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Nanoscale SECM
Published in Allen J. Bard, Michael V. Mirkin, Scanning Electrochemical Microscopy, 2022
Xiang Wang, Gaukhar Askarova, Michael V. Mirkin
When the tip approached the membrane, the iT decreased because the diffusion of the redox mediator (FcTMA+) to its surface occurred only through NPCs and was blocked by the impermeable portion of the NE (Figure 7.20C); however, the current remained significantly higher than the corresponding values for the pure negative feedback (Figure 7.20C, black curve). The permeability constant values were obtained from the fit between the theoretical and experimental approach curves in the presence (blue curve in Figure 7.20C) and absence (red curve) of wheat germ agglutinin (WGA)—a lectin that selectively blocks molecular transport through the peripheral zone of the NPC. The effective radii of the central and peripheral zones were deduced from comparison of the permeability constants measured with and without WGA. The ∼7-nm difference between the radii of the entire nanopore and central route corresponds to the thickness of the peripheral zone, which is comparable to the diameter of WGA (∼5 nm).
Next-Generation Immunoassays
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Valerie Fitzgerald, Paul Leonard
Using eukaryotic hosts, a more sophisticated protein secretion and folding apparatus is provided, as well as the capacity for post-translational modifications, when compared to prokaryotes. While mammalian cells are still the system of choice for the production of antibodies the disadvantages associated with their use which include a requirement for highly specialised dedicated facilities, high cost and lengthy production times mean that other systems have been investigated. Yeast, filamentous fungi, insect cells along with transgenic plants and animals have all been used in the production of recombinant antibodies [2]. Perhaps the most widely used eukaryotic display technology is yeast display. Antibody fragments are displayed on the cell surface as part of a hybrid protein that is made up of the antibody or antibody fragment and the Aga2p (agglutinin) subunit, which is covalently linked by two disulphide bonds to Aga1 (agglutinin) in the yeast cell wall [8]. The use of yeast technology combines the ease-of-culture seen with prokaryotic systems with the capacity for folding and secretion of eukaryotic systems. Other benefits of the system include rapid and quantitative screening facilitated by FACS and convenient evaluation of characteristics such as affinity without the need for soluble expression and purification of each clone of interest [23].
Polymeric Nanoparticles for Tumor-Targeted Drug Delivery
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
Tania Betancourt, Amber Doiron, Lisa Brannon-Peppas
A new approach to targeting is the use of lectins, which are plant proteins that specifically recognize cell surface carbohydrates. The latter function as selective cancer-cell-targeting agents. PLGA nanoparticles of mean diameter 331 nm incorporating isopropyl myristate were used to deliver paclitaxel to malignant A549 and H1299 and normal CCL-186 pulmonary cells in vitro by means of wheat germ agglutinin lectin as the targeting molecule. The in vitro cytotoxicity against A549 and H1299 cells was significantly increased with wheat germ agglutinin-conjugated PLGA nanoparticles containing IPM and paclitaxel compared to controls.77 In a subsequent study, Mo and Lim evaluated in vivo efficacy of the same nanoparticles in a SCID mouse model injected with an A549 tumor nodule.78 One injection of wheat germ agglutinin-conjugated PLGA nanoparticles containing IPM and a paclitaxel dose of 10 mg/kg inhibited tumor growth without appreciable weight loss. Tumor doubling was increased to 25 days compared to 11 days for conventionally formulated paclitaxel.
Antibody separation using lectin modified poly(HEMA-EDMA) hydrogel membranes
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Esra Feyzioğlu Demir, Cansu Ilke Kuru, Murat Uygun, Deniz Aktaş Uygun, Sinan Akgöl
One most widely used affinity chromatography techniques for biomolecules separations is lectin affinity chromatography. In this chromatographic method, various lectin molecules are used as a ligand molecule [24]. Lectin proteins have high affinity and selectivity towards to carbohydrates and carbohydrate bearing molecules [25]. In addition, despite its high specificity toward carbohydrate bearing biomolecules, bonded molecules can be easily eluted by washing with a solution containing a competitive carbohydrate (e.g. D-glucose or α-D-mannose). The main specific carbohydrate units recognized by lectins are N-acetylneuraminic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose, and fructose molecules [26]. Most commonly used lectins for antibody purification studies are concanavalin A (Con A), wheat germ agglutinin, mannan-binding protein and jacalin [27]. Con A is a lectin originally extracted from the jack-bean, Canavalia ensiformis and have been intensively preferred as a lectin molecule for affinity chromatography studies due to their affinities towards glycoconjugates containing α-D-mannopyranosyl, α-D-glucopyranosyl and sterically related residues. Because of these unique properties, Con A is the preferred choice for the separation and adsorption of glycoenzymes and carbohydrate bearing biomolecules [24].
Immobilized RGD concentration and proteolytic degradation synergistically enhance vascular sprouting within hydrogel scaffolds of varying modulus
Published in Journal of Biomaterials Science, Polymer Edition, 2020
Yusheng J. He, Martin F. Santana, Madison Moucka, Jack Quirk, Asma Shuaibi, Marja B. Pimentel, Sophie Grossman, Mudassir M. Rashid, Ali Cinar, John G. Georgiadis, Marcella K. Vaicik, Keigo Kawaji, David C. Venerus, Georgia Papavasiliou
At day 7, hydrogel scaffolds were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, rinsed three times with PBS for 10 min, permeabilized with 1%Triton X-100 in PBS (PBST) for 1 hr, and blocked using 6% normal goat serum in PBST for 1 hr at room temperature and overnight at 4 C. Hydrogel scaffolds were then incubated with 25UI/ml Alexa Fluor 546-phalloidin in 2% normal goat serum PBST to stain F-actin overnight at 4 C. F-actin stained spheroids were imaged using PASCAL laser scanning confocal microscopy (Carl Zeiss MicroImaging, Inc.) to produce a series of z-stack images that could be flattened to obtain z-projected images. F-actin stained images were analyzed with sprout morphology plugin of ImageJ/Fiji to quantify total sprout length and total number of junctions. To determine the cell type contributing to 3 D vascular sprouting and invasion in response to material properties, differential staining of ECs and SMCs was performed at day 7 following spheroid encapsulation in scaffolds. Hydrogels were incubated with fluorescent Ulex Europaeus Agglutinin I (UEAI) and mouse anti-human α-SMA primary antibody (1:20 dilution) in 2% normal goat serum PBST overnight at 4 C to stain for ECs and SMCs, respectively. Subsequently, scaffolds were rinsed 3 times with PBST for 10 min and incubated with a secondary goat anti-mouse smooth muscle cell antibody for 4 h at room temperature and overnight at 4 C. Differentially stained hydrogels were then rinsed 3 times with PBST for 10 min prior to confocal imaging.