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Self-Assembly of Multifunctional Nano-Composites for Multiplex, Multimodal Biosensing

Kim, Jin Woo
University of Arkansas
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Engineering imaging probes by integrating multiple discrete nanoscale materials into single multifunctional nanoscale architecture has the potential to transform many fields of research, ranging from optoelectronics and nanophotonics to biosensors with applications to agriculture, food safety, biosecurity, and biomedicine. This would allow us to implement multiple tasks in parallel or in sequence and to acquire more comprehensive, accurate and reliable information in the same system using multiple sensing modalities.

The goal of the proposed research is to realize the "programmable" self-assembly of multifunctional ensembles of nanoparticles (NPs) that conform to a specific design with specific functions for multiplex and multimodal biosensing applications. Specially, the intent is to develop and implement a versatile means to functionalize DNA to various NPs with precise control over DNA's locations and angles to achieve accurate, scalable and high-rate self-assembly of complex hybrid NP composites with desirable multifunctionalities. The PI proposes to develop, characterize, and implement DNA/NP composite building blocks (termed nBLOCKs) to self-assemble multifunctional hybrid nano-ensembles of plasmonic (e.g., gold [Au]), fluorescent/photoluminescent (e.g., quantum dots [QDs]), and magnetic (e.g., super-paramagnetic iron oxide [SIO]) particles, whose properties are commonly used for the biosensor development.

The specific objectives are to: (1) Design and verify DNA sequences for nBLOCK assemblies to minimize unplanned defects and errors; (2) develop and optimize nBLOCK assembly processes; (3) develop, characterize and optimize multifunctional nanocomposite assembly for multiplex, multicolor and multimodal plasmonic/photoluminescent/magnetic biosensing using three different types of nBLOCKs, i.e., Au NPs, QDs and SIO NPs; (4) evaluate the sensing capabilities of nanocomposites in vitro, including sensitivity and stability; and (5) evaluate in-vitro toxicity of nanocomposites and their implications on agriculture, food, environment, and human. The ultimate significance of the proposed nBLOCK and their nanocomposites is that its development could address the urgent need in the field of nanotechnology for the functional, reliable and scalable techniques for more complicated and controlled multifunctional nanostructures that incorporate various nanocomponents for specific applications.

This technology, if successfully developed, would provide an effective and efficient route to "second-generation" multifunctional nano-architecture with their properties to be "programmable"/"customizable" depending on the application. The proposed technology has high potential to transform many fields of research, including biology, chemistry, physics, and materials science and engineering with applications to agriculture, food safety and biosecurity.

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Non-Technical Summary:
This project will explore the programmable self-assembly of multifunctional composites of nanoparticles into structures, which conform to a specific design, with properties that can be programmed for multiple biosensing tasks in parallel or in sequence. This is achieved with novel technology that controls the number, placement, and orientation of DNA, which will do the assembly, on the nanoparticle. This work has the potential to expand the current state of nanotechnology-based biological research on many fronts and provide out-of-the-box research capability by providing an effective and efficient route to a "second-generation" multifunctional nano-architecture that enables realizing the multiplex, multicolor, and multimodal nanoscale biosensing devices with more capability than existing technologies. It also has high promise to transform many fields of research, including biology, chemistry, physics, material science and engineering with applications to agriculture, food safety and biosecurity.

DNA sequence design:The nearest-neighbor model of DNA duplex thermal stability will be used to design oligonucleotide sequences for the nBLOCKs. We will select sequences using our established computational methodology. The designed DNA will be purchased from vendors with appropriate modifications in 3' and 5' ends as necessary.nBLOCK assembly: The proposed "programmable" and "customized" nBLOCKs will be fabricated using our established aqueous-phase anisotropic DNA functionalization technology. To accomplish the proposed nanocomposite assembly, we will further optimize and refine the functionalization method as well as generalize the functionalization reaction to other types of NPs, including QDs and SIO NPs.
Nanocomposite Assembly: Multifunctional nanocomposites: We propose to assemble the following two nano-ensembles with different overall shapes via DNA-directed self-assembly: (1) Spherical multifunctional shell and (2) Rod-shaped multifunctional shell. They are selected to explore the optical tunability, especially in near-infrared (NIR) range, depending upon the overall geometry of the nanostructure. In each architecture, we will control the spacing between NPs and the thickness of each NP layer in an attempt to tune and enhance the three properties of interest (i.e., plasmonic, fluorescent/luminescent, and paramagnet properties). Specifically, we will use three different shapes of plasmonic Au NPs, including spherical NPs (from vendors), hexagonal dipyramidal NPs and rod-shaped golden carbon nanotubes (synthesized in PI's laboratory). QDs and SIOs in different sizes will be acquired from vendors. Based upon the thickness of each layer and gaps between them, appropriate sets of nBLOCKs will be prepared. The required oligonucleotides will be designed according to our established method and will be ordered from vendors.
Bioconjugation: Complementary sequence of the outer layer of the nanocomposites are linked to antibody (Ab) and specifically hybridized to the nanocomposite. The effectiveness of conjugations will be evaluated by estimating density of Ab on each composite using fluorophore labeled secondary Ab against the primary Ab on the composite. Fluorometeric and epi-fluorescence microscopic analyses will be done to assess the density.
In vitro tests: In vitro tests will be done to verify the capabilities and limitations of the proposed nanocomposites for their practical applications. We will evaluate their stability as well as targeting efficiency using representative pathogenic microorganisms, including Escherichia coli O157:H7 and Staphylococcus aureus according to our established methods.
Cytotoxicity Tests:The manufactured nanocomposites will be examined via in vitro toxicity tests using different types of cells, ranging from microorganisms, yeast, and mammalian cells, including Escherichia coli K-12, Saccharomyces cerevisiae, and the breast cancer cell lines SK-BR-3, MDA-MB-231, and MFC-7.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Escherichia coli