pubs.acs.org/Langmuir © 2009 American Chemical Society
Scleroglucan-Borax Hydrogel: A Flexible Tool for Redox Protein Immobilization Marco Frasconi, Sara Rea, Pietro Matricardi, Gabriele Favero, and Franco Mazzei* Department of Chemistry and Drug Technologies, Sapienza University of Rome, P.le Aldo Moro, 5 00185 Rome, Italy Received April 9, 2009. Revised Manuscript Received June 10, 2009 A highly stable biological film was prepared by casting an aqueous dispersion of protein and composite hydrogel obtained from the polysaccharide Scleroglucan (Sclg) and borax as a cross-linking agent. Heme proteins, such as hemoglobin (Hb), myoglobin (Mb), and horseradish peroxidase (HRP), were chosen as model proteins to investigate the immobilized system. A pair of well-defined quasi-reversible redox peaks, characteristics of the protein heme FeII/FeIII redox couples, were obtained at the Sclg-borax/proteins films on pyrolytic graphite (PG) electrodes, as a consequence of the direct electron transfer between the protein and the PG electrode. A full characterization of the electron transfer kinetic was performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The efficiency of our cross-linking approach was investigated by studying the influence of different borax groups percentage in the Sclg matrix, revealing the versatility of this hydrogel in the immobilization of redox proteins. The native conformation of the three heme proteins entrapped in the hydrogel films were proved to be unchanged, reflected by the unaltered Soret adsorption band and by the catalytic activity toward hydrogen peroxide (H2O2). The main kinetic parameters, such as the apparent Michaelis-Menten constant, for the electrocatalytic reaction were also evaluated. The peculiar characteristics of Sclg-borax matrix make it possible to find wide opportunities as proteins immobilizing agent for studies of direct electrochemistry and biosensors development.
Introduction Many experimental approaches in biology and applications in diagnostic and drug discovery require the immobilization of proteins onto substrates.1-3 Particularly, the immobilization of redox proteins on electrodes materials is a relevant issue to obtain a better understanding of the electron transfer mechanisms involving redox biomolecules both in biological systems,4,5 and as biorecognition element in biosensors and biofuel cells.6-8 The approach to direct electron transfer of proteins requires interfaces that exhibit fast electron transfer kinetics with biocompatibility, namely, without denaturation. Generally, a protein’s electroactive center is deeply embedded in the protein structure, thus an unfavorable orientation of the protein molecules on the electrode surface can prevent their electron transfer toward electrode. Moreover, the adsorption of redox enzymes onto the bare electrode surface resulting in some noteworthy structural and functional changes9,10 eventually leads to their denaturation. Therefore, in the past couple of decades, a burst of research activity has been directed toward the modification of electrodes or proteins for creating accessible electron transfer interfaces. Moreover, numerous applications of immobilization methods addressed achieving a direct rapid electron transfer of redox (1) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40–45. (2) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47–55. (3) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345–1360. (4) Bartlett, P. N. In Bioelectrochemistry: Foundamentals, Experimental Techniques and Applications; Bartlett, P. N., Ed.; Wiley-VCH: Weinheim, Germany, 2008; Chapter 1, pp 1-37. (5) Leger, C.; Bertrand, P. Chem. Rev. 2008, 108, 2379–2438. (6) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180–1218. (7) Zayats, M.; Willner, B.; Willner, I. Electroanalysis 2008, 20, 583–601. (8) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Chem. Rev. 2008, 108, 2439–2461. (9) Holt, R. E.; Cotton, T. M. J. Am. Chem. Soc. 1989, 111, 2815–2821. (10) Heller, A. Acc. Chem. Res. 1990, 23, 128–134.
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proteins have been exploited.7,11-14 Among many different strategies, hydrogel matrices have been widely used as biocompatible material for the physical entrapment of biomolecules and include a number of successful examples, such as the promising proteinbased hydrogels,15,16 or novel composite materials based on chitosan,17-19 or new hybrid molecule hydrogel where a polymerizable monomer is linked to an alginate matrix.20 This considerable technical success in the entrapment of biomolecules is the result of the gentle environment provided by the trapping material, as well as the existence of high porosity that facilitates exchanges with the aqueous solution. The procedures for stabilizing hydrogels and controlling their porosity include chemical cross-linking or physical interactions with other compounds.21,22 Unfortunately, chemical cross-linking agents frequently induce the denaturation of biomolecules or confer other undesirable effects to these materials. Therefore, it is desirable to look for suitable hydrogel matrices where proteins maintain their redox and catalytic activity. (11) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363–369. (12) Rusling, J. F.; Zhang, Z. In Biomolecular Films; Rusling, J. F., Ed.; Marcel Dekker: New York, 2003; pp 1-64. (13) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877–1881. (14) Cosnier, S. Anal. Bioanal. Chem. 2003, 377, 507–520. (15) Wheeldon, I. R.; Barton, S. C.; Banta, S. Biomacromolecules 2007, 8, 2990– 2994. (16) Wheeldon, I. R.; Gallaway, J. W.; Barton, S. C.; Banta, S. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 15275–15280. (17) Lu, X.; Hu, J.; Yao, X.; Wang, Z.; Li, J. Biomacromolecules 2006, 7, 975– 980. (18) Shan, D.; Han, E.; Xue, H.; Cosnier, S. Biomacromolecules 2007, 8, 3041– 3046. (19) Zhang, M.; Smith, A.; Gorski, W. Anal. Chem. 2004, 76, 5045–5050. (20) Abu-Rabeah, K.; Polyak, B.; Ionescu, R. E.; Cosnier, S.; Marks, R. S. Biomacromolecules 2005, 6, 3313–3318. (21) Levy, M. C.; Edwards-Levy, F. J. Microencapsulation 1996, 13, 169–183. (22) Di Pierro, P.; Chico, B.; Villalonga, R.; Mariniello, L.; Damiao, A. E.; Masi, P.; Porta, R. Biomacromolecules 2006, 7, 744–749.
Published on Web 08/20/2009
DOI: 10.1021/la901245z
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Figure 1. Repeating unit of scleroglucan.
In the present work, we explore a new type of composite hydrogel obtained from the polysaccharide scleroglucan (Sclg) and borax as a cross-linking agent. Scleroglucan is a branched homopolysaccharide consisting of a main chain of (1-3)-linked β-D-glucopyranosyl units bearing, every third unit, a single β-Dglucopyranosyl unit linked (1-6) (Figure 1).23 This polysaccharide, which exhibits a triple helix conformation, is resistant to hydrolysis and its solutions show an interesting rheological behavior: viscosity remains practically constant, even at high ionic strength, up to pH = 12 and to 90 °C. Because of its peculiar properties, Sclg was extensively used for various commercial applications (secondary oil recovery, ceramic glazes, food, paints, cosmetics, etc.);24 oxidized and cross-linked derivatives of this polysaccharide have been prepared and studied for pH-controlled delivery systems.25 Borax is actually a cross-linker that can be used for polymers containing hydroxyl groups: adding borax to a Sclg solution a mixed chemical/physical linkage takes place and the intermolecular cross-links are of physical type, therefore, the new system can be better identified as a physical gel.26-28 Herein, we examine the incorporation of three heme proteins, hemoglobin (Hb), myoglobin (Mb), and horseradish peroxidase (HRP) into Sclg-borax film on pyrolytic graphite (PG) electrodes. All protein hydrogel films showed direct, reversible electrochemistry of their heme FeII/FeIII redox couples. The efficiency of this cross-linking approach was investigated by studying the different possible linkage of borax groups with Scgl; the particular influence of borax groups on the interaction between the triple helices of Scgl and the entrapped enzyme, reveals the versatility of this hydrogel matrix in the immobilization of redox proteins. In addition, electrochemical reduction of hydrogen peroxide, catalyzed by the proteins in these films, has been observed confirming the retain of biocatalytical properties. This new immobilization material combines the advantages of the scleroglucan’s gelling properties to entrap enzymes, providing them with a gentle hydrated environment, with high enzyme retention because of
(23) Coviello, T.; Palleschi, A.; Grassi, M.; Matricardi, P.; Bocchinfuso, G.; Alhaique, F. Molecules 2005, 10, 6–33. (24) Giavasis, I.; Harvey, L. M.; McNeil, B. In Biopolymers: Polysaccharides II; De Baet, S.; Vandamme, E. J.; Steinbuchel, A. Eds.; Wiley-VCH: Weinheim, Germany, 2002; Volume 6, pp 37-60. (25) Coviello, T.; Grassi, M.; Rambone, G.; Alhaique, F. Biomaterials 2001, 22, 1899–1909. (26) Palleschi, A.; Coviello, T.; Bocchinfuso, G.; Alhaique, F. Int. J. Pharm. 2006, 322, 13–21. (27) Coviello, T.; Grassi, M.; Palleschi, A.; Bocchinfuso, G.; Coluzzi, G.; Banishoeib, F.; Alhaique, F. Int. J. Pharm. 2005, 289, 97–107. (28) Bocchinfuso, G.; Palleschi, A.; Mazzuca, C.; Coviello, T.; Alhaique, F; Marletta, G. J. Phys. Chem. B 2008, 112, 6473–6483.
11098 DOI: 10.1021/la901245z
the borax as cross-linking agent to the gel, which helps to limit the leakage of the immobilized protein.
Experimental Section Chemicals and Materials. Horseradish peroxidase type VI-A (HRP, MW 42.1 kDa), horse heart myoglobin (Mb, MW 17.8 kDa), and human hemoglobin (Hb, MW 66.0 kDa) were obtained from Sigma and used without further purification (St. Louis, MO). Scleroglucan (Sclg) (MW ≈ 1 10-6) was obtained from Degussa (Germany). Other chemicals were of analytical grade from Sigma-Aldrich and used without further purification. High purity deionized water (resistance = 18.2 MΩ cm at 25 °C; TOC=
