Development of Novel and Highly Speciﬁc ssDNA-Aptamer-Based Electrochemical Biosensor for Rapid Detection of Mercury (II) and Lead (II) Ions in Water

: In this work, we report on the development of an electrochemical biosensor for high selectivity and rapid detection of Hg 2 + and Pb 2 + ions using DNA-based speciﬁc aptamer probes labeled with ferrocene (or methylene blue) and thiol groups at their 5 (cid:48) and 3 (cid:48) termini, respectively. Aptamers were immobilized onto the surface of screen-printed gold electrodes via the SH (thiol) groups, and then cyclic voltammetry and impedance spectra measurements were performed in bu ﬀ er solutions with the addition of HgCl 2 and PbCl 2 salts at di ﬀ erent concentrations. Changes in 3D conformation of aptamers, caused by binding their respective targets, e.g., Hg 2 + and Pb 2 + ions, were accompanied by an increase in the electron transfer between the redox label and the electrode. Accordingly, the presence of the above ions can be detected electrochemically. The detection of Hg 2 + and Pb 2 + ions in a wide range of concentrations as low as 0.1 ng / mL (or 0.1 ppb) was achieved. The study of the kinetics of aptamer / heavy metal ions binding gave the values of the a ﬃ nity constants of approximately 9.10 − 7 mol, which proved the high speciﬁcity of the aptamers used.


Introduction
The detection of toxic metal ions in aquatic environment is an important global issue because these contaminants may have severe effects on plants, animals, and humans as well as on ecosystem [1]. Among the most toxic metallic water pollutants in aquatic environments are mercury and lead [2]. Lead can cause renal malfunction and can inhibit brain development in humans [3]; mercury can cause damage of the brain, heart, and kidneys [4]. The detection of heavy metals in low concentrations is a difficult task; however, it can be achieved with existing advanced analytical methods, such as atomic absorption or atomic emission spectroscopies (AAS, AES), inductively coupled plasma mass spectroscopy (ICP-MS), cold vapor atomic fluorescence spectroscopy (CVAFS), and high-performance liquid chromatography (HPLC) [5]. These methods are extremely sensitive but expensive and require specialized laboratory conditions and highly trained personnel. As a result, both the time and cost of the analysis are very high.
An alternative approach uses biosensors, which can be much simpler and less expensive [6]. Biosensors require bioreceptors that specifically recognise and bind target analyte molecules. Typical natural bio-receptors used in biosensors, e.g., enzymes and antibodies, are highly specific and, in many
The thiol groups (-SH) at the C3 termini were designed to provide strong and oriented binding of the aptamers to screen-printed gold electrodes. The redox functional groups, e.g., ferrocene or methylene blue, were attached to C5 termini to provide distinctive electrochemical properties, such as current peaks on CV characteristics associated with oxidation and reduction reactions.
The other chemicals used (all from Sigma Aldrich) were Hepes and phosphate-binding buffers (HBB and PBB), and 1,4-dithiothreitol (DTT). Hepes binding buffer (HBB) was prepared by dissolving 50 mM Hepes sodium salt, 3 mM MgCl 2 , 120 mM NaCl, and 5 mM KCl in deionized Milli-Q water. The pH of the buffer was adjusted to 7.4. Similarly, phosphate-binding buffer (PBB) was prepared by dissolving 10 mM Na 2 HPO 4 , 1.76 mM KH 2 PO 4 , 3 mM MgCl 2 , 2.7 mM KCl, and 137 mM NaCl. The pH of the buffer was adjusted to 7.4. The addition of MgCl 2 to the buffers was essential to preserve the secondary structure of aptamer from self-coiling. For long-term storage, as-received aptamer was prepared in sterilized, deionized water at 100 µM and stored at −20 • C in small aliquots.

Immobilization of Aptamers
The aptamers were immobilized on a gold surface via thiol groups at the 3 -termini according to the following procedure. Stock solution of the required aptamer was diluted to 1 µM with HBB or PBB supplemented with 1 mM of 1,4-dithiothretiol (DTT) and 3 mM of MgCl 2 . DTT led to the removal of the protecting group from the SH moiety and released the aptamers with free SH end groups that could then bind to the surface of screen-printed gold electrode. Before immobilization, the aptamers solution samples were activated by rapid (1 min) heating up to 95 • C followed by 1 min cooling at 4 • C using a conventional thermocycler polymerase chain reaction unit (TECHNE PCR, version TC-3000). Following the procedure described in [32], the immobilization of aptamers was carried out by casting aptamers solution onto the screen-printed gold electrode surface; the samples were then incubated for 4 h at room temperature in a humidity chamber. The unreacted aptamers were removed from the electrode surface by several rinses with non-folding buffer (HBB), then the screen-printed gold electrode with immobilized aptamers was kept in HBB to prevent aptamers from coiling.

Electrochemical Measurements
All CV electrochemical measurements were carried out on a DropSTAT4000P potentiostat instrument (from DropSens) controlled by Autolab software using DropSens screen-printed gold electrodes (SPGEs). The electrodes have a conventional three electrode configuration with gold working and counter electrodes and a Ag/AgCl pseudo-reference electrode. CV measurements were carried out on electrodes with the immobilized aptamers, firstly, in pure buffer solution (HBB or PBB), then in buffer solutions with the addition of either HgCl 2 or PbCl 2 salts in different concentrations starting from 0.1 ng/mL up to 1µg/mL. Because the addition of heavy metal salts increased the conductivity of buffer solutions, control measurements were carried out on electrodes without aptamers at each concentration of metal salts.
The voltage range between −0.5 and +0.5 V was selected for these measurements to observe the characteristic peaks of both redox labels, e.g., ferrocene and methylene blue. The labels used have different redox potentials: 0.01 V for methylene blue, and 0.4 V for ferrocene. The selection of fixed voltage at ±0.2 V for further analysis allowed for observation of both the oxidation and reduction currents peaks, given their 0.2 V expansion on both sides. The aptamer/metal ions binding kinetics The real samples of water taken from different natural resources in the area were tested using the same screen-printed electrodes with immobilized aptamers. The reference in this case was bottled drinking water. Impedance spectra were measured using an impedance spectroscopy instrument 4000 A and gold interdigitated electrodes (from Metrohm DropSens) containing 250 fringes on each side spaced by 5µm; the overlapping length was 6.76 mm. The AC voltage amplitude was 5 mV with a frequency that varied from 100 mHz to100 kHz; no DC bias was applied. Similarly to CVs, the impedance spectra measurements were carried out on electrodes both coated and non-coated with aptamers in buffer solutions containing different concentrations of metal salts.

ICP-MS Measurements
In addition to electrochemical measurements, control measurements were carried out using PerkinElmer NexION TM 350X ICP mass spectrometer to determine traces of Hg 2+ and Pb 2+ in real water samples. The ICP-MS instrument was equipped with a PE-AS91 auto-sampler. Samples were introduced via a cross-flow nebulizer with a Scott-type spray chamber. The NexION TM 350X, auto-sampler, and peristaltic pump were controlled by the NexION TM 350X Windows NT software and were fully automated.

Design Strategy of the Aptasensor
The strategy in this study (illustrated in Figure 1) relied on the principle that the aptamers could act as chelating factor for the analytes and undergo conformational changes that lead to changes in the electrochemical properties of aptamers containing the redox label [32]. The conformation anti-Hg aptamer turns to a hair-pin structure accommodating several Hg 2+ ions due to T-Hg-T interaction while anti-Pb aptamer forms G-quadruplex, accommodating Pb 2+ ion due to G-Pb-G interaction. In both cases, the redox labels come closer to the electrode surface and increase the electron transfer. The increase in concentration of target analyte can increase the concentration of coiled aptamers on the surface and subsequently increase the electrochemical current. The real samples of water taken from different natural resources in the area were tested using the same screen-printed electrodes with immobilized aptamers. The reference in this case was bottled drinking water. Impedance spectra were measured using an impedance spectroscopy instrument 4000 A and gold interdigitated electrodes (from Metrohm DropSens) containing 250 fringes on each side spaced by 5µm; the overlapping length was 6.76 mm. The AC voltage amplitude was 5 mV with a frequency that varied from 100 mHz to100 kHz; no DC bias was applied. Similarly to CVs, the impedance spectra measurements were carried out on electrodes both coated and non-coated with aptamers in buffer solutions containing different concentrations of metal salts.

ICP-MS Measurements
In addition to electrochemical measurements, control measurements were carried out using PerkinElmer NexION TM 350X ICP mass spectrometer to determine traces of Hg 2+ and Pb 2+ in real water samples. The ICP-MS instrument was equipped with a PE-AS91 auto-sampler. Samples were introduced via a cross-flow nebulizer with a Scott-type spray chamber. The NexION TM 350X, autosampler, and peristaltic pump were controlled by the NexION TM 350X Windows NT software and were fully automated.

Design Strategy of the Aptasensor
The strategy in this study (illustrated in Figure 1) relied on the principle that the aptamers could act as chelating factor for the analytes and undergo conformational changes that lead to changes in the electrochemical properties of aptamers containing the redox label [32]. The conformation anti-Hg aptamer turns to a hair-pin structure accommodating several Hg 2+ ions due to T-Hg-T interaction while anti-Pb aptamer forms G-quadruplex, accommodating Pb 2+ ion due to G-Pb-G interaction. In both cases, the redox labels come closer to the electrode surface and increase the electron transfer. The increase in concentration of target analyte can increase the concentration of coiled aptamers on the surface and subsequently increase the electrochemical current. The screen-printed gold electrodes activated with the anti-Hg 2+ or anti-Pb 2+ aptamers were prepared as described in the Experimental Methodology section. These electrodes, functionalized with Hg 2+ and Pb 2+ -specific aptamers, were used either directly as apta-sensors or stored  The screen-printed gold electrodes activated with the anti-Hg 2+ or anti-Pb 2+ aptamers were prepared as described in the Experimental Methodology section. These electrodes, functionalized with Hg 2+ and Pb 2+ -specific aptamers, were used either directly as apta-sensors or stored submerged in HBB at 4 • C or room temperature for several days without any decrease in sensitivity.

Cyclic Electrochemical Measurements
Typical cyclic voltammograms (CVs) recorded on electrodes with immobilized aptamers in PBB solution with added heavy metal salts in different concentrations are shown in Figure 2. The anti-Hg 2+ aptamer with ferrocene redox group showed well-resolved anodic and cathodic current peaks at about +0.2 V and −0.2 V, respectively, corresponding to oxidation and reduction of ferrocene (see Figure 2A). There was a clear correlation between the amplitudes of the two peaks and the concentrations of HgCl 2 in the buffer solution; the current rose with the increase in Hg 2+ ions contents, as demonstrated in the schematic diagram in Figure 1. Similar results were observed for anti-Pb 2+ aptamer labeled with methylene blue (shown in Figure 2B); however, the current peaks were not as pronounced but rather appeared as shoulders at potentials of about ±0.2 V. Again, a correlation between the values of current and PbCl 2 salt concentration was apparent and demonstrated the apta-sensing concept in Figure 1. Notably, the measurements on samples with immobilized aptamers stored for two to three weeks in the refrigerator (at 4 • C) yielded the same result as freshly deposited aptamers. The stored samples needed re-activation by a heating/cooling cycle in PCR unit before use. The series of CVs of samples with immobilized aptamers stored in the refrigerator for two to three weeks shown in Figure  S1 as supplementary material exhibited no principle differences with the CVs recorded on freshly prepared aptamers (shown in Figure 2); however, the absolute values of the current differed amongst the samples.
Control experiments were carried out on samples without immobilized aptamers. The CV curves presented in Figure 3 showed much larger currents without characteristic peaks when compared with the samples with aptamers, which clearly indicate that the aptamer layer acts as an insulator. The observed current is due to electrochemical processes occurring in the buffer solution. The current increases with the increase in concentrations of both HgCl 2 and PbCl 2 salts.
Chemosensors 2019, 7, x FOR PEER REVIEW 5 of 13 submerged in HBB at 4 °C or room temperature for several days without any decrease in sensitivity.

Cyclic Electrochemical Measurements
Typical cyclic voltammograms (CVs) recorded on electrodes with immobilized aptamers in PBB solution with added heavy metal salts in different concentrations are shown in Figure 2. The anti-Hg 2+ aptamer with ferrocene redox group showed well-resolved anodic and cathodic current peaks at about +0.2 V and −0.2 V, respectively, corresponding to oxidation and reduction of ferrocene (see Figure 2A). There was a clear correlation between the amplitudes of the two peaks and the concentrations of HgCl2 in the buffer solution; the current rose with the increase in Hg 2+ ions contents, as demonstrated in the schematic diagram in Figure 1. Similar results were observed for anti-Pb 2+ aptamer labeled with methylene blue (shown in Figure 2B); however, the current peaks were not as pronounced but rather appeared as shoulders at potentials of about ±0.2 V. Again, a correlation between the values of current and PbCl2 salt concentration was apparent and demonstrated the apta-sensing concept in Figure 1. Notably, the measurements on samples with immobilized aptamers stored for two to three weeks in the refrigerator (at 4 °C) yielded the same result as freshly deposited aptamers. The stored samples needed re-activation by a heating/cooling cycle in PCR unit before use. The series of CVs of samples with immobilized aptamers stored in the refrigerator for two to three weeks shown in Figure S1 as supplementary material exhibited no principle differences with the CVs recorded on freshly prepared aptamers (shown in Figure 2); however, the absolute values of the current differed amongst the samples.
Control experiments were carried out on samples without immobilized aptamers. The CV curves presented in Figure 3 showed much larger currents without characteristic peaks when compared with the samples with aptamers, which clearly indicate that the aptamer layer acts as an insulator. The observed current is due to electrochemical processes occurring in the buffer solution. The current increases with the increase in concentrations of both HgCl2 and PbCl2 salts.
The dependence of the anodic current changes ∆ = − at +0.2 V against the concentrations of heavy metals ions are shown in Figure 4. Therefore, the effect of background currents (Iref) caused by the conductivity of HBB with added heavy metal salts was eliminated. The semi-logarithmic calibration graphs of both sensors in Figure 4 showed monotonic increase of the response in a wide range of concentrations from 0.1 to 1000 ng/mL.   The dependence of the anodic current changes ∆I a = I a − I a re f at +0.2 V against the concentrations of heavy metals ions are shown in Figure 4. Therefore, the effect of background currents (I ref ) caused by the conductivity of HBB with added heavy metal salts was eliminated. The semi-logarithmic calibration graphs of both sensors in Figure 4 showed monotonic increase of the response in a wide range of concentrations from 0.1 to 1000 ng/mL.
The cross-sensitivity tests were carried out by measuring CV curves on samples with immobilized anti-Hg and anti-Pb aptamers to other metal (e.g., Zn, Cu, Cd, and Pb or Hg) chloride salts. The results of such tests presented in Figure 5 showed no responses for non-complementary metals, which confirm high specificity of the aptamers used.
The real samples of water taken from different natural resources in the area were tested by CV measurements on screen-printed electrodes with immobilized anti-Hg and anti-Pb aptamers. The results were arranged on the respective calibration graphs in Figure 4. For the sake of evaluation of Hg and Pb content in these samples, the concentration dependences in Figure 4 were extrapolated to lower concentrations of Hg and Pb ions below the 0.01 ng/mL level. The estimated values for Hg and Pb contents are shown near respective data points. The obtained data for real samples of water are compared in Table 1, with the results of ICP-MS testing of the same water samples. The correlation between the two sets of data is present in terms of the order of increased concentration; however, the ICP-MS values were typically three to 10 times larger than those obtained with CVs.    The observed differences between CV and ICP-MS data could be attributable to several causes. Firstly, some of the samples of very clean water used in this study showed the content of Hg, and particularly Pb, below the calibration limit; accordingly, the concentrations of Hg and Pb ions can be only roughly estimated. Secondly, the methodology of testing samples of water with low contents of heavy metals was not developed properly. The choice of bottled drinking water as a reference might not be suitable. Concurrently, aptamers are known to lose their secondary structure in clear water with no MgCl 2 stabilizer, which might have produced wrong responses.  The cross-sensitivity tests were carried out by measuring CV curves on samples with immobilized anti-Hg and anti-Pb aptamers to other metal (e.g., Zn, Cu, Cd, and Pb or Hg) chloride salts. The results of such tests presented in Figure 5 showed no responses for non-complementary metals, which confirm high specificity of the aptamers used.
The real samples of water taken from different natural resources in the area were tested by CV measurements on screen-printed electrodes with immobilized anti-Hg and anti-Pb aptamers. The results were arranged on the respective calibration graphs in Figure 4. For the sake of evaluation of Hg and Pb content in these samples, the concentration dependences in Figure 4 were extrapolated to lower concentrations of Hg and Pb ions below the 0.01 ng/mL level. The estimated values for Hg and Pb contents are shown near respective data points. The obtained data for real samples of water are compared in Table 1, with the results of ICP-MS testing of the same water samples. The correlation between the two sets of data is present in terms of the order of increased concentration; however, the ICP-MS values were typically three to 10 times larger than those obtained with CVs.  The observed differences between CV and ICP-MS data could be attributable to several causes. Firstly, some of the samples of very clean water used in this study showed the content of Hg, and particularly Pb, below the calibration limit; accordingly, the concentrations of Hg and Pb ions can be only roughly estimated. Secondly, the methodology of testing samples of water with low contents of heavy metals was not developed properly. The choice of bottled drinking water as a reference might not be suitable. Concurrently, aptamers are known to lose their secondary structure in clear water with no MgCl2 stabilizer, which might have produced wrong responses.

Impedance Spectroscopy Measurements
The impedance spectroscopy scans were carried out on interdigitated electrodes with immobilized aptamers (as well as on bare electrodes) in HBB solutions containing heavy metal salts. Typical results are shown as Nyquist plots in Figure 6. Notably, there is a major difference between these two graphs; in the presence of aptamers, the Nyquist plots in Figure 6A shift to the left and reduce in radius. This indicates the decrease in the charge transfer resistance (Rct) of the surface layer with an increase in Hg 2+ concentration; however, in the absence of aptamers, the Nyquist plots in Figure 6B remain almost unchanged, with a small decrease in the radius due to changes in the bulk resistivity of the solution. Also, the nearly perfect circular shape of Nyquist curves indicates the absence of a diffusion limitation of the charge transfer on the electrodes, an obvious result given a very small thickness of aptamer layer was in the range of a few nanometres.

Impedance Spectroscopy Measurements
The impedance spectroscopy scans were carried out on interdigitated electrodes with immobilized aptamers (as well as on bare electrodes) in HBB solutions containing heavy metal salts. Typical results are shown as Nyquist plots in Figure 6. Notably, there is a major difference between these two graphs; in the presence of aptamers, the Nyquist plots in Figure 6A shift to the left and reduce in radius. This indicates the decrease in the charge transfer resistance (R ct ) of the surface layer with an increase in Hg 2+ concentration; however, in the absence of aptamers, the Nyquist plots in Figure 6B remain almost unchanged, with a small decrease in the radius due to changes in the bulk resistivity of the solution. Also, the nearly perfect circular shape of Nyquist curves indicates the absence of a diffusion limitation of the charge transfer on the electrodes, an obvious result given a very small thickness of aptamer layer was in the range of a few nanometres.
The analysis of impedance spectra using an equivalent circuit model (shown as inset in Figure 6A) was carried out. According to the simplified impedance circuit model (when the diffusion impedance Z diff is neglected) [33,34], the part of the impedance at critical points of the Nyquist plot are given as: where R b is the bulk resistance of the electrolyte solution, R ct and C dl connected in parallel are, respectively, the charge transfer resistance and capacitance associated with an electrical charge double layer on the surface of gold electrodes. As seen in Figure 6, the value of R b is very small (typically in single Ohms), while R ct is much larger (above 900 Ω at low concentrations of heavy metals) and decreases upon an increasing concentration of Hg 2+ ions, likely the result of an enhancement of the electron transfer between the ferrocene label and the electrode, in line with the scheme shown in Figure 1. Concurrently, the increase of Hg 2+ concentration without aptamers immobilized on the surface does not show any significant effect on the R ct values, which corresponds well to the data presented in Figure 3.
Similar results were observed for interdigitated electrodes with immobilized anti-Pb 2+ aptamers in solutions containing PbCl 2 . Chemosensors 2019, 7, x FOR PEER REVIEW 9 of 13 The analysis of impedance spectra using an equivalent circuit model (shown as inset in Figure  6A) was carried out. According to the simplified impedance circuit model (when the diffusion impedance Zdiff is neglected) [33,34], the part of the impedance at critical points of the Nyquist plot are given as: where Rb is the bulk resistance of the electrolyte solution, Rct and Cdl connected in parallel are, respectively, the charge transfer resistance and capacitance associated with an electrical charge double layer on the surface of gold electrodes. As seen in Figure 6, the value of Rb is very small (typically in single Ohms), while Rct is much larger (above 900 Ω at low concentrations of heavy metals) and decreases upon an increasing concentration of Hg 2+ ions, likely the result of an enhancement of the electron transfer between the ferrocene label and the electrode, in line with the scheme shown in Figure 1. Concurrently, the increase of Hg 2+ concentration without aptamers immobilized on the surface does not show any significant effect on the Rct values, which corresponds well to the data presented in Figure 3.

The Kinetics of Aptamers (Hg 2+ and Pb 2+ ) Binding
The kinetics of Hg 2+ and Pb 2+ ions binding to specific aptamers were studied by recording the time dependencies of cathodic current (at −0.2 V) of three-electrode assemblies with immobilized aptamers for different concentrations of both metals ions. Cathodic current was chosen because of a smaller shift of the reduction peak upon binding Hg 2+ ions. Typical time dependences for anti-Hg 2+ aptamers are shown in Figure 7 for different concentrations of HgCl 2 salt varied from 0.01 ng/mL to 1 mg/mL. Then, the characteristic time constants (τ) were evaluated for every concentration of Hg 2+ by fitting the respective kinetics curves to rising exponential function. The resulted dependence of 1/τ versus the concentration (C) of Hg 2+ ions is shown in Figure 7 as an inset. our case, Hg 2+ and Pb 2+ ) as: The values of ka and kd can be found, respectively, as the gradient and intercept of the linear dependence 1/τ against C. Then, both the association constant (KA) and affinity constant (KD) can be found as KA = ka/kd and KD = 1/KA [35,36]. The values of ka and kd were calculated as: ka = 0.1471(sec −1 ·ng −1 ·mL) × 271.52(g·mol −1 ) × 10 3 (mL·ng −1 ) ≈ 3.94.104 (sec −1 ·mol −1 ) and kd = 0.0363 (sec −1 ), where 271.52 (g·mol −1 ) is molecular weight of HgCl2, and 10 3 factor was used as conversion from ng/mL to µg/mL. Therefore, KA = ka/kd = 1.1 × 10 6 (mol −1 ) and KD = 9.08 × 10 −7 (mol) for anti-Hg 2+ aptamer.
The obtained KA and KD values for both anti-Hg 2+ and anti-Pb 2+ aptamers correspond well to the aptamers affinity evaluated in the process of their synthesis [37,38], and they are typical for highly specific binding reactions of analytes to aptamers or antibodies. Following the Langmuir adsorption law, the reciprocal time constant (1/τ) depends on the analyte adsorption and desorption rates (k a and k d ) as well as the concentration (C) of analytes (in our case, Hg 2+ and Pb 2+ ) as: The values of k a and k d can be found, respectively, as the gradient and intercept of the linear dependence 1/τ against C. Then, both the association constant (K A ) and affinity constant (K D ) can be found as K A = k a /k d and K D = 1/K A [35,36].
A similar analysis was carried out for binding kinetics of anti-Pb 2+ aptamer, and quite similar values of K A = 1.2 × 10 6 (mol −1 ) and K D = 8.5534 × 10 −7 (mol) were found.
The obtained K A and K D values for both anti-Hg 2+ and anti-Pb 2+ aptamers correspond well to the aptamers affinity evaluated in the process of their synthesis [37,38], and they are typical for highly specific binding reactions of analytes to aptamers or antibodies.

Conclusions and Future Work
The concept of electrochemical apta-sensor for heavy metal ions was proved, and the results obtained were encouraging. The selectivity and sensitivity of this apta-sensor to heavy metals ions, e.g., Hg 2+ and Pb 2+ , is high and thus promising for the development of novel, simple, and cost-effective electrochemical apta-sensors for rapid detection of heavy metals in water.
A series of cyclic voltammogramm and impedance spectroscopy measurements allowed the investigation of the mechanism of aptamer/heavy metal binding. The proposed model electrochemical apta-sensing, based on changing the conformation of the aptamer oligonucleotide chain from a linear to a folded one, thus bringing the redox label closer to metal surface and increasing the electron charge transfer, was proved. A simple detection of anodic (or cathodic) current at fixed voltage corresponding to oxidation (or reduction) peak potential is sufficient for detection of Hg 2+ and Pb 2+ in a wide range of concentrations, as low as 0.1 ng/mL (or 0.1 ppb). The detection of heavy metals in real water samples was attempted and was partially successful; however, further work is required for developing methodology of real samples testing.
The study of aptamer/target binding kinetics yielded the values for the association constant K A = 1.1 × 10 6 (mol −1 ) and the affinity constant K D = 9.08 × 10 −7 (mol) for aptamer/Hg 2+ binding; similar values of the association constant K A = 1.2 × 10 6 (mol −1 ) and the affinity constant K D = 8.33 × 10 −7 (mol) were found for aptamer/Pb 2+ binding. This study proved highly specific interaction between heavy metal ions and their specific aptamers.
Further work could focus on development of the apta-sensor array for detection of other heavy metals (chromium, cadmium, arsenic, nickel, copper, silver, zinc, etc.) using simple DC electrochemical transducers. The different redox-labels can be used in future for simultaneous detection of different heavy metals.
Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9040/7/2/27/s1, Figure S1. The series of CVs of samples with immobilized aptamers stored in the fridge for two to three week.