental 2.1 Apparatus

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Voltammetric studies were conducted with a CHI 660A and CHI 1040A electrochemical workstations (CH Instruments, USA). Electrochemical impedance measurements (EIS) experiments were performed with a CHI 660A electrochemical workstation (CH Instruments, USA). The measurements were performed with a conventional three-electrode system consisting of a gold disk electrode (CH Instruments, USA), an Ag/AgCl/1.0 mol /L KCl reference electrode and a gold wire auxiliary electrode. All potentials are reported versus Ag/AgCl reference electrode at room temperature. EIS was carried out at a dc potential of 200 mV and the ac amplitude was 5 mV. Frequency was in the range from 0.1 Hz to 100 kHz. ESI results were analyzed based on the equivalent circuit consisting of a resistor connected in series to a capacitor. The complex impedance is represented by the following equation:

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Z (jω) = Z' (ω) + jZ'' (ω)

(2) (3) (4)

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where: Z' (ω) = R Z'' (ω)= -1/ωC ω = 2πf (f - frequency)

(1)

2.2 Reagents

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The cyclic voltammetry was conducted at a sweep rate of 100 mV·s-1 in the range of 0.4 to 0.6 V, while the square wave voltammetry was recorded at a pulse amplitude of 25 mV, increment of 4 mV and a frequency of 15 Hz in the range of 0.4 to -0.6 V.

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Tris-HCl, methylene blue, lead nitrate, mercury(II) chloride, calcium nitrate, magnesium nitrate, copper (II) nitrate, cadmium nitrate, iron (III) nitrate, KH2PO4, NaOH and 6mercapto-1-hexanol (MCH) were purchased from Aldrich Chemicals. K3[Fe(CN)6] and K4[Fe(CN)6] were purchased from Fluka. H2SO4, HCl, KCl and H2O2 were purchased from POCh, Poland. All reagents were used without further purification. All solutions were prepared with Milli-Q water. Milli-Q water and all aqueous buffer solutions were sterilized using an autoclave. Deoxyoligonucleotides were purchased from Genomed Sp. z o.o., Poland. The base sequences were as follows (all used probes were thiolated DNA oligonucleotides): Poli T: 5′-SH-(CH2)6- TTT TTT TTT TTT TTT TTT TT -3′ (100% thymine bases) Poli A: 5′-SH-(CH2)6- AAA AAA AAA AAA AAA AAA AA -3′ (100% adenine bases) TBA: 5′-SH-(CH2)6- GGT TGG TGT GGT TGG -3′ TBAC: 5′-SH-(CH2)6- CCT TCC TCT CCT TCC -3′ Oligonucleotide stock solutions were prepared with 10 mmol /L Tris–HCl, (pH 7.5) and stored in a −20o C freezer before use. 2.3 Solutions The following solutions were prepared: piranha solution (H2O2/H2SO4; 1:3), immobilization buffer solution (1 mol /L KH2PO4, pH 4.5), Tris-HCl solution (50 mmol /L, pH 3.0), mercaptohexanol solution (4 µmol /L in immobilization buffer solution), Pb(NO3)2 solution in

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50 mmol /L Tris-HCl solution, Pb(NO3)2/ Cd(NO3)2/ Fe(NO3)3/ HgCl2/ Ca(NO3)2/ Cu(NO3)2/ Mg(NO3)2/ KCl solution in 50 mmol /L Tris-HCl solution containing 50 µmol /L methylene blue, 5 mmol /L K3[Fe(CN)6]/K4[Fe(CN)6] solution in 50 mmol /L Tris-HCl solution containing 100 mmol /L KCl. The voltammetric measurements were carried out in 50 mmol /L Tris-HCl containing 50 µmol /L methylene blue. The EIS measurements were performed in 50 mmol /L Tris-HCl solution containing KCl and equimolar concentrations of K3[Fe(CN)6]/K4[Fe(CN)6].

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2.4 Methods

Δ = (I – I0)/I0

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Before any electrochemical experiments, the gold electrode was polished with alumina powder of grain sizes from 1 to 0.05 µm (CH Instruments, USA). Then, the electrode was washed with demineralized water and sonicated for 2 min at 400 C. Next, the piranha solution was dropped on the working gold disk electrode and incubated for 1 min. After removing the solution, the electrode was washed with demineralized water. The last step of electrode cleaning was its voltammetric cycling in 50 mol /L Tris-HCl solution (pH 3.0), until the CV characteristic for a clean gold was obtained. The DNA recognition monolayer was prepared as described in [25]. Briefly, after the electrode cleaning, 25 µL of 4 µmol /L solution of thiolated ssDNA in 1 mol /L KH2PO4 (pH 4.5) was dropped on the gold working disc electrode. The ssDNA immobilization was carried out for 120 min. Then, the solution was removed and the electrode was washed with 1 mol /L KH2PO4 (pH 4.5). Then (if needed) 25 µL of 4 µmol /L MCH solution was placed on the electrode's surface for 60 min. Finally, the solution was removed and the electrode was washed with immobilization buffer solution and the electrochemical measurements were conducted. The sensor response was defined with use of the following equation: (5)

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where I0 refers to methylene blue reduction current before and I to the methylene blue reduction current after the 5 min incubation in solution containing lead ions. 3. Results and discussion 3.1 Oligonucleotide sequence Preliminary voltammetric (CV, SWV) measurements for DNA-modified electrodes were conducted without any external label, as lead ion shows redox properties. However, this approach was not successful, as a current signal was much lower in comparison to unmodified gold electrodes (data not shown). For further experiments, a cationic redox label, methylene blue was introduced. As it was previously mentioned [21], Pb2+ ions exhibit affinity towards adenine residues in the DNA strand, where it competes with MB. As a result, a decrease of MB current signal was observed, as the label molecules were expelled from the receptor layer. To confirm the interactions between adenine and lead ions, two 20-nucleotide sequences differing in base content were tested: poli A (100% adenine bases) and poli T (100% thymine bases). The oligonucleotide sequences consisted of 20 nucleotides so that higher sensitivity could be obtained in comparison to shorter 5 or 10-mer ssDNA probes [26]. As shown in Table 1, a decrease of analytical signal (the relative difference of MB reduction peak current before and after electrode subjection to the Pb(NO3)2 solution for 5 min) was observed for both ssDNA probes. In case of poli A, the change of current signal was 0.09 higher than for

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poli T (pH 3.0 and pH 4.5) and this can be explained by the fact that Pb2+ ions interacts stronger with adenine than with thymine residues. On the other hand, thymine shows higher affinity towards MB, which is evidenced via comparison of MB reduction current signal before incubation in Pb(NO3)2 solution for both ssDNA probes. Therefore, for poli T sequence, the redox label might not be easily replaced by lead ions. It is also noticeable that at pH 6.0 an increase of current signal (poli A probe) or almost negligible response (poli T) is detected, which can be attributed to the subjection of lead ions to hydrolysis and formation of hydroxyl complexes in pH above 5.0 [27].

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Here Table 1

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According to the literature reports, Pb2+ ion also exhibits strong affinity towards guanine residues, which results in formation of G-quadruplex structure within G-rich sequences [2, 22 - 24]. To confirm these interactions, a set of square wave voltammetry measurements was conducted for electrodes modified with two 15-nucleotide sequences: guanine rich TBA (thrombin binding aptamer), which was also utilized in the detection of lead ions [2] and TBAC (containing cytosine instead of guanine residues) as a reference sequence. As it can be seen in Fig. 1, at pH 4.0, a dramatic change of analytical signal was observed for TBA, while for TBAC the signal change was negligible. It should be emphasized that the MB current increases for these strands after the incubation with sample solution containing Pb2+ ions. In sharp contrast to these results, current decrease was observed for poli A and poli T strands, as well as for other DNA-modified electrodes selective towards metal cations [28,29]. This unexpected sensor behavior can be explained by the following mechanism: after immersion of oligonucleotide-modified electrode in the sample solution containing lead cations, the Gquadruplex structure is formed and the oligonucleotide strands are folded. As a result, DNA strand is closer to the electrode surface and the electron transfer between methylene blue and the electrode occurs more easily (Scheme 1). Moreover, it was reported that guanine bases are exposed when G-quadruplexes is formed, which leads to the enhanced accumulation of MB molecules, as they exhibit strong affinity towards guanine [30].

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Scheme 1. Schematic illustration of the mechanism of biosensor response. 3.2 Impedimetric investigations of Pb2+ ions - DNA interaction To verify the existence of interaction between lead ions and G-quadruplex structure, electrochemical impedance spectroscopy experiments were performed. The Nyquist plot,

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derived from impedimetric data, presents the subsequent steps of gold electrode modification. For these measurements, a mixed monolayer (ssDNA/MCH) was utilized. The receptor layer modified with TBA or TBAC is negatively charged, thus a repulsion of Fe(CN)6 3-/4- redox couple can be observed. In the case of TBA, the Rct value decreases after incubation of ssDNA/MCH monolayer in Pb2+ solution (Fig. 2a). This can be attributed to the formation of G-quadruplexes and subsequent folding of the ssDNA strand. Therefore, the Fe(CN)6 3-/4marker can get closer to the electrode surface. In addition, Pb2+ cation bound to the DNA layer diminishes negative charge of the receptor layer. In contrast, for the TBAC – modified electrode, the Rct value does not change after the subjection of sensor to the Pb(NO3)2 solution (Fig. 2b). This can be explained by the fact that Pb2+ do not exhibit strong affinity towards cytosine or thymine residues and, as a result, no secondary structures within recognition monolayer are formed. Based on the results of preliminary studies, TBA sequence was chosen for the development of ssDNA-based biosensor for Pb2+ ions detection. Here Fig. 2

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3.3 Calibration curve

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To prepare the calibration curve for the proposed sensor, the Δ values were plotted against lead ion concentration. It is evident from Fig. 3 that the analytical signal rises together with the increase of Pb2+ concentration. The sensor response is linear in the Pb2+ concentration ranging from 0.05 to 1 µmol /L, however for higher concentrations, calibration curve flattening is apparent. The lower limit of detection, calculated as 3δ (δ – standard deviation of blank sample), is 34.7 nmol /L. While the LOD towards Pb2+ of the proposed sensor is rather high, it is slightly below the maximum contaminant level of lead ion in drinking water, determined by US EPA (0.015 mg /L, 72 nmol /L) [31].

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3.4 Selectivity Finally, the selectivity of proposed biosensor was investigated against some common metal ions (Cu2+, Cd2+, Ca2+, Fe3+, Hg2+, Mg2+) at the concentration of 1 µmol /L. As it is shown in Fig. 4a, the most significant response was observed for Pb2+ ions ( = 0.54) and the strongest interference was registered for Hg2+ ions ( = 0.14). The interference of Hg2+ ions cannot be excluded, as they interact specifically with thymine, which is followed by formation of thymine - Hg2+ - thymine bridges. The influence of mercury ions can be reduced by addition of masking agents e.g. cysteine, mercaptopropionic acid, CN- or SCN-. The sensor response towards other metal ions is negligible in comparison to the signal obtained for lead ions. Similar measurements were performed with the use of TBAC probe (Fig. 4b). It is evident that for all cations, except Hg2+, the sensor response is minor, including lead ions. The signal obtained for mercury ions can be explained similarly to the case of TBA probe, namely by the presence of thymine in ssDNA sequence. Here Fig. 4 Here Fig. 5

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It is well known that G-quadruplexes can be also formed in the presence of potassium ions [32] and therefore it is necessary to distinguish signals from lead and potassium ions. The biosensor response in the presence of K+ ions was tested for both ssDNA sequences (Fig. 4). For TBA probe it was observed that the relative current difference is comparable with the response for other interferences, while for TBC it was hardly visible. The binding of Pb2+ ions in presence of potassium ions was also tested and it was observed that the relative current difference was slightly higher than for Pb2+ ions (Fig. 5). Nevertheless, the influence of K+ ions could be reduced with the application of masking agents such as phytic acid [33]. It can be reasoned that lead ions form more stable structure with aptamer than K+ ions. Consequently, the binding affinity of Pb2+ towards TBA was calculated and compared with the value for K+ ions. The binding constant was of 1.7 μmol /L and it was obtained from the calibration curve, which was transformed into the Langmuir isotherm (see Fig. ESM 1 in Electronic Supplementary Material). On the contrary, the binding constant for K+ was determined to be 5 μmol /L [34]. It is therefore evident that lead ion exhibit higher affinity towards thrombin binding aptamer than potassium ion. Moreover, there might be a competition between K+ and methylene blue, which also exhibits affinity towards oligonucleotide sequence (MB can interact with ssDNA electrostatically and with guanine residues). 3.5 Analysis of real samples

Here Table 2 4. Conclusions

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The utility of the proposed biosensor was tested by the detection of Pb2+ ions in tap water. The tap water sample contained 50 mmol /L Tris and 50 μmol /L methylene blue and it was spiked with Pb2+ ions to the final concentration of 1 μmol /L. The relative current change was of 0.53 +/- 0.06 and it corresponded to the lead ion concentration of 0.99 μmol /L. This result indicates the real-life usefulness of the developed aptasensor.

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The results of this study reveal that gold electrodes modified with short oligonucleotides can be employed for the voltammetric determination of Pb2+ ion. The developed ssDNA-based biosensor operates via strong interaction between guanine residues and Pb2+, resulting in the formation of G-quadruplex structure, which was evidenced by EIS measurements. An external redox label – methylene blue was employed to generate current signal. Due to the stabilization of G-quadruplexes by lead ions, the oligonucleotide strand folding occurred and, as a consequence, enhancement of electron transfer between MB and electrode surface was observed. The resulting MB reduction current increases proportionally to the Pb2+ ion concentration. The proposed oligonucleotide - based biosensor distinguishes itself with a limit of detection of 34.7 nmol /L and exhibits high selectivity towards lead ions, with Hg2+ being the only meaningful interferent. The obtained results are promising in comparison to other aptasensors for the detection of lead ions (Table 2), however due to the relatively high LOD value, the developed sensor can have still only limited use for the determination of Pb2+ in real samples. Nevertheless, the direct relationship between the oligonucleotide sequence and the selectivity of its interaction with metal cations was observed. This eventually extends the group of analytes, which can be determined with the application of the ssDNA – based biosensors. Though the sensitivity and selectivity can be recognized as not fully satisfactory, this is an interesting study of binding properties of the system consisting of oligonucleotide

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ACCEPTED MANUSCRIPT sequence, Pb2+ and redox probe. The utility of the sensor was also tested in the analysis of real sample. 5. Acknowledgements

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This work was financially supported by the Polish Ministry of Science and Higher Education (grant No. IP2012 064972) and by Warsaw University of Technology.

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Fig. 1. Comparison of electrochemical response towards lead ion of sensors containing TBA or TBAC oligonucleotide probe. I0 refers to MB reduction current before and I to MB reduction current after incubation in Pb(NO3)2 solution. Experiments were conducted for 1 μmol /L Pb2+ in 50 mmol·/L Tris-HCl buffer solution containing 50 µmol·/L methylene blue (pH 4.0) for incubation time of 5 min.

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Fig. 2. Nyquist plots of a: (a) TBA/MCH – modified gold electrode, (b) TBAC/MCH – modified electrode. (I) bare gold electrode; (II) electrode with ssDNA/MCH receptor layer; (III) analysis after 5 min incubation in 50 mmol L-1 Tris-HCl buffer solution containing 5 µmol /L Pb(NO3)2 (pH 4.0). All measurements were conducted in 50 mmol·L-1 Tris-HCl buffer solution containing 5 mmol /L K3[Fe(CN)6]/K4[Fe(CN)6] and 100 mmol /L KCl (pH 4.0). Z' is the real impedance component and - Z'' is the imaginary impedance component.

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Fig. 3. Calibration curve for TBA – modified gold electrode towards Pb2+ cation (sample incubation time of 5 min). The calibration equation is y=0.4438x + 0.0897, where x refers to lead ion concentration ([Pb2+]/µmol L-1) and y to ((I-I0)/I0).

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Fig. 4. The selectivity of electrodes modified with a) TBA, b) TBAC probe. All cations were at the concentration of 1 mol /L and the measurements were performed in 50 mmol /L TrisHCl buffer solution containing 50 µmol L methylene blue (pH 4.0)

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Fig. 5. Biosensor’s response for 1 mol /L Pb2+, K+ and the mixture of both ions for TBA sequence.

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Figure 1

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a)

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b)

Figure 2

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Figure 3

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Ca2+

Cu2+

Fe3+

Mg2+

Cd2+

Hg2+

K+

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Pb2+

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a)

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b)

Pb2+

Ca2+

Cu2+

Fe3+

Mg2+

Cd2+

Hg2+

K+

Figure 4

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K+

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Pb2+

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Figure 5

Mixture

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ACCEPTED MANUSCRIPT Table 1. Comparison of change in MB reduction current for two ssDNA probes. Io refers to

MB reduction current before and I to MB reduction current after incubation in Pb(NO3)2 solution. is determined by the equation  = (I – I0)/I0. Measurements were conducted in 50 μmol L-1

I0/ μA

I / μA

3

-5.66 +/- 1.13

-5.13 +/-1.29

4.5

-12.80 +/- 0.08

6

-15.20 +/- 1.51

3

5′-SH-(CH2)6-

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methylene blue solution containing 50 mmol L-1 Tris /and 0.4 μmol L-1 Pb(NO3)2 at pH 3.0, 4.5 and 6.0.  

- 0.13 +/- 0.04

-9.87 +/- 0.79

- 0.23 +/- 0.03

-16.63 +/- 1.23

0.08 +/- 0.07

-11.25 +/- 4.99

-10.86 +/- 4.89

- 0.04 +/- 0.01

4.5

-20.65 +/- 1.08

-17.86 +/- 1.11

- 0.14 +/- 0.01

6

-31.24 +/- 3.65

-30.76 +/- 3.62

- 0.02 +/- 0.01

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(A)20-3′ (poli A)

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(T)20-3′ (poli T)

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ACCEPTED MANUSCRIPT Table 2. Comparison of the aptasensors for the detection of lead ions. Technique

Analysis

Selectivity

LOD / mol L-1

Linear range / mol L-1

Reference

[2]

time /min ND

High

6 · 10-9

0 - 1.2 · 10-7

Fluorescence with the use

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High

2.7 · 10-11

1· 10-11 - 1· 10-6

FRET

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Average

3 · 10-10

RS

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Good

4 · 10-11

EIS

ND

High

5 · 10-10

DPV

50

Good

7.5 · 10-13

SWV

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High

3.47 · 10-8

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Fluorescence

5 · 10-10 – 3 · 10-8

[33]

8 · 10-11 – 4.2 4 · 10-8

[36]

5 · 10-10 - 5· 10-5

[37]

1· 10-10 – 1 · 10-7

[38]

5 · 10-8 – 1 · 10-6

This paper

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of AuNPs

[35]

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ACCEPTED MANUSCRIPT Highlights

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The formation of G-quadruplex structure in presence of Pb2+ ions was shown. The increase of MB current was proportional to Pb2+ concentration from 0.05 to 1 µM. The sensor is selective towards lead ions with minor influence of other cations. The ssDNA- based sensor can be used for the analysis of real samples.

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