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Nanocrystalline zinc oxide as a biosensor platform
Nanocrystalline ZnO is a desirable material for a biosensing platform that may enable real-time detection of surface binding events.
14 February 2007, SPIE Newsroom. DOI: 10.1117/2.1200701.0533
Robust biosensing with increased sensitivity and selectivity could provide real-time pathogen detection with significant medical and biological applications. Nanoscale materials provide large surface areas and, together with the thermal and mechanical properties of semiconductor metal oxide nanoparticles, make such an advance feasible. Biomolecular recognition elements (MREs) introduced onto the surface of such particles could be employed to create a novel biosensor platform.
The basic idea for such a platform has already been demonstrated with porous silicon films, which are easy to make and possess inherent optical properties. They can also be chemically functionalized.1 A limitation, however, is that pore size must be tailored to accommodate the receptor-ligand pair of interest. Other common sensing platforms take an indirect approach to pathogen detection,2 requiring one or more components to be labeled with a fluorescent ‘reporter’ molecule in order to detect the binding event. Additional disadvantages of these systems include increased complexity, lengthy sample preparation, time-consuming analysis, and limited sensitivity. To overcome these limitations, we have focused on nanocrystalline ZnO (nano-ZnO) as the material template for designing an optically responsive biosensor platform.3
Nano-ZnO has precisely the desirable qualities indicated above: large surface area, mechanical and thermal stability, and an inherent photoluminescence signal.4 The signal consists of two emission peaks, one in the UV, owing to near-band-edge emission, and another in the visible region, due to oxygen vacancies (see Figure 1). Nano-ZnO eliminates the need for fluorescent labeling and provides an opportunity to detect real-time binding events through UV or visible peak emission intensity changes, emission-maximum shifts,and peak proportionality changes.
Figure 1. Nanocrystalline zinc oxide, left, in the form of nanorods with diameters ranging from 10 to 100nm (scanning electron micrograph). Right, inherent photoluminescence of ZnO nanorods. The photoluminescence signal consists of two emission peaks. One peak is in the UV and the other is in the visible (green) region.
To develop nano-ZnO as an optical sensing platform, the nanorod surfaces first must be chemically altered for biomolecular complexing. Surface modification should encompass UV and/or visible emission stabilization and some level of chemical functionality for subsequent bio- or chemical conjugation. Our surface passivation approach addresses both these issues.
We chose a heterobifunctional organosilane linker, 11-triethoxysilylundecanal, to covalently attach to the nano-ZnO surface and to introduce an amine-reactive functional group that would allow for biofunctionalization. Energy-dispersive X-ray spectroscopy (EDS) mapping of silicon atoms verified the presence of the cross-linking agent, and analysis confirmed a high concentration of Si on the nanorod surface. After organosilane surface modification, a series of washing steps ensured removal of any noncovalently attached silane agent. Because noncovalently attached Si atoms were removed during the washing process, EDS confirmed both the presence of the silane agent and covalent attachment. Analysis of the surface-modified nano-ZnO indicates that the complex is remarkably stable, with no silane dissociation after more than six months in storage at room temperature within a desiccator.
After surface modification, the amine-reactive group must be stable and maintain reactive functionality in order to form biomolecule complexes. To determine whether the aldehyde group is available for subsequent biofunctionalization, we used a hydrazide derivative of a common fluorophore, Texas Red (TR). After derivatization, the nanopowders exhibited obvious and visible differences. The unmodified nano-ZnO remained white, while the surface-modified nano-ZnO was a deep purple, as expected with successful attachment of TR (see Figure 2, inset). These powders, also investigated through fluorescent probing, clearly showed the presence of the silane linker on the surface-modified nano-ZnO. The unmodified nanopowder displayed minimal incorporation of TR, indicating that nonspecific binding was negligible (Figure 2). Most important, because the hydrazide form of TR is specifically designed for covalent attachment to amine-reactive aldehyde groups, these results illustrate that the chemically reactive functionality of the aldehyde, while in its immobilized state, is retained
Figure 2. Texas Red-derivatized nanopowders of unmodified nano-ZnO and silane-modified nano-ZnO. The nanopowders exhibit obvious differences (inset). Fluorescent probing indicates the aldehyde functional group of the silane-modified nano-ZnO is chemically reactive and available for attachment of amine-containing biomolecules. Texas Red hydrazide: excitation = 582nm, emission = 602nm.
Introduction of chemical functionality and retention of reactivity illustrate our ability to create a platform for forming biomolecular complexes on the nano-ZnO surface. This optically responsive biosensing platform can serve as a template for immobilizing MREs, including antibodies, aptamers, enzymes, and peptides. Although the silane linker investigated here introduced an amine-reactive aldehyde, other linkers could be employed to introduce alternative reactive groups. This could facilitate multiplexing through deposition of several different linkers at once to introduce multiple reactive groups and toimmobilize MREs specific to a series of target pathogens.
Diane M. Steeves, Jason W. Soares
US Army Natick Soldier Research Development and Engineering Center
3. J. W. Soares, D. M. Steeves, D. Ziegler, B. S. DeCristofano, Surface modification of nanocrystalline ZnO for biosensing applications, Proc. SPIE 6370, pp. 637011, 2006. Nanomaterial Synthesis and Integration for Sensors, Electronics, Photonics, and Electro-Optics
4. D. Banerjee, J. Y. Lao, D. Z. Wang, J. Y. Huang, D. Steeves, B. Kimball, Z. F. Ren, Synthesis and photoluminescence studies on ZnO nanowires, Nanotechnology 15, no. 3, pp. 404-409, 2004.