Nonenzymatic Sensor for Lactate Detection in Human Sweat

Letter pubs.acs.org/ac

Cite This: Anal. Chem. 2017, 89, 11198-11202

Nonenzymatic Sensor for Lactate Detection in Human Sweat Nikolay V. Zaryanov, Vita N. Nikitina, Elena V. Karpova, Elena E. Karyakina, and Arkady A. Karyakin* Chemistry Faculty of M.V. Lomonosov Moscow State University, 119991, Moscow, Russia S Supporting Information *

ABSTRACT: For noninvasive diagnostics of hypoxia, we propose the nonenzymatic sensor based on screen-printed structures with the working surface modified in course of electropolymerization of 3-aminophenylboronic acid (3-APBA) with imprinting of lactate. Impedimetric sensor allows lactate detection in the range from 3 mM to 100 mM with the detection limit of 1.5 mM; response time is 2−3 min. Sensor sensitivity remains unchanged within 6 months of storage unpacked in dry state at a room temperature, which is unachievable for enzyme based devices. Analysis of human sweat with poly(3-APBA) based sensor is possible due to (i) much higher lactate content compared to other polyols and (ii) high sensor −2 selectivity (Kglucose lactate < 3 × 10 ). Successful detection of lactate in human sweat by means of the poly(3-APBA) based sensor has been confirmed using the highly specific reference method based on lactate oxidase enzyme (correlation coefficient r > 0.9). The attractive performance characteristics of poly(3-APBA) based enzyme-free sensors justify their future use for noninvasive clinical analysis and sports medicine.

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powered sensor,24 omitting, unfortunately, validation by any standard analytical method. Lactate monitoring in undiluted sweat became possible after engineering of the lactate oxidase enzyme in the course of shielding its active site upon immobilization.25 Despite a success of biosensors for lactate detection in sweat,19,25 poor stability of lactate oxidase causes an increasing interest to elaboration of chemical sensors. The reported amperometric devices, however, require high anodic potential (>1 V),26 which is obviously not applicable for analysis of biological liquids due to the presence of easily oxidizable compounds.27 Among synthetic receptors the phenylboronic acid is particularly attractive due to binding selectivity to compounds possessing 1,2- or 1,3-diol functionalities, common structural elements of saccharides and hydroxy acids. The underlying chemistry has been thoroughly investigated28−31 including elaboration of sensors.32−36 The corresponding reagentless sensor has been made on the basis of boronate-substituted polyaniline, which is able to generate an increase in its conductivity as a result of binding with polyols.37 Phenylboronic acid is also used to capture glycosylated enzymes through their sugar residues.38 We report on successful lactate detection in human sweat by means of nonenzymatic sensor based on boronate-functionalized polyaniline. In order to improve selectivity toward the target analyte, the polymer has been imprinted with lactate. Successful detection of lactate in human sweat has been

-Lactate, concerning its urgency for clinical diagnostics, can be considered as the second low-molecular weight metabolite after glucose. Lactate being the product of glycolysis, anaerobic glucose metabolism, is known as a marker of hypoxia.1,2 It is controlled in the blood of patients with lactic acidosis,3,4 and in sport medicine it is used for calculating of lactate anaerobic thresholds.5−7 Not surprisingly, soon after the discovery of glucose biosensors,8,9 lactate biosensors have also been elaborated.10 Noninvasive diagnostics is highly important in both clinics and sport medicine because it does not allow either blood vessel injury or even damaging of the skin surface. Noninvasively collected sweat is already used in clinical practice, and its conductivity indicates cystic fibrosis.11,12 We’ve shown that an increase of lactate concentration in blood upon the physical loading test causes the corresponding increase in sweat lactate content,13 offering a prospect for noninvasive monitoring of hypoxia. An importance of lactate detection in biological liquids provides elaboration of the corresponding biosensors.10,14−21 The most convenient enzyme is lactate oxidase (EC 1.1.3.2), which generates hydrogen peroxide as a side product. Lactate oxidase is a rather active enzyme: its specific activity is approximately only 2 times lower compared to glucose oxidase. Optimization of its immobilization protocols19 resulted in extremely high sensitivity (0.3 A M−1 cm−2).22 The use of biosensors for lactate detection in human sweat became possible substituting platinum (which is irreversibly inactivated by sweat content, mainly, by peptides) to Prussian Blue as hydrogen peroxide transducer.19 The “electrochemical tattoo” biosensor pioneered studies on sweat lactate monitoring23 and was developed into stretchable biofuel cell as self© 2017 American Chemical Society

Received: September 7, 2017 Accepted: October 24, 2017 Published: October 24, 2017 11198

DOI: 10.1021/acs.analchem.7b03662 Anal. Chem. 2017, 89, 11198−11202

Letter

Analytical Chemistry confirmed by the lactate oxidase based reference method. In contrast to enzyme-based devices, the reported poly(3-APBA) based sensor retained its sensitivity upon 6 months storage in a dry state at room temperature.

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EXPERIMENTAL SECTION Materials. Experiments were carried out with Millipore Milli-Q water. Potassium L-lactate and 3-aminophenylboronic acid hydrochloride were purchased from Sigma-Aldrich (Germany). Inorganic salts and acids were obtained of the highest purity from Reachim (Moscow, Russia). Pilocarpine chloride (1%) was purchased from Ferein (Russia). Planar screen-printed three-electrode sensor structures (Rusens Ltd., Russia) had a carbon working electrode (diameter 1.8 mm) encircled with carbon auxiliary electrode (Figure S1, Supporting Information). Instrumentation. Cyclic voltammetry was carried out using a μAUTOLAB III (Metrohm, The Netherlands). Impedance spectra were recorded using a Solartron 1255 frequency response analyzer (Solartron, U.K.) with a homemade low noise electrochemical interface. Skin electrophoresis was made using the Potok-1 (Russia). Methods. Cyclic voltammetry was carried out in a threecompartment electrochemical cell (with isolated spaces for all electrodes) containing a glassy carbon auxiliary and Ag/AgCl in 1 M KCl reference. Impedance spectra were recorded in an electrochemical cell with the common space of working and auxiliary electrodes. The latter was a platinum cylinder encircling the working electrode. Sweat samples were collected from 7 healthy human volunteers using the Macroduct Sweat Collector (USA) during 30 min after activation of the skin spot with 1% pilocarpine solution by means of electrophoresis. The informed content was obtained from all subjects. Samples were stored frozen at −18 °C. For standardization of the analytical procedure, sweat samples were twice diluted with buffer to a final phosphate concentration of 50 mM (pH 6.0) and 0.1 M KCl. Analysis was carried in a drop of 50−100 μL on the surface of planar threeelectrode sensors.

Figure 1. Electropolymerization of 3-APBA (0.15 M) in the presence of 0.9 M L-lactate and 0.3 M H2SO4, sweep rate 40 mV s−1. Inset: cyclic voltammogram of modified electrodes in pH 1.2 (dash-dot), 3.5 (dashed), and 6.0 (solid); sweep rate 40 mV s−1.

unsubstituted polyaniline are observed. This indicates that the obtained polymer is also of polyaniline structure. Impedance spectra of the screen-printed electrode modified with lactate imprinted poly(3-APBA) are shown in the inset in Figure 2. They have been successfully fit to the simplest

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Figure 2. Relative resistance decrease as a function of L-lactate concentration; 0.05 M phosphate pH 6.0 with 0.1 M KCl. Inset: Impedance spectra of the electropolymerized 3-APBA in the presence of lactate: (□) initial, (●) in the presence of 50 mM L-lactate, (Δ) in buffer after lactate detection; ΔE = 5 mV, Edc = 100 mV. The equivalent circuit used.

RESULTS AND DISCUSSION The lactate sensor has been elaborated by electropolymerization of 3-aminophenylboronic acid (3-APBA) onto the working electrode of planar sensor structures. Common polymerization of 3-APBA has been carried out in the presence of fluoride ions.39 Polymer growth on planar screen-printed electrodes (Figure S2, Supporting Information) has been found to be rather similar to electropolymerization onto glassy carbon disks.37 The improvement of binding constant toward the target analyte is possible by means of molecular imprinting. Accordingly, to improve affinity toward lactate, the latter has been imprinted in the polymer in the course of its synthesis. A possibility for fluoride-free electropolymerization of 3-aminophenylboronic acid in the presence of hydroxy acids has been shown in ref 40. Polymer growth in cyclic voltammetric conditions is displayed in Figure 1. As seen, the current at 0.9 V is increased upon cycling, thus indicating the growth of conducting polymer. The inset in Figure 1 displays cyclic voltammograms of the modified electrode in solutions of different pH. In 0.1 M HCl (pH 1.2), the two sets of peaks reminding conventional

equivalent circuit valid for conducting polymers,41 where Rp represents the film resistance (Figure 2, inset). The film resistance can also be found from the diameter of the highfrequency semicircles (Figure 2, inset). As seen, after addition of L-lactate film resistance is reversibly decreased. Impedance spectra of unmodified sensors were close to pure capacitive behavior. No response to either lactate or saccharides addition has been observed (Figure S3, Supporting Information). Calibration graph of the lactate imprinted poly(3-APBA) based sensor in model solution has been recorded soaking the sensor in increasing concentrations of lactate (Figure 2). The relative decrease of the film resistance is plotted as a response. As seen, lactate detection is possible in the range from 3 mM to 100 mM, which corresponds to its concentration in human sweat. 11199

DOI: 10.1021/acs.analchem.7b03662 Anal. Chem. 2017, 89, 11198−11202

Letter

Analytical Chemistry

× 10−2. The corresponding pH dependences are displayed in Figure S6 (Supporting Information). Similarly to potentiometry, we determine the selectivity coefficient as the ratio of binding constants for the interfering compound and for the target analyte. Obviously, the lower coefficient (compared to unity) corresponds to the higher selectivity. Considering glucose as an interfering compound for lactate detection, at pH 6.0 the selectivity coefficient (Kglucose lactate ) is less than 3 × 10−2. Considering both the excessively high lactate content in human sweat (which is 100 times higher compared to other −2 polyols) and an improved selectivity (Kglucose lactate < 3 × 10 ), we conclude that there is the possibility for lactate detection in sweat by means of the proposed poly(3-APBA) based sensor. Analysis of human sweat was carried out recording impedance spectrum of its drop onto the surface of the poly(3-APBA) based planar sensor. Lactate concentration in human sweat was calculated from the calibration graph (see Figure 2). For comparison, the same sweat samples were analyzed by means of the flow-injection system equipped with the biosensor based on lactate oxidase enzyme and Prussian Blue as a transducer.19,43 Figure 3 presents validation of the proposed nonenzymatic sensor for analysis of human sweat. As seen, the values of

From the dependence of polymer resistance on analyte concentration (Figure 2) it is possible to evaluate the binding constant for lactate according to28,37 R p , relative =

Rp R p0

=

1 + K app[lactate](R p∞/R p0) 1 + K app[lactate]

(1)

R∞ p

In eq 1 and are film resistances at zero and infinite lactate concentrations, and Kapp is the apparent binding constant. The latter for lactate imprinted poly(3-APBA) onto the working surface of the three-electrode structures reaches the value of Kapp= 43 ± 4 M−1 (Figure 2). Optimization of growing conditions (specifically, L-lactate-to-3-APBA ratio as well as sulfuric acid concentration) in order to achieve the highest apparent binding constant is shown in Figure S4, Supporting Information. For comparison, the apparent binding constant for nonimprinted poly(3-APBA) onto the screen-printed structures is almost 3 times lower (15 ± 3 M−1, Figure S5, Supporting Information). Since the sensitivity of the sensor is determined by the binding constant, the lactate imprinted poly(3-APBA) is the preferable material for lactate detection in human sweat. Sensor sensitivity according to eq 1 is equal to the apparent 0 binding constant multiplied by (R0p − R∞ p )/Rp. The sensitivity (S) evaluated from the calibration graph shown in Figure 2 is of 23 ± 3 M−1. The lower detection limit has been found to be 1.5 mM. Lactate detection with the proposed enzyme-free sensor is possible in the range from 3 mM to 100 mM. The response time to dynamic changes in lactate concentration is 2−3 min. Reproducibility of analytical performance characteristics is of major importance for future commercialization. Commonly (in particular for linear calibration graphs) reproducibility is estimated on the basis of fitting parameters. The parameters of eq 1, however, are not truly independent. Hence, such estimation is not correct. Reproducibility of the calibration graphs for poly(3-APBA) based planar sensors was estimated as follows. From n similar sensors, one was randomly chosen. Then for the fixed concentration (C1) the relative resistance (sensor response, R1) was found. Finally, from the calibration graphs of the remaining n − 1 sensors, the concentrations (Ci) corresponding to the particular response R1 were evaluated. Relative standard deviation (RSD) of Ci from the concentration C1 gave an estimation for reproducibility of the calibration graph. For poly(3-APBA) based planar sensors, RSD was always in the frame of 5%, thus allowing the use of the elaborated sensors without their precalibration. Despite phenylboronic acid based materials have broad selectivity to compounds with 1,2- and 1,3-diol functionalities, the corresponding sensors seem to be applicable for lactate detection in human sweat due to the following reasons. First, lactate concentration in human sweat is 100 times higher compared to glucose and 1000 times higher than fluoride ion content.42 Other sweat components either appear in much lower concentrations or are unable to bind to phenylboronic functionality (Table S1, Supporting Information). Second, selectivity of phenylboronic based sensors among saccharides and hydroxy acids is dependent on solution pH. As known, in weak acidic and neutral solutions the binding constants for saccharides are increased, whereas for hydroxy acids they are decreased with the raising of the solution pH.28 For lactate imprinted poly(3-APBA) modified glassy carbon electrodes at pH 6.0 the ratio of glucose-to-lactate binding constants is of 2.7 R0p

Figure 3. Lactate concentration in 17 sweat samples collected from 7 healthy human subjects measured using poly(3-APBA) sensor and with the flow-injection system equipped with the biosensor.

lactate concentration in sweat obtained with the nonenzymatic poly(3-APBA) sensor are in good correlation with the values obtained by the reference method. The Pearson correlation coefficient exceeds 0.9 and the slope of the regression line is 1.0. Hence, the proposed nonenzymatic sensor based on poly(3-APBA) is valid for lactate detection in human sweat and is able to replace lactate oxidase and lactate dehydrogenase based biosensors. As mentioned, a crucial point for application of biosensors is an inherent instability of biomolecules. This dramatically affects the sensor shelf life or storage stability, which determines commercialization of disposable single-used test strips. Avoiding the use of biomolecules one can significantly improve the sensor shelf life. Figure 4 shows the sensitivity of the nonenzymatic poly(3-APBA) based sensors upon storage in a Petri plate at room temperature in a dry state. Even after 6 months of such storage, the sensitivity remains unchanged. For comparison, lactate oxidase based biosensors made according to the improved enzyme immobilization protocol22 in similar 11200

DOI: 10.1021/acs.analchem.7b03662 Anal. Chem. 2017, 89, 11198−11202

Analytical Chemistry

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Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03662. Figures of planar screen-printed tree-electrode structure used for sensor elaboration, polymerization of 3-aminophenylboronic acid in the presence of fluoride, impedance spectra of bare screen-printed structure, optimization of the lactate imprinted polymer synthesis, calibration graph for sensor modified with poly(3aminophenylboronic acid) in the presence of fluoride, and pH-dependences of the apparent binding constants and one table presenting composition of human sweat (PDF)

Figure 4. Relative sensitivity of planar sensors based on electropolymerized 3-APBA after storage at room temperature in air in a dry state.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

conditions lose almost half of their sensitivity within the first 10 days. Except the dramatically improved shelf life, the proposed poly(3-APBA) based sensor is much cheaper. Indeed, considering the cost of raw materials according to the SigmaAldrich catalog, lactate oxidase enzyme is 730 times more expensive than 3-aminophenylboronic acid. The discovered applicability of the nonenzymatic poly(3APBA) based sensor for lactate detection in human sweat (its perfect correlation with the standard method) and its excellent storage stability unreachable for biosensors would open horizons for enzyme-free noninvasive monitoring of hypoxia.

ORCID

Arkady A. Karyakin: 0000-0002-0457-7638 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the Russian Science Foundation through Grant No. 16-13-00010 is greatly acknowledged. REFERENCES

(1) Cain, S. M. Am. J. Physiol. 1967, 213, 57−63. (2) Lee, D. C.; Sohn, H. A.; Park, Z.-Y.; Oh, S.; Kang, Y. K.; Lee, K.m.; Kang, M.; Jang, Y. J.; Yang, S.-J.; Hong, Y. K.; Noh, H.; Kim, J.-A.; Kim, D. J.; Bae, K.-H.; Kim, D. M.; Chung, S. J.; Yoo, H. S.; Yu, D.-Y.; Park, K. C.; Yeom, Y. I. Cell 2015, 161, 595−609. (3) Bakker, J.; Gris, P.; Coffernils, M.; Kahn, R. J.; Vincent, J.-L. Am. J. Surg. 1996, 171, 221−226. (4) Mizock, B. A.; Falk, J. L. Crit. Care Med. 1992, 20, 80−93. (5) Brooks, G. A. Med. Sci. Sports Exercise 1985, 17, 22−31. (6) Jacobs, I. Sports Med. 1986, 3, 10−25. (7) Svedahl, K.; MacIntosh, B. R. Can. J. Appl. Physiol. 2003, 28, 299−323. (8) Clark, L. C.; Lyons, C. Ann. N. Y. Acad. Sci. 1962, 102, 29−45. (9) Updike, S. J.; Hicks, J. P. Nature 1967, 214, 986−988. (10) Williams, D. L.; Doig, A. R.; Korosi, A. Anal. Chem. 1970, 42, 118−121. (11) Barben, J.; Casaulta, C.; Spinas, R.; Schoeni, M. H. Swiss Med. Wkly. 2007, 137, 192−198. (12) Naehrlich, L. Klin. Paediatr. 2007, 219, 70−73. (13) Sakharov, D. A.; Shkurnikov, M. U.; Vagin, M. Y.; Yashina, E. I.; Karyakin, A. A.; Tonevitsky, A. G. Bull. Exp. Biol. Med. 2010, 150, 83− 85. (14) Schindler, J. G.; Vongulich, M. Fresenius' Z. Anal. Chem. 1981, 308, 434−436. (15) Mascini, M.; Moscone, D.; Palleschi, G. Anal. Chim. Acta 1984, 157, 45−51. (16) Hajizadeh, K.; Halsall, H. B.; Heineman, W. R. Talanta 1991, 38, 37−47. (17) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451−2457. (18) Suman, S.; Singhal, R.; Sharma, A. L.; Malthotra, B. D.; Pundir, C. S. Sens. Actuators, B 2005, 107, 768−772. (19) Yashina, E. I.; Borisova, A. V.; Karyakina, E. E.; Shchegolikhina, O. I.; Vagin, M. Y.; Sakharov, D. A.; Tonevitsky, A. G.; Karyakin, A. A. Anal. Chem. 2010, 82, 1601−1604.

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CONCLUSIONS We conclude on the applicability of the poly(3-APBA) based sensor for analysis of human sweat. Pretreatment of biological samples (blood or blood serum, for instance) by their dilution with the desired buffer solution is quite common in clinical analysis. From this point of view, the proposed here one-to-one dilution of human sweat for analyte standardization can be considered as acceptable. Despite its broad specificity recognizing 1,2- and 1,3 diol functionalities, the lactate imprinted poly(3-APBA) based sensor is suitable for lactate detection in human sweat. First, the concentration of lactate in sweat is 100 times higher compared to glucose. Second, the corresponding selectivity coefficient is lower than 3 × 10−2. Finally, successful detection of lactate in human sweat has been confirmed using the highly specific reference method based on lactate oxidase enzyme; the corresponding Pearson correlation coefficient exceeds 0.9. Except cost efficiency, the most attractive advantage of the enzyme-free sensors is their stability. Particularly, the poly(3APBA) based sensor exhibits excellent shelf life with its analytical parameters remaining unchanged upon keeping for half a year in a dry state at room temperature. Lactate biosensors in similar conditions lose almost half of their sensitivity within the first 10 days. Concerning other biological liquids, their lactate content is similar or even lower compared to glucose. Hence, the use of poly(3-APBA) based sensors in other biofluids is possible relying only on the improved selectivity of the sensor. 11201

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Analytical Chemistry (20) Hirst, N. A.; Hazelwood, L. D.; Jayne, D. G.; Millner, P. A. Sens. Actuators, B 2013, 186, 674−680. (21) Nesakumar, N.; Thandavan, K.; Sethuraman, S.; Krishnan, U. M.; Rayappan, J. B. B. J. Colloid Interface Sci. 2014, 414, 90−96. (22) Pribil, M. M.; Cortes-Salazar, F.; Andreyev, E. A.; Lesch, A.; Karyakina, E. E.; Voronin, O. G.; Girault, H. H.; Karyakin, A. A. J. Electroanal. Chem. 2014, 731, 112−118. (23) Jia, W.; Bandodkar, A. J.; Valdes-Ramirez, G.; Windmiller, J. R.; Yang, Z.; Ramirez, J.; Chan, G.; Wang, J. Anal. Chem. 2013, 85, 6553− 6560. (24) Jeerapan, I.; Sempionatto, J. R.; Pavinatto, A.; You, J.-M.; Wang, J. J. Mater. Chem. A 2016, 4, 18342−18353. (25) Pribil, M. M.; Laptev, G. U.; Karyakina, E. E.; Karyakin, A. A. Anal. Chem. 2014, 86, 5215−5219. (26) Schmitt, R. E.; Molitor, H. R.; Wu, T. Int. J. Electrochem. Sci. 2012, 7, 10835−10841. (27) Scheller, F. W.; Pfeifer, D.; Schubert, F.; Reneberg, R.; Kirstein, D. In Biosensors: Fundamental and Applications; Turner, A. P. F., Karube, I., Wilson, J. S., Eds.; Oxford University Press: Oxford, U.K., 1987. (28) Martínez-Aguirre, M. A.; Villamil-Ramos, R.; Guerrero-Alvarez, J. A.; Yatsimirsky, A. K. J. Org. Chem. 2013, 78, 4674−4684. (29) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291−5300. (30) Bosch, L. I.; Fyles, T. M.; James, T. D. Tetrahedron 2004, 60, 11175−11190. (31) Peters, J. A. Coord. Chem. Rev. 2014, 268, 1−22. (32) Wu, Q.; Wang, L.; Yu, H.; Wang, J.; Chen, Z. Chem. Rev. 2011, 111, 7855−7875. (33) Yang, X.; Cheng, Y.; Jin, S.; Wang, B. In Artificial Receptors for Chemical Sensors; Mirsky, V. M., Yatsimirsky, A. K., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA, 2010; pp 169−189. (34) Wu, X.; Li, Z.; Chen, X.-X.; Fossey, J. S.; James, T. D.; Jiang, Y.B. Chem. Soc. Rev. 2013, 42, 8032−8048. (35) Bull, S. D.; Davidson, M. G.; van den Elsen, J. M. H.; Fossey, J. S.; Jenkins, A. T. A.; Jiang, Y.-B.; Kubo, Y.; Marken, F.; Sakurai, K.; Zhao, J.; James, T. D. Acc. Chem. Res. 2013, 46, 312−326. (36) Takahashi, S.; Kurosawa, S.; Anzai, J.-i. Electroanalysis 2008, 20, 816−818. (37) Andreyev, E. A.; Komkova, M. A.; Nikitina, V. N.; Zaryanov, N. V.; Voronin, O. G.; Karyakina, E. E.; Yatsimirsky, A. K.; Karyakin, A. A. Anal. Chem. 2014, 86, 11690−11695. (38) Reuillard, B.; Le Goff, A.; Holzinger, M.; Cosnier, S. J. Mater. Chem. B 2014, 2, 2228−2232. (39) Nicolas, M.; Fabre, B.; Marchand, G.; Simonet, J. Eur. J. Org. Chem. 2000, 2000, 1703−1710. (40) Nikitina, V. N.; Zaryanov, N. V.; Kochetkov, I. R.; Karyakina, E. E.; Yatsimirsky, A. K.; Karyakin, A. A. Sens. Actuators, B 2017, 246, 428−433. (41) Inzelt, G.; Lang, G. G. In Electropolymerization: Concepts, Materials and Applications; Cosnier, S., Karyakin, A. A., Eds.; WileyVCH: Weinheim, Germany, 2010; pp 51−76. (42) Harvey, C. J.; LeBouf, R. F.; Stefaniak, A. B. Toxicol. In Vitro 2010, 24, 1790−1796. (43) Karpova, E. V.; Karyakina, E. E.; Karyakin, A. A. J. Electrochem. Soc. 2017, 164, B3056−B3058.

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DOI: 10.1021/acs.analchem.7b03662 Anal. Chem. 2017, 89, 11198−11202