Highly Sensitive Biomolecular Fluorescence Detection Using Nanoscale ZnO Platforms

Highly Sensitive Biomolecular Fluorescence Detection Using Nanoscale ZnO Platforms
Received December 2, 2005
In Final Form: March 28, 2006
Web Release Date: April 21, 2006
Adam Dorfman, Nitin Kumar, and Jong-in Hahm*
ACS Publications
Copyright? 2006 American Chemical Society
Department of Chemical Engineering, The Pennsylvania State University, 160 Fenske Laboratory, University Park, Pennsylvania 16802
Fluorescence detection is currently one of the most widely used methods in the areas of basic biological research, biotechnology, cellular imaging, medical testing, and drug discovery. Using model protein and nucleic acid systems, we demonstrate that engineered nanoscale zinc oxide structures can significantly enhance the detection capability of biomolecular fluorescence. Without any chemical or biological amplification processes, nanoscale zinc oxide platforms enabled increased fluorescence detection of these biomolecules when compared to other commonly used substrates such as glass, quartz, polymer, and silicon. The use of zinc oxide nanorods as fluorescence enhancing substrates in our biomolecular detection permitted sub-picomolar and attomolar detection sensitivity of proteins and DNA, respectively, when using a conventional fluorescence microscope. This ultrasensitive detection was due to the presence of ZnO nanomaterials which contributed greatly to the increased signal-to-noise ratio of biomolecular fluorescence. We also demonstrate the easy integration potential of zinc oxide nanorods into periodically patterned nanoplatforms which, in turn, will promote the assembly and fabrication of these materials into multiplexed, high-throughput, optical sensor arrays. These zinc oxide nanoplatforms will be extremely beneficial in accomplishing highly sensitive and specific detection of biological samples involving nucleic acids, proteins and cells, particularly under detection environments involving extremely small sample volumes of ultratrace-level concentrations.
Fluorescence detection of biomolecules is a heavily relied on technique in gene profiling, proteomics, drug discovery, disease diagnostics, and environmental analysis. Novel methods which enable rapid, high-throughput, ultrasensitive, and specific optical detection are in great demand for the burgeoning areas of genomics and proteomics.1-4 For many biomolecular detection techniques exploiting fluorescence, enhancing detection sensitivity and increasing the signal-to-noise ratio still remain as major challenges in carrying out the much needed, system-wide study of proteins and population-level genetic screening. To improve the fluorescence detection capability and resolution, numerous research efforts have been made in parallel on three main aspects of biomolecular fluorescence detection: (1) molecular design of better fluorophores, (2) development of improved detection apparatus, and (3) engineering of advanced substrates. New organic, inorganic, and hybrid labels were developed to prevent photobleaching of fluorescing dyes while allowing measurements of multiple fluorophores with a single excitation source at very low concentration levels.5-10 Advanced confocal optics and more reliable miniaturized detection devices were developed in order to increase detection sensitivity, in some cases down to the single molecule level.11-16 The use of metallized substrates has been explored to increase quantum yield and photostability of fluorophore labels.17-19 Focusing on the development of novel bioarray substrates suitable for fluorescence detection, we herein report for the first time the development and utilization of nanoscaled zinc oxide (ZnO) platforms for use as attractive substrates in enhanced fluorescence detection of biomolecules such as proteins and DNA.
ZnO thin films and micro/nano structures have received considerable attention in the past particularly due to their desirable optical properties, which include a wide band gap of 3.37 eV and a large exciton binding energy of 60 meV at room temperature. ZnO has been previously demonstrated as a candidate material for use in a broad range of optical and optoelectric applications. Examples of ZnO applications in these areas include short-wavelength light-emitters,20,21 field-emitters,22 luminescence,23 and UV lasers.24 In addition to their rich potential in these applications, sensitization of ZnO by organic dye molecules has been extensively studied for use as highly efficient solar cells.25-30 However, biosensing applications of wide band gap ZnO have not been previously demonstrated although nanometer scale ZnO is stable in typical biomolecular detection environments and an easily processed material which is ideal for aiding optical detection of target bioconstituents. In this paper, we demonstrate that engineered nanoscale ZnO structures can significantly enhance fluorescence detection capability toward sensing proteins and nucleic acids without any need for amplification. In addition, we demonstrate that these nanostructured ZnO building blocks can be easily assembled into biosensor platforms of predetermined dimensions during their synthesis while eliminating additional postsynthetic processes. Our results suggest that ZnO nanoplatforms can be conveniently fabricated into fluorescence enhancing substrates and straightforwardly assembled into a tailor-made array format which, in turn, will promote highly sensitive, multiplexed, high-throughput, fluorescence detection of target biomolecules.
We first determined the suitability of the wide band gap ZnO nanomaterials as optical signal enhancing platforms in biomolecular detection. The ZnO-mediated fluorescence signal was measured and compared with other nanoscale materials such as silicon nanorods (SiNRs) as well as other commonly used biosensor substrates such as glass slides, quartz slides, silicon surfaces, and polymers. The overall experimental designs to examine various substrates systematically are illustrated schematically in Figure 1. ZnO (Figure 1a,b) and SiNRs (Figure 1c) were grown on Si wafers using chemical vapor deposition on Ag and Au catalysts, respectively. For regularly patterned ZnO synthesis (Figure 1b), microcontact printing techniques were employed to deliver the catalysts selectively at predetermined locations on Si wafers which subsequently led to directed ZnO growth only on patterned areas. The average diameter of as-grown ZnO and SiNRs measured by scanning electron microscopy (SEM) are 677 ? 65 and 547 ? 17 nm, respectively. Ultrathin films of poly(methyl methacrylate) (PMMA) were produced by spincasting and subsequent thermal annealing under Ar environment. The average thickness of the PMMA films, determined by ellipsometry, is 98 ? 2 nm. Nanoimprint lithographic techniques were used to generate periodic, micrometer scale patterns on these polymeric films (Figure 1d). These substrates were then incubated with fluorophore-labeled proteins or DNA to allow direct adsorption of biomolecules onto surfaces. A commercially available confocal microscope was used for fluorescence detection, and the excitation and detection wavelengths were chosen according to the specific emission properties of the fluorophores that were employed in our proof-of-concept experiments. As our experimental scheme involves nonspecific adsorption of these biomolecules onto substrates, biomolecules were randomly distributed over the entire surface area upon deposition.
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