Article pubs.acs.org/ac
Single Electrode Genosensor for Simultaneous Determination of Sequences Encoding Hemagglutinin and Neuraminidase of Avian Influenza Virus Type H5N1 Iwona Grabowska,† Kamila Malecka,† Anna Stachyra,‡ Anna Góra-Sochacka,‡ Agnieszka Sirko,‡ Włodzimierz Zagórski-Ostoja,‡ Hanna Radecka,† and Jerzy Radecki*,† †
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, Olsztyn, Warmian-Masurian, 10-747 Poland ‡ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, Warsaw, Masovian, 02-106 Poland S Supporting Information *
ABSTRACT: The duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au−S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its 5′-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of H5 hemagglutinin from H5N1 virus. The second probe, decorated on its 5′-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 ± 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNAFc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the “signal on” mode of analytical signal generation. The peak at −250 ± 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the “signal off” mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase. (ELISA),4 PCR tests,5,6 quartz crystal microbalance (QCM),7 and the refractometric method.8,9 However, it must be pointed out that those methods are labor-intensive and time-consuming especially for large numbers of clinical samples. The need for rapid and convenient methods for pathogen detection has attracted the scientist’s interest to the methods based on detection of complementary nucleotide sequences (genosensors). A variety of optical,10−13 acoustic,14 gravimetric,15 and electrochemical16−18 types of genosensors has been reported. The change of accessibility of redox markers present in the sample solution toward an electrode surface modified with ssDNA probe upon hybridization is a base of ion-channel mimetic genosensors developed by Umezawa group.17,18 The sensing platform consisting of hairpin DNA molecular beacons (MB) has been studied for sensing applications based on
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vian influenza virus (AIV) primarily spread by migration of wild birds particularly ducks, gulls, and shorebirds have plagued the poultry industry for decades with millions of dollars in losses.1 Influenza virus belongs to the orthomyxoviridae family which consists of A, B, and C types. Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are present on the surface and play important roles in infecting the host cell. The H5N1 strain of AIV causes much concern because it is one of the most virulent and the deadliest. Humans do not become easily infected with the H5N1; however, it is possible, and in such cases, severe respiratory diseases can be observed.2,3 Experts are worried that the virus could one day mutate and become easily human-transmissible. To hold back the disease progress at an early stage of an outbreak and prevent the worldwide transmission, sensitive and easy to handle diagnostic tools for rapid and accurate identification of H5N1 infected carriers play a critical role. So far, a variety of methods have been developed for the detection and identification of the avian influenza virus, such as enzyme-linked immunosorbent assays © 2013 American Chemical Society
Received: May 23, 2013 Accepted: September 25, 2013 Published: September 25, 2013 10167
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fluorescence measurement of the attached fluorophore/ quencher pair.19,20 The relatively low cost of handling of such genosensors make them very attractive tools for diagnosis of infectious agents or testing DNA damage by toxicants and pollutants.21 Electrochemical DNA (E-DNA) hybridization biosensors rely on the conversion of the DNA base-pair recognition event into a useful analytically electrical signal. There are several different ways of electrochemical detection of DNA hybridization. One of them is based on monitoring of the decrease or increase of oxidation/reduction peak current of the electroactive labels which selectively bind dsDNA or ssDNA.22−24 The organic dyes, metal complexes, enzymes, or metal nanoparticles are most frequently used for such purpose. E-DNA sensors which consist of an oligonucleotide probe modified on one terminus with a redox reporter and attached to an electrode at the other end belong to the next group. Such types of genosensors could be divided into two subgroups. The “signal off” architecture of DNA genosensors is based on the stronger electrochemical response observed for the flexible single strand DNA, and a weak response is obtained for the rigid duplex with the complementary DNA analyte.22,25−28 In the “signal on” subgroup, the presence of the target oligonucleotide generates the increasing electrochemical signaling current.29 The DNA sensors, based on the changes of oxidation/ reduction of peak current of electroactive oligonucleotide bases treated as an analytical signal, were proposed by Palecek and co-workers.30,31 In addition, the modification of electrode surface in order to improve efficiency of nucleobases oxidation was reported in recent years.32,33 The rapid progress of nanomaterial-based electrochemical biosensors suggests their huge potential as diagnostics tools for many diseases. Wang et al.34 demonstrated the use of gold nanoparticle tracers for stripping-based electrochemical detection of DNA hybridization and antibody−antigen interactions. The inorganic nanoparticles offer an electrochemical diversification of the electrical tags population, which is very desirable for multiplexed clinical testing. This was demonstrated by Liu and Wang.35,36 who used different inorganic-nanocrystal tracers for a multitarget electronic detection of proteins or DNA. Four nanoparticles (cadmium sulfide, zinc sulfide, copper sulfide, and lead sulfide) were applied to differentiate the signals coming from four proteins or DNA targets in connection with sandwich immunoassay or hybridization assay, respectively, along with stripping voltammetry of the corresponding metals. Recently, several papers describing miniaturizing integrated systems consisting of the sets of microelectrodes modified with different sequences of nucleotides have been published.37−39 Such analytical systems, suitable for simultaneous determination of a few different targets, could fulfill demands of the modern medical diagnosis. Their weak points include relatively complicated technology of preparation and expensive instruments needed for integration of the signals coming from each electrode. The groups of Fred Listad40 and Nils Metzler-Nolte41 described the possibilities of detection of two ssDNA targets on one electrode surface modified with two different ssDNA probes. Such a system was suitable for simultaneous detection of two different ssDNA strands decorated with two different redox labels. Moreover, Xiang et al.42 described disposable genosensor for detection of two different ssDNA targets using only one modified screen printed carbon electrode. The electrode was
modified with two gene biomarkers from a Salmonella pathogen decorated with two different redox labels. The analytical signal in this multiplexed genosensor is based on changes in current intensity from two redox-tags conjugated to stem-loop probes immobilized at the surface of the electrode. This sensor was working in “signal off” mode with the sensitivity in the 10 nM range. In this work, we have introduced a novel, single electrodebased, dual E-DNA sensor generating two kinds of analytical signals, according to “signal off” and “signal on” modes. We demonstrated its suitability for simultaneous detection of two different ssDNA sequences derived from HA- and NAencoding sequences from H5N1 AIV, respectively. The genosensor presented fulfills the demand of systems for simultaneous determination of multiple markers of diseases. Taking into account the well-known efficiency of RNA/DNA hybridization, we feel that the presented results with ssDNA targets are very promising from detection of the influenza virus genome point of view
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EXPERIMENTAL SECTION Reagents and Materials. 6-Mercaptohexanol (HS(CH2)6OH), sodium perchlorate (NaClO4), and sodium phosphate (Na2HPO4) were purchased from Sigma-Aldrich (Poznan, Poland). KOH, H2SO4, ethanol, and methanol were obtained from ABChem, Gliwice, Poland. All aqueous solutions were prepared with deionized and charcoal-treated water (resistivity of 18.2 MΩcm) purified with a Milli-Q reagent grade water system (Millipore, Bedford, MA). Two modified oligonucleotide probes: SH-ssDNA-Fc (5′ferrocene-ATT TGG AGC TAT AGC AGG TT-SH-3′) and SH-ssDNA-MB (5′-methylene blue-AAT GGG ACT GTC AAA GAC AG-SH-3′), obtained from biomers.net, GmbH, Germany, were used as probes for immobilization on a surface of gold electrodes. The SH-ssDNA-Fc probe contains the DNA sequence characteristic for gene encoding hemagglutinin of H5N1 AIV, whereas the SH-ssDNA-MB probe contains the DNA sequence characteristic for gene encoding neuraminidase of the same virus. Both probes cover the relatively well conserved regions encoding HA and NA. The complementary oligonucleotides were obtained from the same source. Electrode Preparation. Gold disk electrodes of 2 mm2 area (Bioanalytical Systems (BAS), West Lafayette, IN) were used for the experiments. The electrodes were polished with wet 0.3 and 0.05 μm alumina slurry (Alpha and Gamma Micropolish, Buehler, Lake Bluff, IL) on a flat pad for at least 10 min and rinsed repeatedly with water and finally sonicated (30 s). The polished electrodes were then dipped in 0.5 M KOH solution deoxygenated by purging with argon for 15 min, and the potential was cycled between −400 and −1200 mV (versus Ag/AgCl reference electrode) with a scan rate of 100 mVs−1 until the CV no longer changed. On the polished electrodes, 10 μL of the following solution was dropped: 1 μM SH-ssDNA-Fc and 1 μM SH-ssDNA-MB and 0.1 μM 6mercaptohexanol in buffer (0.1 M NaClO4, 2.5 mM Na2HPO4, pH 7.0) at room temperature for 3 h. Then, the electrodes were rinsed with a solution of 0.1 M NaClO4 and 2.5 mM Na2HPO4, pH 7.0. In the next step, gold electrodes were immersed in 1 mM 6-mercaptohexanol in 0.1 M NaClO4 and 2.5 mM Na2HPO4 for 1 h. Next, after rinsing with the same buffer, the electrodes were left in the refrigerator for 24 h and stored in a buffer solution (0.1 M NaClO4, 2.5 mM M Na2HPO4, pH 7.0) at 4 °C until use. 10168
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Immobilization on the Electrode Surface of ssDNA Probes Complexed with ssDNA Targets. The appropriate mixtures were prepared in solutions in the following way. 1 μM SH-ssDNA-Fc or 1 μM SH-ssDNA-MB was mixed with the corresponding complementary ssDNA in buffer (0.1 M NaClO4, 2.5 mM Na2HPO4, pH 7.0) and left at room temperature for 18 h. After this time, 10 μL of 1 μM SHssDNA-Fc complex and/or 1 μM SH-ssDNA-MB complex were dropped on the surface of gold electrodes and left at room temperature for 3 h. Then, in the next step, gold electrodes were immersed in 1 mM 6-mercaptohexanol in 0.1 M NaClO4 and 2.5 mM Na2HPO4 for 1 h and stored as described above. Electrochemical Measurements. All electrochemical measurements were performed with a potentiostat-galvanostat AutoLab (Eco Chemie, Utrecht, Netherlands) with a threeelectrode configuration. Potentials were measured versus the Ag/AgCl electrode, and a platinum wire was used as an auxiliary electrode. Osteryoung square-wave voltammetry (OSWV) was performed with a potential scanned from 500 to −500 mV and with a step potential of 10 mV, a square-wave frequency of 100 Hz, and an amplitude of 25 mV.
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RESULTS AND DISCUSSION Characterization of Gold Electrodes Modified with Either SH-ssDNA-Fc or SH-ssDNA-MB Probes and Their Responses toward Complementary Oligonucleotides. At the first step of preparation of genosensor for simultaneous detection of two different sequences of ssDNA, the position of peak current for electrodes modified with each probe separately was tested. Figure 1A line “a” illustrates results obtained for an electrode modified with a mixed layer consisting of SH-ssDNAFc + 6-mercaptohexanol. Only a very low and broad peak at about +250 mV was recorded. The voltammogram obtained in the case of an electrode modified with a mixed layer of SHssDNA-MB + 6-mercaptohexanol represented the well shaped peak current at −245 ± 3 mV and was presented in Figure 1B line “a”. To confirm that SH-ssDNA-Fc is attached at the surface of the electrode modified with the mixed layer, we have treated it with one concentration, 1 μM, of complementary oligonucleotide without any redox marker. As a result, the very well-defined peak current appeared at +400 ± 4 mV (Figure 1A line “b”). A similar experiment was performed with an electrode modified with a mixed layer consisting of SH-ssDNA-MB probe and the corresponding complementary oligonucleotide. In this case, the decrease of peak current at −245 mV was observed and its position did not change (Figure 1B line “b”). These results suggested that the surface environment of the modified electrodes allowed for the observation of specific interaction between the probes and the complementary ssDNA targets. In order to prove it, the electrodes were modified separately with appropriate probe/reactant mixtures prepared in solution (see Experimental Section). The results (data not presented) indicated the same positions of the peaks as observed in a previous experiment after hybridization with the complementary oligonucleotides (Figure 1). This experiment confirmed that the applied modification procedure of the electrode allowed one to observe the hybridization process for both modified SH-ssDNA probes, SH-ssDNA-Fc and SHssDNA-MB. In the second step, in order to test the system sensitivity toward complementary oligonucleotides, we have evaluated the response of each electrode, separately modified with single
Figure 1. Representative Osteryoung square wave voltammograms recorded with electrodes modified with: (A) SH-ssDNA-Fc probe line (a) and after hybridization with 1 μM complementary oligonucleotide sequence line (b); (B) SH-ssDNA-MB probe line (a) and after hybridization with 1 μM complementary oligonucleotide sequence line (b) in 0.1 M NaClO4 and 2.5 mM Na2HPO4, pH 7.0; frequency: 100 Hz; step potential: 0.105 V/s; amplitude: 0.025 V.
probe, to different concentrations (from 0.1 to 500 nM) of the respective complementary 20-mer ssDNA targets. The results for SH-ssDNA-Fc + 6-mercaptohexanol and for SH-ssDNAMB + 6-mercaptohexanol modified electrodes are presented in Figure 2A,B, respectively. Both electrodes responded to their complementary ssDNA targets in a concentration-dependent manner. In the case of electrode modified with SH-ssDNA-Fc, the lowest target concentration resulting in the peak at +400 mV was 10 nM. The peak increased with the further increase of the target concentration, which is in agreement with the “signal on” mode of analytical signal generation. In the case of electrode modified with SH-ssDNA-MB, the increasing concentration of the complementary target resulted in a decrease of current value of the peak current at −245 mV (Figure 2B). This indicated the generation of electrochemical signal according to “signal off” mode. Both probes optimal sensitivity stays in the same concentration range. It seems that, in this range, the signals are dependent on concentration of the target. The similar sensitivities of both probes may result from their identical length (20 oligomers) and signals for specific full 10169
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length hybrid formation between ssDNA probe and target analyte. Some differences in the sensitivity could originate from the different density of the probes on the electrode surface as well as different electrochemical properties of markers: ferrocene and methylene blue. In the literature, the E-DNA sensors based on the ss-DNA-Fc system are described, and in the majority of them, analytical signal is generated according to “signal off” scheme.22,43−46 In the research presented, we have observed responses of the SH-ssDNA-Fc modified electrode generated according to “signal on” mode. The explanation of this phenomenon might be as follows. In the case of the oligonucleotide sequence characteristic for hemagglutinin of the influenza H5N1 virus (SH-ssDNA-Fc), there are possibilities for formation of dimers (ssDNA-Fc−ssDNA-Fc) between the immobilized DNA strands. The energy of its formation is −12.87 kcal/mol (Net Primer, PREMIER Biosoft lnternational, www.premierBiosoft.com, Supporting Information, Figure S-1). As a consequence of this, the redox probe was kept relatively far from the electrode surface (Scheme 1). After hybridization with complementary strain, the dimer is decomposed and one strand of it formed a helix. Therefore, the distance between the redox probe and electrode surface increases, and this should cause a decrease of current. However, at the same time, the second component of dimer recovered its natural flexibility, and as a consequence, Fc probes could get closer to the electrode surface; this leads to the increase of current. Our experimental data showed that after stimulation of electrode with target ssDNA at concentration ≥10 nM the second effect was dominating and the electrode generated analytical signal according to the “signal on” scheme. A similar mechanism based on strand displacement, in which target binding released a flexible single strand modified with redox tag, was used by Yang et al. for improvement of ssDNA detection.47−51 In the case of the oligonucleotides sequence characteristic for neuraminidase of the influenza H5N1 virus (SH-ssDNA-MB), the formation of above mentioned dimers (ssDNA-MB and ssDNA-MB) is less probable because of two reasons. The energy of this process could be only −7.47 kcal/mol (Supporting Information, Figure S-2), and additionally immobilization of SH-ssDNA-MB via 3′ end creates the steric obstacle for dimerization. Also, the creation of hairpins is possible with the energy of −2.27 kcal/mol (Supporting Information, Figure S-2). As a consequence, the electrochemical response of this type of electrode was generated
Figure 2. Representative Osteryoung square wave voltammograms recorded with electrodes modified with: (A) SH-ssDNA-Fc probe and (B) SH-ssDNA-MB probe before hybridization line (a) and after hybridization with 0.1 nM (b), 1 nM (c), 10 nM (d), 50 nM (e), 100 nM (f), and 500 nM (g) of oligonucleotide sequence complementary to SH-ssDNA-Fc (A) and to SH-ssDNA- MB (B). For measuring conditions, see Figure 1.
Scheme 1. Schematic Representation of Working Principle of the “Signal On” and “Signal Off” Mode of the Genosensor Consisting of Two Different ssDNA Probes Decorated with Ferrocene (Fc) and Methylene Blue (MB)
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according to the “signal off” scheme (Scheme 1). In our case, both probes are attached to the electrode surface via Au−S bonds. Therefore, the accessibility of appropriate nucleotides is limited. Because of this, only a few base pairs could participate in the creation of dimer: a maximum of 8 in the case of SHssDNA-Fc (Supporting Information, Figure S-1) and a maximum of 5 in the case of SH-ssDNA-MB (Supporting Information, Figure S-2). According to the literature data, both types of signal generation, “signal on” or “signal off”, could be related to the difference in the length of the probe. The conformational change in a Fc-modified 15-base aptamer upon binding of thrombin caused the increase of redox probe current, and “signal on” was observed by Radi and co-workers.52 The opposite phenomena, the inhibition of electron transfer and “signal off”, was reported by Plaxco and co-workers when the thrombin was bound to MB-modified 32-base aptamer.53 In our work, both probes have the same 20-base length and only SH-ssDNA-Fc probe generates the signal according to “signal on” mode. Therefore, in our opinion, the formation of dimer between neighboring ssDNA-Fc probes attached to the surface of electrode is the explanation of this phenomena. The dimer formation was recently reported by Lai and coworkers.47,51 Simultaneous Determination of Two Appropriate Complementary Oligonucleotides Using Single Gold Electrode Modified with Both SH-ssDNA-Fc and SHssDNA-MB Probes. In the first step, the electrode modified with both, SH-ssDNA-Fc and SH-ssDNA-MB, sequences was exposed to a solution of complementary ssDNA to the SH-ssDNA-Fc probe with 1 μM concentration. The current peak appeared at the position of +400 mV ± 2 mV. The position and height of the peak observed at −250 ± 4 mV, characteristic for the SH-ssDNA-MB probe, stayed without any changes (Figure S-3, Supporting Information). In the next step, the same electrode was treated with a solution of 20-mer ssDNA target complementary to the SH-ss-DNA-MB probe with the same concentration (1 μM) as in the previous experiment. As a result, we have observed the decrease of current peak at position −250 mV, but no peak appeared at position +400 mV. The same tendency was observed when the order of the treatment was opposite in the experiment; namely, in the first step, electrode was treated with a solution of complementary 20-mer ssDNA target to SH-ssDNA-MB and, in the second, with a solution of complementary 20-mer ssDNA target to SHssDNA-Fc (Figure S-4, Supporting Information). The obtained results indicated that the prepared duo-genosensor was able to selectively respond toward each of the targets. In order to prove that the observed peaks originated from two different complexes, we have modified the single electrode with the appropriate two complexes previously prepared in the solution (see the Experimental Section). The position of two observed peaks was the same as in the previous experiment, in which the complexes were created directly on the electrode surface. This indicated that we can observe the hybridization of two different sequences of ssDNA by means of a single electrode. In the last step of the experiment, the electrode modified with both ssDNA probes was stimulated with a mixture of two targets consisting with two components: one was complementary to SH-ssDNA-Fc and another to SH-ssDNA-MB. The concentration range of each target was from 8 to 100 nM. The representative voltammograms are presented in Figure 3. The
Figure 3. Representative Osteryoung square wave voltammograms recorded with electrodes modified with SH-ssDNA-Fc probe and SHss-DNA-MB probe (a) and after hybridization with 8 nM (b), 10 nM (c), 20 nM (d), 40 nM (e), 60 nM (f), 80 nM (g), and 100 nM (h) oligonucleotide sequences complementary to SH-ssDNA-Fc and SHssDNA-MB. For measuring conditions, see Figure 1.
changes of height and the positions of the peaks have the same tendency as in the case of using the single targets. The relation of value of peak current vs concentration is presented in Figure 4. Both types of signals were in the linear relations to the target
Figure 4. The relation of changes of peak current recorded for electrodes modified with SH-ssDNA-Fc probe (■) and SH-ssDNAMB probe (●) vs concentration of particular complementary oligonucleotide sequences: from 8 to 100 nM (n = 4).
ssDNA concentration in the range of 20−80 nM. The detection limits for both complementary sequences were 21 and 18 nM for SH-ssDNA-MB and SH-ssDNA-Fc probes, respectively. In the case of influenza virus, sensor should be adapted for the detection of HA and NA RNAs after deproteinization of biological material. Alternatively, viral RNAs isolated from the biological samples have to be processed by reverse-transcription to cDNAs. The sensitivity of virus detection will depend on the appropriate methodology of sample preparation. In our opinion, the limit of detection in the nM range will fulfill this need. 10171
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ACKNOWLEDGMENTS This work was supported by Innovative Economy Program, Grant No. WND-POIG.01.01.02-00-007/08, and the Statutory Fund Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland.
Therefore, the proposed analytical system, consisting of a single electrode, is suitable for simultaneous detection of two different oligonucleotide sequences at nM levels. Such properties make the system a very promising tool for diagnostic applications.
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CONCLUSIONS The genosensor consisting of two different ssDNA probes decorated with ferrocene and methylene blue, respectively, was suitable for simultaneous determination of two different complementary oligonucleotide sequences derived from genes encoding hemagglutinin and neuraminidase of the H5N1 type of AIV. The duo-genosensor was selective with similar (in the range of 18−21 nM) limits of detection for both targets. The presence of oligonucleotide sequences complementary to SHssDNA-Fc probe does not influence the function of the probe decorated with methylene blue and vice versa. The presence of ssDNA complementary to the SH-ssDNA-Fc probe resulted in the increase of ferrocene redox current, which indicates that this probe worked according to the “signal on” mode. On the other hand, the presence of the ssDNA complementary to the SH-ssDNA-MB probe resulted in a decrease of redox current of methylene blue, which indicates that this probe worked according to the “signal off” mode. The duo-genosensor is able to detect selectively and with good sensitivity sequences characteristic for genes encoding hemagglutinin (ssDNA-Fc) and neuraminidase (ssDNA-MB) of the influenza H5N1 virus simultaneously by means of single measurement. To our knowledge, this is the first example of a duo-genosensor working in the dual mode: “signal on” and “signal off”. Detection of influenza virus by means of genosensors may be hampered (put in doubt) by a high mutational rate of the viral genome. With a standard single probe-DNA sensor, especially directed toward a highly variable hemagglutinin gene, this may lead to an increase in false negative results. The duo-sensor would reduce such misreadings, as the probability of simultaneous appearance of double mutant in two restricted regions of separated targets is rather low. Duo-sensor also should be advantageous by diminishing false positive readings which may appear in the case of nonperfect hybridization with component(s) present in noninfected host samples. The probability for existence in native samples of components efficiently interacting with two independent DNA probes is limited.
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ASSOCIATED CONTENT
S Supporting Information *
Supporting information concerning the energy calculation of ssDNA-Fc-ssDNA-Fc and ssDNA-MB-ssDNA-MB dimers formation in solution and the representative OSWV voltammograms recorded with an electrode modified with SH-ssDNA-Fc and SH-ssDNA-MB probes before and after hybridization with target oligonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +48895234612. Fax: +48895240124. E-mail: j.
[email protected]. Notes
The authors declare no competing financial interest. 10172
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Analytical Chemistry
Article
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dx.doi.org/10.1021/ac401547h | Anal. Chem. 2013, 85, 10167−10173
