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Dip-pen nanolithography (DPN) is used to precisely position core/thick-shell ("giant") quantum dots (gQDs; ≥10 nm in diameter) exclusively on top of silicon nanodisk antennas (≈500 nm diameter pillars with a height of ≈200 nm), resulting in periodic arrays of hybrid nanostructures and demonstrating a facile integration strategy toward next-generation quantum light sources. A three-step reading-inking-writing approach is employed, where atomic force microscopy (AFM) images of the pre-patterned substrate topography are used as maps to direct accurate placement of nanocrystals. The DPN "ink" comprises gQDs suspended in a non-aqueous carrier solvent, o-dichlorobenzene. Systematic analyses of factors influencing deposition rate for this non-conventional DPN ink are described for flat substrates and used to establish the conditions required to achieve small (sub-500 nm) feature sizes, namely: dwell time, ink-substrate contact angle and ink volume. Finally, it is shown that the rate of solvent transport controls the feature size in which gQDs are found on the substrate, but also that the number and consistency of nanocrystals deposited depends on the stability of the gQD suspension. Overall, the results lay the groundwork for expanded use of nanocrystal liquid inks and DPN for fabrication of multi-component nanostructures that are challenging to create using traditional lithographic techniques.
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The development of homogenously nano-patterned chemically modified surfaces that can be used to initiate a cellular response, particularly stem cell differentiation, in a highly controlled manner without the need for exogenous biological factors has never been reported, due to that fact that precisely defined and reproducible systems have not been available that can be used to study cell/material interactions and unlock the potential of a material driven cell response. Until now material driven stem cell (furthermore any cell) responses have been variable due to the limitations in definition and reproducibility of the underlying substrate and the lack of true homogeneity of modifications that can dictate a cellular response at a sub-micron level that can effectively control initial cell interactions of all cells that contact the surface. Here we report the successful design and use of homogenously molecularly nanopatterned surfaces to control initial stem cell adhesion and hence function. The highly specified nano-patterned arrays were compared directly to silane modified bulk coated substrates that have previously been proven to initiate mesenchymal stem cell (MSC) differentiation in a heterogenous manner, the aim of this study was to prove the efficiency of these previously observed cell responses could be enhanced by the incorporation of nano-patterns. Nano-patterned surfaces were prepared by Dip Pen Nanolithography (DPN) to produce arrays of 70 nm sized dots separated by defined spacings of 140, 280 and 1000 nm with terminal functionalities of carboxyl, amino, methyl and hydroxyl and used to control cell growth. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and will change the capabilities for stem cell definition in vitro and then cell based medical therapies. In addition to highlighting the ability of the materials to control stem cell functionality on an unprecedented scale this research also introduces the successful scale-up of DPN and the novel chemistries and systems to facilitate the production of homogeneously patterned substrates (5 mm2) that are applicable for use in in vitro cell conditions over prolonged periods for complete control of material driven cell responses.
Assuntos
Nanotecnologia/métodos , Células-Tronco/citologia , Adesão Celular , Técnicas de Cultura de Células , Separação Celular , Citometria de Fluxo , Vidro , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Fenótipo , Engenharia Tecidual/métodosRESUMO
BACKGROUND: Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed. RESULTS: In this proof-of-concept study, we have designed a miniaturized planar recombinant (single-chain Fragment variable). antibody array technology platform for multiplexed profiling of non-fractionated, directly labelled serum samples. The size of the individual spot features was reduced 225-times (78.5 µm(2)/spot) and the array density was increased 19-times (38,000 spots/cm(2)). These miniaturized, multiplexed arrays were produced, using a desktop nanofabrication system based on dip-pen nanolithography technology, and interfaced with a high-resolution fluorescent-based scanner. The reproducibility, sensitivity, specificity, and applicability of the set-up were demonstrated by profiling a set of well-characterized serum samples. CONCLUSION: The designed antibody array platform opens up new possibilities for large-scale, multiplex profiling of crude proteomes in a miniaturized fashion.
Assuntos
Anticorpos/química , Proteínas Sanguíneas/análise , Miniaturização , Análise Serial de Proteínas , Anticorpos/imunologia , Proteínas Sanguíneas/imunologia , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels ("enhanced excitation") and the spatially biased funneling of fluorophore emissions through coupling to PC resonances ("enhanced extraction"), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couple fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 min. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM.
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Biomarcadores Tumorais/análise , Imunoensaio/métodos , Lasers , MicroRNAs/análise , Fótons , Proteínas/análise , Silício , Imunoensaio/instrumentação , Interleucina-3/análise , Análise em Microsséries , Fenômenos Ópticos , Espectrometria de Fluorescência , Fator de Necrose Tumoral alfa/análiseAssuntos
Cristalização/métodos , Microscopia de Força Atômica/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Silício/química , Tinta , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
This article reveals the enabling aspects of nanografting (an atomic force microscopy-based lithography technique) in surface physical chemistry. First, we characterize self-assembled monolayers and multilayers using nanografting to place unknown molecules into a matrix with known structure or vice versa. The availability of an internal standard in situ allows the unknown structures to be imaged and quantified. The same approaches are applied to reveal the orientation and packing of biomolecules (ligands, DNA, and proteins) upon immobilization on surfaces. Second, nanografting enables systematic investigations of size-dependent mechanics at the nanometer scale by producing a series of designed nanostructures and measuring their Young's modulus in situ. Third, one can investigate systematically the influence of ligand local structure on biorecognition and protein immobilization by precisely engineering ligand nanostructures. Finally, we also demonstrate the regulation of the surface reaction mechanism, kinetics, and products via nanografting.
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Nanoestruturas/química , Fenômenos Bioquímicos , Bioquímica , Fenômenos Químicos , Físico-Química , Humanos , Cinética , Modelos Biológicos , Nanoestruturas/ultraestrutura , Propriedades de SuperfícieRESUMO
We discuss a nanoengineering approach for supramolecular chemistry and self assembly. The collective properties and biofunctionalities of molecular ensembles depend not only on individual molecular building blocks but also on organization at the molecular or nanoscopic level. Complementary to "bottom-up" approaches, which construct supramolecular ensembles by the design and synthesis of functionalized small molecular units or large molecular motifs, nanofabrication explores whether individual units, such as small molecular ligands, or large molecules, such as proteins, can be positioned with nanometer precision. The separation and local environment can be engineered to control subsequent intermolecular interactions. Feature sizes as small as 2 x 4 nm(2) (32 alkanethiol molecules) are produced. Proteins may be aligned along a 10-nm-wide line or within two-dimensional islands of desired geometry. These high-resolution engineering and imaging studies provide new and molecular-level insight into supramolecular chemistry and self-assembly processes in bioscience that are otherwise unobtainable, e.g., the influence of size, separation, orientation, and local environment of reaction sites. This nanofabrication methodology also offers a new strategy in construction of two- and three-dimensional supramolecular structures for cell, virus, and bacterial adhesion, as well as biomaterial and biodevice engineering.
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Nanotecnologia , Ligação Proteica , Animais , Reações Antígeno-Anticorpo , Aderência Bacteriana , Sítios de Ligação , Bovinos , Adesão Celular , Substâncias Macromoleculares , Camundongos , Microscopia de Força Atômica , Microscopia de Tunelamento , CoelhosRESUMO
A molecular-level approach is developed to prevent or inhibit the degradation processes of alkanethiol self-assembled monolayers (SAMs). Previous studies revealed two degradation pathways: direct desorption and oxidation-desorption. By use of scanning tunneling microscopy (STM) and atomic force microscopy (AFM), in situ and time-dependent imaging reveals and confirms that degradations of alkanethiol SAMs on gold mainly initiate at defect sites, such as domain boundaries and vacancy islands, and then propagate into the ordered domains. Our approach targets at attaching small molecules with preferred adhesion to the defects. The best candidates are aqueous media containing a small amount of amphiphilic surfactant molecules, such as N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). High-resolution studies demonstrate that DMSO and DMF molecules attach to SAM surfaces and more favorably at defect sites, forming relatively stable adsorbates. This attachment increases the activation energy sufficiently to inhibit both degradation pathways. The robustness of this approach has been investigated as a function of surfactant concentration, solution temperature, and the stirring condition. Molecular-level mechanisms and energetics for degradation inhibition of SAMs are also discussed in detail.