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Enzymatic Biosensors Based on Electrodeposited Alginate Hydrogels
Available online at www.sciencedirect.com
ScienceDirect Procedia Engineering 168 (2016) 622 – 625
30th Eurosensors Conference, EUROSENSORS 2016
Enzymatic biosensors based on electrodeposited alginate hydrogels A. Márquez-Maquedaa*, J.M. Ríos-Gallardoa, N. Viguésb, F. Pujolb, M. Díaz-Gonzáleza, J. Masb, C. Jiménez-Jorqueraa, C. Domíngueza, X. Muñoz-Berbela a Institut de Microelectrònica de Barcelona, (IMB-CNM, CSIC), Bellaterra, Barcelona, Spain Department of Genetics and Microbiology. Autonomous University of Barcelona (UAB), Bellaterra, Barcelona, Spain
b
Abstract In the current work, we study the use of electrodeposited calcium alginate hydrogels in the development of enzymatic biosensors. Alginate electrodeposition involves the rupture of CaCO3 particles by protons from water electrolysis into Ca2+ and HCO3-. The released calcium ions cross-link alginate forming the hydrogel. Horseradish peroxidase (HRP, used as model enzyme) was electrodeposited on screen printed gold electrode (SPGE) and the activity of the enzyme was analyzed amperometrically using 3,3’,5,5’-Tetramethylbenzidine (TMB) as the mediator. The obtained gels showed good adherence to the electrode surface and stability, and did not extend more than 0.5 mm far from the desired area of deposition, allowing high control of the electrodeposition. The biosensors showed good analytical characteristics to H 2O2 determination, with short response times, sensitivity, low limit of detection and long lineal range. © 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
© 2016 The Authors. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference. Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference
Keywords: Biosensor; Enzyme immobilization.
1. Introduction Sensing technologies allow the determination and/or monitoring of parameters of key relevance in several strategic sectors including environmental monitoring, clinical diagnosis or food and beverage industry [1]. Among other technologies, biosensors are taking a prominent position by the use of biological molecules (e.g. enzymes) as recognition elements, which provide them with high sensitivity and selectivity. Biological molecules, however, are
* Corresponding author. Tel.: +34 935 94 77 00 - 2433. E-mail address: [email protected]
1877-7058 © 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference
doi:10.1016/j.proeng.2016.11.229
623
A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
very sensitive to the environmental factors and the immobilization protocol may affect their activity. Biomolecules immobilization is thus a critical process in biosensors development. Classical immobilization protocols such as physisorption, covalent bonding or cross-linking rely on poor stability of the biomolecule in the sensor surface or loss of activity due to steric hindrance or reaction with amino acids from the active site. Biomolecules on biocompatible matrices, emerges as an auspicious alternative for (i) enabling the immobilization of high amount of biocomponent in a 3D architecture, (ii) avoiding orientation problems or inactivation of the biomolecule for reaction with the recognition or active centres, (iii) providing a wet environment (hydrogels) to improve biomolecules stability and (iv) acting as a diffusion barrier that minimizes the poisoning of the electrode and allows working with real samples without pre-treatments (waste water, milk, blood, etc.). Assessing this advantages, in the present work the electrodeposition of calcium alginate hydrogel was performed to immobilize HRP on a SPGE to assemble a H2O2 biosensor. The electrodeposition of the matrix presents two more advantages: selectivity and reproducibility. Selectivity is achieved because the matrix grows only over the working electrode surface. Furthermore, applying a constant current is enough to growth the matrix, so maintaining the intensity and duration makes the process reproducible. Electrodeposited alginate matrix characteristics, mediator diffusion through the matrix and amperometric response to H2O2 of the biosensor are discussed in this work. 2. Results and discussion 2.1. Alginate gel electrodeposition Electrodeposition of calcium alginate hydrogels [2] was optimized by considering the magnitude of the applied current and the duration of the electrodeposition step. The proposed mechanism consists on the rupture of CaCO 3 particles due to the protons generated by water electrolysis and the subsequent cross-linking reaction between different alginate molecules by Ca2+ ions: ࡴ ࡻ ՜ ࡻ ࡴା ࢋି
(1)
ࡴା ࢇࡻ ՜ ࢇା ࡴࡻି
(2)
ࢍࢇ࢚ࢋࢋ࢙࢘ ࢇା ՜ ࢍࢇ࢚ࢋ െ ࢇା െ ࢍࢇ࢚ࢋ
(3)
The texture of the hydrogel depended on the applied current, being more rigid when applying higher currents (more cross-linking events). The duration of the electrodeposition step, on the other hand, showed high correlation with the presence of bubbles –due to the co-generation of O2 during electrodeposition- in the final gel. Hence, electrodeposition times above 60 s were discarded for presenting high amounts of bubbles in the gel, which made the gel heterogeneous and even induce their detachment from the electrode surface. In optimal conditions (60 s of electrodeposition at 100 µA) homogenous gels were obtained in the working electrode area. The precursor composition was fixed in 1 % w/v of sodium alginate and 0.5 % w/v of CaCO 3. Alginate gel is susceptible to cation chelating agents, such as phosphate, so PBS buffer use was declined for the electrochemical measurements as it would dissolve the matrix. Alternatively, Imidazol-HCl and MES-NaOH buffers were used instead of PBS, and showed perfect compatibility with the gel. MES-NaOH was finally chosen in combination with KCl 0.145 M, to emulate physiological samples, and CaCl 2 0.01 M to enhance the gel cohesion. MES pH was corrected with NaOH 10 M to a pH of 5.5.
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A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
2.2. Gel diffusion properties The diffusion of molecules through the gel was evaluated using TMB as model molecule, which was also the mediator of the enzymatic reaction. The hydrogel limited TMB diffusion requiring around 5 min to completely cross the membrane –stabilization in cyclic voltammograms in Figure 1-, which may delay the response of the system.
Fig. 1. a) TMB 0.5 mM diffusion through the alginate gel. Consecutive cyclic voltammetries show the increment of the TMB redox peaks. Dashed line represents a cyclic voltammetry in buffer without TMB. The arrow indicates the TMB addition. b) Temporal evolution of current density values of the redox peaks noted in the voltammogram. Buffer: MES-NaOH pH 5.5 0.1 M, KCl 0.145 M, CaCl2 0.01 M. Scan rate: 50 mV·s-1. 2.3. Enzymatic detection Horseradish peroxidase catalyzes the following reaction: ࢀࡹ࢘ࢋࢊ ࡴ ࡻ ՜ ࢀࡹ࢞ ࡴ ࡻ
(4)
The resultant oxidized TMB can be reduced electrochemically on the transducer surface if an appropriate potential is applied. In the studied cell, the cyclic voltammetries show that a potential lower than 250 mV (vs. Ag) induces the complete reduction of TMB (figure 1a). In the voltammetry appear two subsequent redox events due to the two-step oxidation process of TMB, where a semi-quinone imine is the intermediate [3]. 11 µg·ml-1 of HRP was mixed with the gel precursor and then immobilized during the electrodeposition step on the working electrode surface. Once oxidized by HRP, TMB was detected at the reducing potential of 0.1 V (vs. Ag), where the current background was just about zero. TMB detection required 7 min of stabilization of the measurement (Figure 2a). There is a good correlation between the H2O2 final concentration in the sample and the amperometric response (Figure 2b). The linear regression equation is j (µA cm-2) = -0.019 – 0.022 [H2O2] (µM) with a correlation coefficient of 0.9961 (n=8). 3. Experimental 3.1. Reagents and materials Screen printed gold electrodes (Dropsens, 220BT) with Au counter electrode and Ag pseudo-reference electrode, Alginic acid, sodium salt (Aldrich), MES (Sigma, ≥99%), NaOH (Sigma-Aldrich, ≥98%), CaCl2 (Sigma-Aldrich,
A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
625
≥99%), KCl (Fluka, ≥99%), DMSO (Panreac, 99.5%) TMB (Sigma, ≥98%), HRP (Sigma, Type VI-A), H2O2 (SigmaAldrich, 30% wt. in H2O). All the solutions were prepared with distilled water. TMB was previously dissolved in DMSO due to its hydrophobicity (50 mM). 3.2. Electrochemical measurements For the electrochemical measurements, a Dropsens µSTAT8000 potentiostat controlled with Dropview 8400 software was used. Alginate electrodeposition and H2O2 detection were performed in a 50 µl drop over the SPGEs while the TMB diffusion experiment was performed with the SPGEs immersed in a 20 ml solution (previously covered with electrodeposited alginate gel).
Fig. 2. a) Amperometric response to different H2O2 concentrations. The arrow indicates the moment of H2O2 addition. b) Average values of stable responses as function of H2O2 concentration (n = 8). Medium: MES-NaOH 0.1 M pH 5.5, KCl 0.145 M, CaCl2 0.01 M, TMB 5·10-4 M. Applied potential: 0.1 V (vs. Ag). 4. Conclusions A simple and geometrically selective electrodeposition technique of alginate hydrogel was developed to entrap horseradish peroxidase close to the transducer of an electrochemical biosensor in a biocompatible enviroment. This biosensor shows a linear amperometric response to H2O2 (from 0 to 50 µM, using TMB as mediator) and a limit of detection of 29 nM (3 times the standard deviation of the blank). Acknowledgements This work was supported in part by the Spanish R & D National Program (MEC Project TEC2014-54449-C3-1-R). X.M.-B also wants to acknowledge the “Ramón y Cajal” program from the Spanish Government. A.M.-M. is also grateful to the MICINN for the award of a research studentship from FPI program. References [1] A. Sassolas, L. Blum, B. Leca-Bouvier, Inmobilization strategies to develop enzymatic biosensors. Biotechnology Advances 30 (2012), 489511. [2] Y. Cheng, X. Luo, J. Betz, G. F. Payne, W. E. Bentley, G. W. Rubloff (2011): Mechanism of anodic electrodeposition of calcium alginate. Soft Matter, 2011, 7, 5677-5684 [3] G. Volpe, D. Compagnone, R. Draisci, G. Palleschi, 3,3’,5,5’-Tretramethylbenzidine as electrochemical substrate for horseradish peroxidase based enzyme immunoassays. A comparative study, Analyst, June 1998, Vol. 123 (1303-1307).
ScienceDirect Procedia Engineering 168 (2016) 622 – 625
30th Eurosensors Conference, EUROSENSORS 2016
Enzymatic biosensors based on electrodeposited alginate hydrogels A. Márquez-Maquedaa*, J.M. Ríos-Gallardoa, N. Viguésb, F. Pujolb, M. Díaz-Gonzáleza, J. Masb, C. Jiménez-Jorqueraa, C. Domíngueza, X. Muñoz-Berbela a Institut de Microelectrònica de Barcelona, (IMB-CNM, CSIC), Bellaterra, Barcelona, Spain Department of Genetics and Microbiology. Autonomous University of Barcelona (UAB), Bellaterra, Barcelona, Spain
b
Abstract In the current work, we study the use of electrodeposited calcium alginate hydrogels in the development of enzymatic biosensors. Alginate electrodeposition involves the rupture of CaCO3 particles by protons from water electrolysis into Ca2+ and HCO3-. The released calcium ions cross-link alginate forming the hydrogel. Horseradish peroxidase (HRP, used as model enzyme) was electrodeposited on screen printed gold electrode (SPGE) and the activity of the enzyme was analyzed amperometrically using 3,3’,5,5’-Tetramethylbenzidine (TMB) as the mediator. The obtained gels showed good adherence to the electrode surface and stability, and did not extend more than 0.5 mm far from the desired area of deposition, allowing high control of the electrodeposition. The biosensors showed good analytical characteristics to H 2O2 determination, with short response times, sensitivity, low limit of detection and long lineal range. © 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
© 2016 The Authors. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference. Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference
Keywords: Biosensor; Enzyme immobilization.
1. Introduction Sensing technologies allow the determination and/or monitoring of parameters of key relevance in several strategic sectors including environmental monitoring, clinical diagnosis or food and beverage industry [1]. Among other technologies, biosensors are taking a prominent position by the use of biological molecules (e.g. enzymes) as recognition elements, which provide them with high sensitivity and selectivity. Biological molecules, however, are
* Corresponding author. Tel.: +34 935 94 77 00 - 2433. E-mail address: [email protected]
1877-7058 © 2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference
doi:10.1016/j.proeng.2016.11.229
623
A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
very sensitive to the environmental factors and the immobilization protocol may affect their activity. Biomolecules immobilization is thus a critical process in biosensors development. Classical immobilization protocols such as physisorption, covalent bonding or cross-linking rely on poor stability of the biomolecule in the sensor surface or loss of activity due to steric hindrance or reaction with amino acids from the active site. Biomolecules on biocompatible matrices, emerges as an auspicious alternative for (i) enabling the immobilization of high amount of biocomponent in a 3D architecture, (ii) avoiding orientation problems or inactivation of the biomolecule for reaction with the recognition or active centres, (iii) providing a wet environment (hydrogels) to improve biomolecules stability and (iv) acting as a diffusion barrier that minimizes the poisoning of the electrode and allows working with real samples without pre-treatments (waste water, milk, blood, etc.). Assessing this advantages, in the present work the electrodeposition of calcium alginate hydrogel was performed to immobilize HRP on a SPGE to assemble a H2O2 biosensor. The electrodeposition of the matrix presents two more advantages: selectivity and reproducibility. Selectivity is achieved because the matrix grows only over the working electrode surface. Furthermore, applying a constant current is enough to growth the matrix, so maintaining the intensity and duration makes the process reproducible. Electrodeposited alginate matrix characteristics, mediator diffusion through the matrix and amperometric response to H2O2 of the biosensor are discussed in this work. 2. Results and discussion 2.1. Alginate gel electrodeposition Electrodeposition of calcium alginate hydrogels [2] was optimized by considering the magnitude of the applied current and the duration of the electrodeposition step. The proposed mechanism consists on the rupture of CaCO 3 particles due to the protons generated by water electrolysis and the subsequent cross-linking reaction between different alginate molecules by Ca2+ ions: ࡴ ࡻ ՜ ࡻ ࡴା ࢋି
(1)
ࡴା ࢇࡻ ՜ ࢇା ࡴࡻି
(2)
ࢍࢇ࢚ࢋࢋ࢙࢘ ࢇା ՜ ࢍࢇ࢚ࢋ െ ࢇା െ ࢍࢇ࢚ࢋ
(3)
The texture of the hydrogel depended on the applied current, being more rigid when applying higher currents (more cross-linking events). The duration of the electrodeposition step, on the other hand, showed high correlation with the presence of bubbles –due to the co-generation of O2 during electrodeposition- in the final gel. Hence, electrodeposition times above 60 s were discarded for presenting high amounts of bubbles in the gel, which made the gel heterogeneous and even induce their detachment from the electrode surface. In optimal conditions (60 s of electrodeposition at 100 µA) homogenous gels were obtained in the working electrode area. The precursor composition was fixed in 1 % w/v of sodium alginate and 0.5 % w/v of CaCO 3. Alginate gel is susceptible to cation chelating agents, such as phosphate, so PBS buffer use was declined for the electrochemical measurements as it would dissolve the matrix. Alternatively, Imidazol-HCl and MES-NaOH buffers were used instead of PBS, and showed perfect compatibility with the gel. MES-NaOH was finally chosen in combination with KCl 0.145 M, to emulate physiological samples, and CaCl 2 0.01 M to enhance the gel cohesion. MES pH was corrected with NaOH 10 M to a pH of 5.5.
624
A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
2.2. Gel diffusion properties The diffusion of molecules through the gel was evaluated using TMB as model molecule, which was also the mediator of the enzymatic reaction. The hydrogel limited TMB diffusion requiring around 5 min to completely cross the membrane –stabilization in cyclic voltammograms in Figure 1-, which may delay the response of the system.
Fig. 1. a) TMB 0.5 mM diffusion through the alginate gel. Consecutive cyclic voltammetries show the increment of the TMB redox peaks. Dashed line represents a cyclic voltammetry in buffer without TMB. The arrow indicates the TMB addition. b) Temporal evolution of current density values of the redox peaks noted in the voltammogram. Buffer: MES-NaOH pH 5.5 0.1 M, KCl 0.145 M, CaCl2 0.01 M. Scan rate: 50 mV·s-1. 2.3. Enzymatic detection Horseradish peroxidase catalyzes the following reaction: ࢀࡹ࢘ࢋࢊ ࡴ ࡻ ՜ ࢀࡹ࢞ ࡴ ࡻ
(4)
The resultant oxidized TMB can be reduced electrochemically on the transducer surface if an appropriate potential is applied. In the studied cell, the cyclic voltammetries show that a potential lower than 250 mV (vs. Ag) induces the complete reduction of TMB (figure 1a). In the voltammetry appear two subsequent redox events due to the two-step oxidation process of TMB, where a semi-quinone imine is the intermediate [3]. 11 µg·ml-1 of HRP was mixed with the gel precursor and then immobilized during the electrodeposition step on the working electrode surface. Once oxidized by HRP, TMB was detected at the reducing potential of 0.1 V (vs. Ag), where the current background was just about zero. TMB detection required 7 min of stabilization of the measurement (Figure 2a). There is a good correlation between the H2O2 final concentration in the sample and the amperometric response (Figure 2b). The linear regression equation is j (µA cm-2) = -0.019 – 0.022 [H2O2] (µM) with a correlation coefficient of 0.9961 (n=8). 3. Experimental 3.1. Reagents and materials Screen printed gold electrodes (Dropsens, 220BT) with Au counter electrode and Ag pseudo-reference electrode, Alginic acid, sodium salt (Aldrich), MES (Sigma, ≥99%), NaOH (Sigma-Aldrich, ≥98%), CaCl2 (Sigma-Aldrich,
A. Márquez-Maqueda et al. / Procedia Engineering 168 (2016) 622 – 625
625
≥99%), KCl (Fluka, ≥99%), DMSO (Panreac, 99.5%) TMB (Sigma, ≥98%), HRP (Sigma, Type VI-A), H2O2 (SigmaAldrich, 30% wt. in H2O). All the solutions were prepared with distilled water. TMB was previously dissolved in DMSO due to its hydrophobicity (50 mM). 3.2. Electrochemical measurements For the electrochemical measurements, a Dropsens µSTAT8000 potentiostat controlled with Dropview 8400 software was used. Alginate electrodeposition and H2O2 detection were performed in a 50 µl drop over the SPGEs while the TMB diffusion experiment was performed with the SPGEs immersed in a 20 ml solution (previously covered with electrodeposited alginate gel).
Fig. 2. a) Amperometric response to different H2O2 concentrations. The arrow indicates the moment of H2O2 addition. b) Average values of stable responses as function of H2O2 concentration (n = 8). Medium: MES-NaOH 0.1 M pH 5.5, KCl 0.145 M, CaCl2 0.01 M, TMB 5·10-4 M. Applied potential: 0.1 V (vs. Ag). 4. Conclusions A simple and geometrically selective electrodeposition technique of alginate hydrogel was developed to entrap horseradish peroxidase close to the transducer of an electrochemical biosensor in a biocompatible enviroment. This biosensor shows a linear amperometric response to H2O2 (from 0 to 50 µM, using TMB as mediator) and a limit of detection of 29 nM (3 times the standard deviation of the blank). Acknowledgements This work was supported in part by the Spanish R & D National Program (MEC Project TEC2014-54449-C3-1-R). X.M.-B also wants to acknowledge the “Ramón y Cajal” program from the Spanish Government. A.M.-M. is also grateful to the MICINN for the award of a research studentship from FPI program. References [1] A. Sassolas, L. Blum, B. Leca-Bouvier, Inmobilization strategies to develop enzymatic biosensors. Biotechnology Advances 30 (2012), 489511. [2] Y. Cheng, X. Luo, J. Betz, G. F. Payne, W. E. Bentley, G. W. Rubloff (2011): Mechanism of anodic electrodeposition of calcium alginate. Soft Matter, 2011, 7, 5677-5684 [3] G. Volpe, D. Compagnone, R. Draisci, G. Palleschi, 3,3’,5,5’-Tretramethylbenzidine as electrochemical substrate for horseradish peroxidase based enzyme immunoassays. A comparative study, Analyst, June 1998, Vol. 123 (1303-1307).