A self-assembling peptide scaffold functionalized for use with neural stem cells

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2005.Includes bibliographical references (leaves 33-35).The performance of a biological scaffold formed by the self-assembling peptide RADA16 is comparable to the most commonly used synthetic materials employed in the culture of neural stem cells. Furthermore, improvements in the performance of RADA16 have recently been made by appending the self-assembling peptide sequence with various functional motifs from naturally occurring proteins. The focus of this work is to further analyze the performance of these functionalized self-assembling peptide scaffolds when used for the culture of neural stem cells, and to characterize these newly developed materials for comparison with RADA16. The effect of the functional motifs on the structure of the peptide scaffold was evaluated with circular dichroism and scanning electron microscopy, and the mechanical properties of the peptide scaffolds were examined through theological analysis. The functionalized peptides were found to have lower percentages of beta-sheet structure as well as reduced storage moduli in comparison with RADA16. SEM images confirmed the ability of the functionalized peptides to form three-dimensional nanofiber scaffolds capable of encompassing, neural stem cells. Three-dimensional cell culture techniques were used to evaluate the ability of the functionalized peptide scaffolds to promote neural stem cell proliferation, and a scaffold formed by the combination of different functionalized peptides was found to increase the proliferation of neural stem cells in comparison to non-functionalized RADA 16.by Angus M. Hucknall.S.M

Micro- and nano-structured poly(oligo(ethylene glycol)methacrylate) brushes grown from photopatterned halogen initiators by atom transfer radical polymerization.

Photolithographic techniques have been used to fabricate polymer brush micro- and nanostructures. On exposure to UV light with a wavelength of 244 nm, halogens were selectively removed from films of chloromethylphenyltrichlorosilane and 3-(2-bromoisobutyramido)propyl-triethoxysilane on silicon dioxide. Patterning was achieved at the micrometer scale, by using a mask in conjunction with the incident laser beam, and at the nanometer scale, by utilizing interferometric lithography (IL). Friction force microscopy images of patterned surfaces exhibited frictional contrast due to removal of the halogen but no topographical contrast. In both cases the halogenated surface was used as an initiator for surface atom-transfer radical polymerization. Patterning of the surface by UV lithography enabled the definition of patterns of initiator from which micro- and nanostructured poly[oligo(ethylene glycol)methacrylate] bottle brushes were grown. Micropatterned brushes formed on both surfaces exhibited excellent resistance to protein adsorption, enabling the formation of protein patterns. Using IL, brush structures were formed that covered macroscopic areas (approximately 0.5 cm²) but exhibited a full width at half maximum height as small as 78 nm, with a period of 225 nm. Spatially selective photolytic removal of halogens that are immobilized on a surface thus appears to be a simple, rapid, and versatile method for the formation of micro- and nanostructured polymer brushes and for the control of protein adsorption

A versatile diffractive maskless lithography for single-shot and serial microfabrication

Abstract: We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of ~700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity

A Novel Immunoassay Platform Enabled by Non-fouling Poly(OEGMA) Surfaces

<p>The primary barriers to multiplexed point of care immunoassays are: (1) cost; (2) response time; and (3) sample handling. Described here is a self-contained, multiplexed immunoassay platform for point of care detection that leverages a number of enabling technologies to address these barriers. This platform is referred to as the "D4" assay, as it is composed of the following four sequential, concerted events (Figure 1): (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse across surface; (4) Detect binding event. </p><p>The D4 assay process begins when a finger-stick is administered and the resulting droplet of blood is applied to the surface of a detector chip. Hydrophobic ink printed onto the surface of the chip confines the blood droplet to a non-fouling region containing soluble, labile spots of detection antibodies and insoluble, non-labile spots of capture antibodies. As the soluble detection antibodies are dissolved from their printed spots by the droplet of blood, three serial events occur to generate signal (Figure 2): (1) the first half of the detection complex is formed by the binding of analytes present in blood to the stable capture agent spots; (2) diffusion of the blood laterally through the polymer brush, resulting in the dissolution and diffusion of soluble detection antibody spots; (3) solubilized detection antibodies bind to their respective analyte-capture agent spots, completing the detection complex and resulting in signal generation at the position of the non-labile capture antibody spots. </p><p>This assay relies upon the ability of labeled detection antibodies, printed into a nonfouling brush as "labile spots", to be carried by blood flow to adjacent rows of stably immobilized capture antibodies by diffusion of the analyte solution (Figure 2). Generation of signal at a given capture spot location provides identification of individual analytes (positives). Quantification of the concentration of the different analytes is carried out identically to a conventional fluorescence immunoassay by pre-calibration of the system using a dilution series of the analyte spiked into whole blood.</p><p>The D4 assay addresses several critical needs in point of care testing as follows: First, the cost of testing is reduced through miniaturization, multiplexing and one-step, on-site processing of undiluted whole blood obtained from a finger stick. Second, in order to simplify the immunoassay process, the D4 relies on diffusion to bring spatially localized reagents together to create a functional assay and thereby eliminate the need for liquid transfer steps, microfluidic manipulation of sample or reagents, and wash steps. Third, this multiplexed platform is capable of screening for a panel of markers in a single drop of blood with no sample preprocessing. Fourth, the assay is fast, which alleviates the difficulties often associated with communicating the outcome of diagnostic tests. A prototype of the D4 assay is shown in Figure 3 below.</p>Dissertatio

Direct Fluorescence Detection of RNA on Microarrays by Surface-Initiated Enzymatic Polymerization

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly­(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3′–OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3′–OH of an RNA–DNA hybrid. We found that the 3′-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format

Nanoparticle–Film Plasmon Ruler Interrogated with Transmission Visible Spectroscopy

The widespread use of plasmonic nanorulers (PNRs) in sensing platforms has been plagued by technical challenges associated with the development of methods to fabricate precisely controlled nanostructures with high yield and characterize them with high throughput. We have previously shown that creating PNRs in a nanoparticle–film (NP–film) format enables the fabrication of an extremely large population of uniform PNRs with 100% yield using a self-assembly approach, which facilitates high-throughput PNR characterization using ensemble spectroscopic measurements and eliminates the need for expensive microscopy systems required by many other PNR platforms. We expand upon this prior work herein, showing that the NP–film PNR can be made compatible with aqueous sensing studies by adapting it for use in a transmission localized surface plasmon resonance spectroscopy format, where the coupled NP–film resonance responsible for the PNR signal is directly probed using an extinction measurement from a standard spectrophotometer. We designed slide holders that fit inside standard spectrophotometer cuvettes and position NP–film samples so that the coupled NP–film resonance can be detected in a collinear optical configuration. Once the NP–film PNR samples are cuvette-compatible, it is straightforward to calibrate the PNR in aqueous solution and use it to characterize dynamic, angstrom-scale distance changes resulting from pH-induced swelling of polyelectrolyte (PE) spacer layers as thin as 1 PE layer and also of a self-assembled monolayer of an amine-terminated alkanethiol. This development is an important step toward making PNR sensors more user-friendly and encouraging their widespread use in various sensing schemes