Biomacromolecules 2004, 5, 1869-1876
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Enzymatic Copolymerization Alters the Structure of Unpolymerized Mixtures of the Biomimetic Monomers: The Amphiphilic Decyl Ester of L-Tyrosine and L-TyrosineamidesAn AFM Investigation of Nano- to Micrometer-Scale Structure Differences Kenneth A. Marx*,† Center for Intelligent Biomaterials, Department of Chemistry, University of Massachusetts Lowell, One University Avenue, Lowell, Massachusetts 01854
Jun S. Lee Center for Advanced Materials, University of Massachusetts Lowell, One University Avenue, Lowell, Massachusetts 01854
Changmo Sung School of Nano Engineering, Inje University, Gimhae, Kyungnam, South Korea Received April 6, 2004; Revised Manuscript Received June 21, 2004
Previously, we have shown that the amphiphilic decyl esters of both D- and L-tyrosine (DELT) self-assemble in aqueous solution above their critical micelle concentration values to form long rodlike structures that can be enzymatically polymerized. In the current study, we have examined the self-assembled structures of unpolymerized and enzymatically (horseradish peroxidase) copolymerized 1:1 molar mixtures of DELT with the nonamphiphilic comonomer L-tyrosineamide. The structures were examined following adsorption to gold-coated mica surfaces using optical microscopy and scanning electron microscopy, but primarily noncontact atomic force microscopy. Both unpolymerized and copolymerized 1:1 comonomer mixture aggregates produced amorphous to spherical shaped structures, exhibiting increased flexibility that contrasted with our previous observations of the more highly ordered long rodlike structures seen with the pure DELT. The unpolymerized comonomer aggregates were amorphous and of varying size. Interestingly, they contained occasional novel structuresssmooth, sharp, nipplelike features that rose hundreds of nanometers above the smooth aggregate surface. However, upon enzymatic copolymerization, the structures are altered, forming nearly hemispherical aggregates in contact with each other on the surface. These structures possessed diameters of 1.51 ( 0.24 µm. The copolymerized structures lacked any evidence of the sharp nipplelike features observed in the unpolymerized sample, but they did exhibit nanometer-scale detailed surface features, indicative of a higher degree of internal organization. The measured surface roughness of the copolymerized comonomer mixture was more than 10 times greater than the surface roughness of the unpolymerized comonomer mixture. Introduction Phenols represent an important class of substrates for peroxidases, such as horseradish peroxidase (HRP), an enzyme widely used to carry out polymerizations of derivatized phenolic and aniline substrates.1 This class of monomers also represents important starting materials for thin films formed on conducting surfaces via electropolymerization. Both enzymatic polymerization and electropolymerization are examples of attractive green chemistry approaches to creating oligomeric and polymeric products using environmentally friendly solutes and water as the solvent. * To whom correspondence should be addressed. E-mail:
[email protected]. † The author dedicates this research study to the memory of the late Prof. Walter H. Stockmayer, a world-renowned polymer chemist and a former colleague and mentor.
There have been numerous reports of derivatized phenols being used as the starting monomers for film formation by electropolymerization and enzymatic polymerization. Each method has its advantages. Electropolymerization has the advantage that the film formation is localized to the electrode surface, where synthesis conditions are under electrochemical control. Nonconducting polymer growth, resulting from polymerizing simple phenolic monomers, is self-limiting, resulting in thin insulating films (10-100 nm) that can possess permselective properties.2-5 These thin films can be used to physically entrap enzymes such as HRP to form biosensors, or using amperometry via the electrode surface they can be used to directly detect analytes such as H2O2.5 More complex thin films can be formed using amphiphilic derivatives of phenol, such as the decyl esters of D- and L-
10.1021/bm049793y CCC: $27.50 © 2004 American Chemical Society Published on Web 07/29/2004
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tyrosine (respectively, DEDT and DELT, the latter being used in this study).5 These amphiphilic tyrosine derivatives self-assemble onto Pt and Au electrode surfaces and form films via a combination of adsorption and electropolymerization.6-8 Phenolic monomers can be enzymatically polymerized via a free radical based ring-ring polymerization mechanism to produce products possessing a high level of conjugation. These reactions can be carried out in a variety of formats that include reversed micelles,9 at the Langmuir trough airwater interface,9 and in nonaqueous media.10,11 Most often, HRP is the enzyme used.1 However, other peroxidases such as soybean peroxidase, possessing superior thermal stability,12 have also been employed for polymerizations of phenolic compounds. Peroxidases have been the subject of recent investigations to genetically engineer them to possess enhanced catalytic and stability properties.13 Oxidases such as laccase have also been used for polymerization of phenolic monomers.14 Being an oxidase, laccase has the distinct advantage that molecular oxygen can be used in place of H2O2. In addition to nonbiological phenolic monomers, a number of important biological phenols are involved in structures in the cell, some of which possess great commercial importance. For example, in plants, phenolic compounds containing an o-methoxy group form polymers called lignins, via oxidative ether linkages, that have structural importance for plant cell walls.15 Industrially, peroxidases and laccase are being investigated for use in the enzymatic delignification of pulp waste in the wood pulp and paper industry. Other o-methoxy linked phenolic polymers exist in microorganisms that are formed via enzymatic reactions. When cross-linked, these polymers form the basis for biologically important cell surface adhesives.16 Where the phenolic monomers are amphiphilic, possessing pendant aliphatic chains, products tend to be oligomeric in size where HRP is the catalyst.17,18 In the case of oligomerization of 4-(hexadecyloxy)phenol, the conjugated product also possessed interesting electrical and third-order nonlinear optical properties.19 Pure amphiphilic DELT and DEDT monomers possess the ability to self-assemble in aqueous solution. Using fluorescence light scattering, we have measured their pH-dependent critical micelle concentration (cmc) values, and at concentrations above these the monomers self-assemble to form long rodlike or platelike structures (>100 µm) with widths around 2 µm.20,21 Similar rodlike structures have been observed in a variety of self-assembling small molecule and polymer amphiphilic systems.22 In the self-assembled DELT and DEDT systems, there is little significant change visible at the SEM level of resolution in these structures before and after HRP polymerization, except that the structures following HRP polymerization are far more robust mechanically to breakage into short fragments. The quartz crystal microbalance (QCM) has been utilized to sensitively measure mass changes of biopolymer systems on metal surfaces as a result of binding events or electrochemical processes.23 Using the QCM approach, significant changes have been recorded in studies of the gold surface binding of the self-assembled DEDT aggregates prior to and following HRP polymerization.6,7 These self-assembling
Marx et al.
monomers were shown to bind the gold QCM surface at increasing pH. The time course of enzymatic polymerization in these optically opaque solutions was followed continuously via QCM parameters that resulted from increases in the aggregates’ viscoelastic properties during polymerization. Separately, in an X-ray photoelectron spectroscopy (XPS) study, the binding kinetics and pH-dependent behavior of the self-assembling DELT isomer binding to a gold surface was carried out and the optimum binding pH identified.8 In the current study, we have examined the effect of adding a nonamphiphilic biomimetic comonomer, L-tyrosineamide, to the DELT isomer to form a 1:1 molar mixture of comonomers. The structures of an unpolymerized and an enzymatically (HRP) copolymerized 1:1 mixture were examined via scanning electron microscopy (SEM) and noncontact atomic force microscopy (NC-AFM). There is a significant difference between the pure DELT rodlike structures studied earlier and the globular structures of both the unpolymerized and enzymatically copolymerized (1:1) mixtures studied here. In addition, there were significant differences in average structure properties as well as evidence for more regular structures possessing high-resolution nanometer-scale surface features observed after polymerization. Experimental Section The comonomers we used in this study are L-tyrosineamide, the amide derivative of L-tyrosine and the DELT, prepared as previously described.7,20,21 The copolymerization was carried out at a comonomer molar ratio of 1:1, at 0.3 mM/0.3 mM of each monomer in phosphate buffer, pH 7.0, using HRP (Sigma) and added hydrogen peroxide as previously described.21 Following a standard 24-h copolymerization period, the copolymerized comonomer system was then examined on gold-coated mica substrates allowing 24 h for saturation binding at 20 °C to the gold-coated mica surface. In the case of the unpolymerized comonomer system, a similar 24-h period was allowed for binding to the goldcoated mica in the NC-AFM imaged samples. However, in the case of one of the optical microscopy samples, 15 min was allowed for adsorption to compare with the 24-h sample. Mica was gold-coated by sputtering in the vacuum chamber of an XPS device (ESCALAB MK II). AFM is a unique type of microscopy that allows investigation of small features on a specimen that cannot be observed by SEM. In addition, AFM provides three-dimensional information about surface morphology. There are several modes to scan the sample morphology. NC-AFM is the best candidate to investigate the overall geometry and small features of samples due to large height variations. An advantage of this method is that there is no physical contact between the tip and sample. NC-AFM is particularly useful in investigating soft polymeric or biological materials, where distortion or damage of the sample surface can, thus, be avoided. Using the NC-AFM method, we selected a cantilever based on its spring constant. The noncontact ULTRASHARP silicon cantilever (NSC15 series, MirkoMasch) with Al coating on the laser-reflection side was used in this study. The dimensions of the cantilever are 125 µm × 35
Unpolymerized Mixtures of Biomimetic Monomers
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Figure 1. Schematic view of steps involved in the self-assembly process of the comonomer mixture and its enzymatic copolymerization. States I and II represent the species we visualized in this study, where n ) m (a 1:1 molar ratio of comonomers). State II is a hypothetical structure of the final copolymer formed where the o versus m linkage has not been specified.
µm × 4 µm. Its spring force constant is 40 N/m and a resonant frequency, 325 kHz, was used. The tip dimension was small, possessing
