B3056 Journal of The Electrochemical Society, 164 (5) B3056 B3058 (2017) JES FOCUS ISSUE ON BIOSENSORS AND MICRO NANO FABRICATED ELECTROMECHANICAL SYSTEMS Communication—Accessing Stability of Oxidase[.]
B3056 Journal of The Electrochemical Society, 164 (5) B3056-B3058 (2017) JES FOCUS ISSUE ON BIOSENSORS AND MICRO-NANO FABRICATED ELECTROMECHANICAL SYSTEMS Communication—Accessing Stability of Oxidase-Based Biosensors via Stabilizing the Advanced H2 O2 Transducer Elena V Karpova, Elena E Karyakina, and Arkady A Karyakinz Chemistry Faculty of M V Lomonosov Moscow State University, 119991 Moscow, Russia Operational stability of biosensors is of particular importance especially for wearable devices Prussian Blue (PB) based advanced hydrogen peroxide transducer, 1000 times more active and selective than platinum, is deposited onto screen-printed structures and stabilized with nickel hexacyanoferrate (NiHCF), both in open circuit mode Operational stability of PB-NiHCF bilayer based biosensors and labile lactate oxidase is significantly improved in terms of twice longer half inactivation and ≈3.5 times lower inactivation constant The dynamic range of PB-NiHCF based biosensors is similar to it for conventional PB based ones, which allows using the former for similar purposes drastically improving their performance characteristics © The Author(s) 2017 Published by ECS This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited [DOI: 10.1149/2.0091705jes] All rights reserved Manuscript submitted October 12, 2016; revised manuscript received December 27, 2016 Published January 18, 2017 This paper is part of the JES Focus Issue on Biosensors and Micro-Nano Fabricated Electromechanical Systems Biosensors after their discovery1,2 have found wide practical applications in various areas of human life The biosensor market is still growing: from billion $ in 20053 to 15 billion $ in 2013.4 Current trend in clinical diagnostics as well as sports medicine is continuous monitoring of metabolites Accordingly, various biosensor based wearable devices are elaborated requiring high operational stability of the biosensors Oxidases serve as terminal ones for more than 90% of enzyme based biosensors As shown already in 70-s, the most progressive way to couple the oxidase-catalyzed and the electrochemical reactions allowing to achieve the lowest detection limit is to detect hydrogen peroxide (H2 O2 ), their side product.5 However, H2 O2 oxidation on platinum electrodes most widely used nowadays suffers from parasitic signal produced by easily oxidizable compounds More than 20 years ago we discovered Prussian Blue (iron hexacyanoferrate) as selective electrocatalyst for hydrogen peroxide reduction allowing its low-potential detection in the presence of oxygen.6,7 In neutral aqueous media, favorable for applications in life science and for biosensors, Prussian Blue (PB) is three orders of magnitude more active, and three orders of magnitude more selective compared to the commonly used platinum.8 Nano-structuring the electrocatalyst onto an inert electrode supports results in elaboration of the electrochemical sensor with record performance characteristics.9,10 Despite a number of non-iron transition metal hexacyanoferrates were also proposed as suitable transducers for oxidase-based biosensors, these materials are catalytically inactive; their apparent catalytic activity in H2 O2 reduction is due to the presence of Prussian Blue as defects in their structure.11 Combining novel enzyme immobilization protocols with apparently the best electrocatalyst for H2 O2 reduction, the advanced biosensors for glucose, glutamate, lactate have been elaborated.12–15 Among advantages of the PB based biosensors is their applicability for wearable devices, for example, for monitoring of undiluted sweat.15 We note that the use of noble metals including platinum for analysis of sweat is impossible because this excreted liquid contains various peptides irreversibly inactivating these electrocatalysts Main efforts for improvement stability of the biosensors were devoted to stabilization of the enzymes, the generally accepted ‘weak’ elements in biosensors However, the electrocatalyst upon action (including Prussian Blue16 ) is also able to degrade We report that operation stability of the Prussian Blue based biosensors is also determined by stability of the electrocatalyst Stabilizing the latter it is possible to significantly prolong the biosensor lifetime z E-mail: aak@analyt.chem.msu.ru Experimental Experiments were carried out with Millipore Milli-Q water All inorganic salts were obtained at the highest purity from Reachim (Moscow, Russia) and used as received D-Glucose was purchased from ICN Biomedicals, USA Sodium lactate, 40% solution, was purchased from ICN Glucose oxidase (EC 1.1.3.4) from Aspergillus niger (lyophilized powder, activity 270 IU) was purchased from Sigma, Germany Lactate oxidase (EC 1.1.3.2) from Pediococcus sp (lyophilized powder, activity 72 IU) was from Sorachim, Switzerland Planar 3-electrode structures made by screen-printing (Rusens Ltd, Russia) contained carbon working electrode (Ø = 1.8 mm) PalmSens potentiostat (Netherlands) interfaced to PC was used Interfacial synthesis of Prussian Blue was made by dipping a droplet of 2–4 mM K3 [Fe(CN)6 ] and 2–4mM FeCl3 in 0.1 M HCl and 0.1 M KCl and initiating by addition of H2 O2 to a final concentration of 50–200 mM Deposition of Nickel Hexacyanoferrate was made using 0.5 M KCl and 0.1 M HCl as a background electrolyte Concentration of precursors (Ni2+ , [Fe(CN)6 ]3− ) was varied in the range 0.5–2 mM After deposition modified electrodes were annealed at 100◦ C during h Biosensors were made casting an enzyme containing drop (2 μL) onto the transducer surface with subsequent drying at a room temperature for one hour Glucose oxidase casting mixture was prepared suspending aqueous enzyme (10 mg/mL) by 0.3% Nafion analogue in 85% isopropanol Lactate oxidase was suspended by 2% γ-aminopropyltriethoxysilane in 90% isopropanol Results and Discussion Among a number of approaches used for stabilization of Prussian Blue: covering with organic polymers,17,18 entrapment in sol-gel19–21 or conductive polymer matrixes22,23 – building of multilayers with non-iron hexacyanoferrates isostructural to Prussian Blue seems to be the most progressive.24 We already reported on the open circuit interfacial deposition of Prussian Blue22 allowing to avoid electrochemical techniques, highly required for cost-effective mass production In contrast to,22 we’ve chosen hydrogen peroxide as a reductant for ferric-ferrocyanide ([FeFe(CN)6 ]) complex8 to synthesize Prussian Blue film with the highest catalytic activity Obviously, labile electrocatalyst (PB) has to be covered with stabilization layer (nickel hexacyanoferrate (NiHCF)) Both high operational stability and electrocatalytic activity are desirable Hence the reasonable optimization parameter is the sensitivity (S), evaluated from the slope of the calibration graph, multiplied by the time during which the modified electrode remains its current at the level >95% Downloaded on 2017-01-24 to IP 5.189.201.32 address Redistribution subject to ECS terms of use (see ecsdl.org/site/terms_use) unless CC License in place (see abstract) Journal of The Electrochemical Society, 164 (5) B3056-B3058 (2017) 20 -1 S⋅t95%, A⋅min⋅M ⋅cm -2 10 B3057 -3 -2 1⋅10 M -40 j, μA⋅cm 10 -3 5⋅10 M 10 j, μA⋅cm -2 15 -4 5⋅10 M -20 [P B] ,n m ol⋅ cm -4 1⋅10 M -2 H [N i -5 5⋅10 M -2 -5 1⋅10 M m ol⋅c nm , ] CF 10 10 Figure Sensitivity multiplied by t95% in mM H2 O2 as a function of iron- and nickel hexacyanoferrates surface coverages; 0.0 V Ag|AgCl, 0.05 M phosphate buffer pH 6.0 with 0.1 M KCl, upon stirring (t95% ) from its initial value Operational stability has been investigated under hard conditions: in mM H2 O2 upon stirring The 3-D plot displaying the optimization parameter as a function of hexacyanoferrate surface coverages (Figure 1), has an absolute maximum corresponding to 2.0 ± 0.2 nmol cm−2 of Prussian Blue and 2.4 ± 0.3 nmol cm−2 of nickel hexacyanoferrate This particular point also corresponds to the highest operational stability of the electrocatalyst In hard conditions under mM H2 O2 the electrode does not displays any decay in current response during more than one hour The proposed approach is also characterized by a high reproducibility: variation in both sensitivity and t95% among 10 different modified electrodes is less than 10% Since lactate oxidase is much less stable than glucose oxidase, operational stability of lactate biosensors has been investigated Response of the biosensor made on the basis of PB-NiHCF is approximately 1.5 times less compared to the lactate-sensitive electrode using common PB as a transducer (Figure 2) However, the response current of the bilayer based biosensor is much more stable: the time of half inactivation (≈7.5 hours) is almost twice of it for conventional Prussian Blue based lactate biosensor (≈4.0 hours) The current-time dependencies in Figure seem to obey the pseudo first order inactivation after approximately hours The corresponding inactivation constants, evaluated replotting Figure in semi-logarithmic plots, in case of PB-NiHCF bilayer for monitoring of 0.25 mM and 0.5 mM lactate are of kin ≈ 1.2 · 10−3 min−1 and kin ≈ 1.8 · 10−3 min−1 , respectively Lactate biosensors made on the basis of conventional PB display inactivation constants of kin ≈ 4.1 · 10−3 min−1 in 0.25 mM -30 PB PB-NiHCF 50%⋅j0 0 -6 10 -5 10 -4 t, s 200 10 300 -3 [Lactate], M Figure Calibration graphs for lactate biosensors made on the basis of common Prussian Blue (o) and PB-NiHCF bilayer (•);0.0 V Ag|AgCl, 50 mM phosphate, pH 6.0, with 0.1 M KCl, upon stirring Inset: response of the biosensor based on PB-NiHCF bilayer to lactate lactate and of kin ≈ 6.0 · 10−3 min−1 in 0.5 mM lactate Hence, the improved operational stability of lactate biosensors based on PB-NiHCF can be characterized in terms of ≈3.5 times decreased inactivation constants Not surprisingly, for more stable enzyme, glucose oxidase, the operational stability can also be improved using the more stable transducer The time for twofold decrease of the current response for the bilayer based biosensor (≈27.5 hours) is also twice of it for conventional Prussian Blue based glucose biosensor (≈14.0 hours) The corresponding inactivation constants for PB-NiHCF and for PB based transducers are kin ≈ 3.7 · 10−4 min−1 and 8.1 · 10−4 min−1 , respectively Such improvement of the operational stability is of particular importance for continuous monitoring of metabolites with wearable devices Calibration graphs of lactate biosensors in batch mode are displayed in Figure Despite lactate biosensor made on the basis of PB-NiHCF bilayer displays lower response, for both biosensors the dynamic range is similar: from μM to mM Hence, a slightly lower sensitivity of the biosensor based on PB-NiHCF bilayer does not affect the dynamic range, and the biosensor is suitable for similar tasks as the lactate sensitive electrode based on conventional Prussian Blue As expected, no current decay between injections of the analyte can be registered (Figure 3, inset) A noise at high lactate concentrations is due to solution turbulence around planar sensor structure upon stirring The dynamic ranges for Prussian Blue based and PB-NiHCF bilayer based glucose biosensors are similarly prolonged from μM to 10 mM analyte concentrations Summary -2 j, μA⋅cm -15 -1 t, h Figure Operational stability of lactate biosensors made on the basis of PB and PB-NiHCF bilayer; 0.0 V Ag|AgCl, 0.25 mM lactate in 50 mM phosphate, pH 6.0, with 0.1 M KCl, upon stirring Operational stability of the oxidase-based biosensors, which is of particular importance especially for wearable devices, can be significantly improved stabilizing the transducer used Prussian Blue (PB) based advanced hydrogen peroxide transducer, 1000 times more active and selective than platinum, which allows H2 O2 detection by reduction in the presence of oxygen, and in contrast to Pt is suitable for analysis of excretory liquids like sweat, is deposited onto screen-printed electrode structures and stabilized with nickel hexacyanoferrate (NiHCF), both in open circuit mode Operational stability of PB-NiHCF bilayer based biosensors and even apparently the most labile lactate oxidase is significantly improved in terms of twice longer half inactivation and ≈3.5 times lower inactivation constant The dynamic range of PB-NiHCF based biosensors is similar to it for conventional PB based ones, which allows using the former for similar purposes drastically improving their performance characteristics Downloaded on 2017-01-24 to IP 5.189.201.32 address Redistribution subject to ECS terms of use (see ecsdl.org/site/terms_use) unless CC License in place (see abstract) B3058 Journal of The Electrochemical Society, 164 (5) B3056-B3058 (2017) Acknowledgments Financial support through Russian Science Foundation grant # 16-13-00010 is greatly acknowledged References 10 11 L C Clark and C Lyons, Ann NY Acad Sci., 102, 29 (1962) S J Updike and J P Hiks, Nature, 214, 986 (1967) J D Newman and A P F Turner, Biosens Bioelectron., 20, 2435 (2005) A P F Turner, Chem Soc Rev., 42, 3184 (2013) G G Guilbault and G J Lubrano, Anal Chim Acta, 64, 439 (1973) A A Karyakin, O V Gitelmacher, and E E Karyakina, Anal Lett., 27, 2861 (1994) A A Karyakin, O V Gitelmacher, and E E Karyakina, Anal Chem., 67, 2419 (1995) A A Karyakin, Electroanalysis, 13, 813 (2001) A A Karyakin, E A Puganova, I A Budashov, I N Kurochkin, E E Karyakina, V A Levchenko, V N Matveyenko, and S D Varfolomeyev, Anal Chem., 76, 474 (2004) A A Karyakin, E A Puganova, I A Bolshakov, and E E Karyakina, Angew Chem Int Ed., 46, 7678 (2007) N A Sitnikova, M A Komkova, I V Khomyakova, E E Karyakina, and A A Karyakin, Anal Chem., 86, 4131 (2014) 12 A A Karyakin, E E Karyakina, and L Gorton, Anal Chem., 72, 1720 (2000) 13 A A Karyakin, E A Kotel’nikova, L V Lukachova, E E Karyakina, and J Wang, Anal Chem., 74, 1597 (2002) 14 E I Yashina, A V Borisova, E E Karyakina, O I Shchegolikhina, M Y Vagin, D A Sakharov, A G Tonevitsky, and A A Karyakin, Anal Chem., 82, 1601 (2010) 15 M M Pribil, G U Laptev, E E Karyakina, and A A Karyakin, Anal Chem., 86, 5215 (2014) 16 A A Karyakin, E E Karyakina, and L Gorton, Electrochem Commun., 1, 78 (1999) 17 J J GarciaJareno, J NavarroLaboulais, and F Vicente, Electrochim Acta, 41, 2675 (1996) 18 L V Lukachova, E A Kotel’nikova, D D’Ottavi, E A Shkerin, E E Karyakinia, D Moscone, G Palleschi, A Curulli, and A A Karyakin, IEEE Sens J., 3, 326 (2003) 19 S Bharathi and O Lev, Appl Biochem Biotechnol., 89, 209 (2000) 20 Y Z Guo, A R Guadalupe, O Resto, L F Fonseca, and S Z Weisz, Chem Mater., 11, 135 (1999) 21 A Salimi and K Abdi, Talanta, 63, 475 (2004) 22 A V Borisova, E E Karyakina, S Cosnier, and A A Karyakin, Electroanalysis, 21, 409 (2009) 23 R Koncki and O S Wolfbeis, Anal Chem., 70, 2544 (1998) 24 N A Sitnikova, A V Borisova, M A Komkova, and A A Karyakin, Anal Chem., 83, 2359 (2011) Downloaded on 2017-01-24 to IP 5.189.201.32 address Redistribution subject to ECS terms of use (see ecsdl.org/site/terms_use) unless CC License in place (see abstract) ... nmol cm? ?2 of nickel hexacyanoferrate This particular point also corresponds to the highest operational stability of the electrocatalyst In hard conditions under mM H2 O2 the electrode does not displays... oxidase, the operational stability can also be improved using the more stable transducer The time for twofold decrease of the current response for the bilayer based biosensor (? ?27 .5 hours) is also twice... lactate oxidase is much less stable than glucose oxidase, operational stability of lactate biosensors has been investigated Response of the biosensor made on the basis of PB-NiHCF is approximately