We report that engineered nanoscale zinc oxide structures can be effectively used for the identification of the biothreat agent, Bacillus anthracis by successfully discriminating its DNA sequence from other genetically related species. We explore both covalent and non-covalent linking schemes in order to couple probe DNA strands to the zinc oxide nanostructures. Hybridization reactions are performed with various concentrations of target DNA strands whose sequence is unique to Bacillus anthracis. The use of zinc oxide nanomaterials greatly enhances the fluorescence signal collected after carrying out duplex formation reaction. Specifically, the covalent strategy allows detection of the target species at sample concentrations at a level as low as a few femtomolar as compared to the detection sensitivity in the tens of nanomolar range when using the non-covalent scheme. The presence of the underlying zinc oxide nanomaterials is critical in achieving increased fluorescence detection of hybridized DNA and, therefore, accomplishing rapid and extremely sensitive identification of the biothreat agent. We also demonstrate the easy integration potential of nanoscale zinc oxide into high density arrays by using various types of zinc oxide sensor prototypes in the DNA sequence detection. When combined with conventional automatic sample handling apparatus and computerized fluorescence detection equipment, our approach can greatly promote the use of zinc oxide nanomaterials as signal enhancing platforms for rapid, multiplexed, high-throughput, highly sensitive, DNA sensor arrays.

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.

Proteins are the key components of the cellular machinery responsible for the processing of detailed biological functions decoded from genetic information. The rapid pace in the discovery of new gene products by large-scale genomics demands significant improvements in current technology pertaining to quantitative and functional proteomics. Specifically, the design of alternative strategies for analyzing protein functions via novel high-throughput approaches is highly warranted in this information-rich age of whole genome biology. Biomolecular fluorescence is the most widely used detection mechanism in both laboratory-scale and high-throughput proteomics research, as evidenced by its use in essential techniques such as enzyme-linked immunosorbent assays, fluorescent gel staining, and protein arrays.So far, the most recognizable contribution of nanoscience to the field of biomolecular fluorescence detection has been made mainly in the areas of developing new fluorescent probes. A well-known example of this contribution is the design of semiconductor nanocrystals and quantum dots whose emission spectrum at a specific wavelength can be tuned by simply changing the size of the nanomaterials. Continuing research efforts in these fields have led to improved fluorophores that are less subject to photobleaching while displaying high quantum yields. [1][2][3][4][5][6][7][8] An alternative step forward to promoting the study of proteins may be achieved by innovative assembly and fabrication of nanomaterials for use as advanced biosensor substrates in biomolecular fluorescence detection. As-grown nanomaterials have not yet been demonstrated as potentially suitable substrates for improved fluorescence detection when used in conjunction with target biomolecules. In order to design such substrates comprised of nanomaterials, four key characteristics of the candidate nanoscale materials should be carefully considered: i) signal enhancement, ii) ease of fabrication, iii) stability, and iv) surface chemistry. Candidate nanomaterials should exhibit an appropriate optical property to foster the fluorescence signal from fluorophore-linked biomolecules in order to promote detection at low concentrations, even at an ultratrace level. In addition, simple and straightforward synthesis and assembly routes should yield the successful growth and fabrication of these nanomaterials in order to facilitate high-throughput screening of interacting proteins. These nanomaterials should be biocompatible and chemically inert at detection environments involving most protein-protein interactions. Lastly, chemical reactions applicable to derivatize the surfaces of nanomaterials covalently should be widely available in order to link specific protein molecules on nanomaterials and maximize specificity of protein detection.ZnO nanostructures have received considerable attention particularly due to their desirable optical properties, which include a wide bandgap of 3.37 eV and a large exciton binding energy of 60 meV at room temperature. ZnO has been previ...

Novel methods for affixing functional proteins on surfaces with high areal density have the potential to promote basic biological research as well as various bioarray applications. The use of polymeric templates under carefully balanced thermodynamic conditions enables spontaneous, self-assembled protein immobilization on surfaces with spatial control on the nanometer scale. To assess the full potential of such nanometer-scale protein platforms in biosensing applications, we report for the first time the biological activity of proteins on diblock copolymer platforms. We utilized horseradish peroxidase, mushroom tyrosinase, enhanced green fluorescent protein, bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine IgG, and protein G as model systems in our protein activity studies. When specific catalytic functions of HRP and MT, immobilized on selective domains of microphase-separated PS-b-PMMA, are evaluated over a long period of time, these enzymes retain their catalytic activity and stability for well over 3 months. By performing confocal fluorescence measurements of self-fluorescing proteins and interacting protein/protein systems, we have also demonstrated that the binding behavior of these proteins is unaffected by surface immobilization onto PS-b-PMMA diblock copolymer microdomains. Our polymer platforms provide highly periodic, high-density, functional, stable surface-bound proteins with spatial control on the nanometer scale. Therefore, our diblock copolymer-guided protein assembly method can be extremely beneficial for high-throughput proteomic applications.

We developed a straightforward method to produce hexagonal ZnO nanorods and microrods using a novel biocatalyst, Magnetospirillum magnetotacticum. ZnO nanorods were synthesized homogeneously on growth substrates when the bacterial catalysts were deposited uniformly on substrates whereas ZnO microrods were formed when the catalysts were introduced to selective areas of growth substrates using microcontact printing. X-ray diffraction measurements reveal that these ZnO structures exhibit Wurtzite structures with preferential growth along [0001] direction. Room-temperature photoluminescence spectra of the as-synthesized ZnO nanorods and microrods show extremely strong and sharp UV emission at 390 nm and negligible green emission at 510 nm. Our results demonstrate that Magnetospirillum magnetotacticum is an effective catalyst for the growth of nanometer- and micrometer-sized ZnO structures with exceptionally high-quality optical properties. These defect-free ZnO nano-and micro-materials, when combined with microcontact printing techniques to achieve patterned growth over large areas of substrates, can facilitate their photonic-based applications as optoelectronic devices and chemical/biological sensors.