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Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization
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20.(i) Chips were incubated in 0.15 M of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and 0.03 M of -hydroxysulfosuccinimide dissolved in 0.1 M 2-[morpholino]-ethanesulfonic acid buffer ( 4.7) (Pierce Biotechnology, Inc., Rockland, IL) for . (ii) They were then incubated for in fluorescent molecule 5-(aminoacetamido)fluorescein (AAAF) (Invitrogen Corp., Carlsbad, CA) [0.01 mM in PBST ( Triton X-100), 7.3] or protein A (Sigma-Aldrich Corp., St. Louis, MO) ( in PBST, 7.3).
21.Greg T. Hermanson, Bioconjugate Techniques (Academic, San Diego, CA, 1996).
22.Olympus BX51 upright epifluorescence microscope (Olympus America, Inc., Center Valley, PA) with CoolSnapHQ monochrome CCD camera (Photometrics, Tuscon, AZ) and filter set with excitation: , dichroic mirror: , emission: (Chroma Technology Corp., Rockingham, VT) was used. A sample without fluorescent molecule was used as negative control and did not show any fluorescence signal.
23.(i) Surface reactive groups were quenched by incubating in ethylenediamine (0.5 M in PBST) for (ii) rinsed three times in starting block buffer with 0.05% Tween-20 (Pierce Biotechnology). Rabbit antimouse IgG labeled with Alexa-488 (Invitrogen) was used as probe antibody for .
See EPAPS Document No. for fluorescence images of the hydrogel features made by Scheme I after aqueous immobilization process. (a) Immobilized fluorescent molecule 5-(aminoacetamido) fluorescein (b) and protein A detected by fluorescently labeled probe antibody. Scale bar represents
in all panels. This document can be reached via a direct link in the online article’s HTML reference section or via the EPAPS homepage (http://www.aip.org/pubservs/epaps.html
26.Thermally grown silicon oxide surface was treated for with 3% (v/v) solution of 3-methacryloxypropyl(trimethoxysilane) (Gelest, Inc., Morrisville, PA) dissolved in stock solution of 95% ethanol, 4.7% water, and 0.3% acetic acid.
27.80% acrylamide, 10% methylenebisacrylamide, and 10% acrylic acid (w/w) were dissolved in 40% glycerol (v/v) (Sigma-Aldrich) to make the final monomer content of 5% (w/v). Free radicals were generated by addition of of 25% (w/v) of ammonium persulfate and of -tetramethylethylenediamine (Bio-Rad Laboratories, Hercules, CA) per milliliter of gel precursor solution. of prepolymerized hydrogel solution was dispensed on each diameter silicon oxide wafer and covered with a sacrificial silicon wafer and allowed to polymerize for . The sacrificial wafer was then lifted-off mechanically by hand and a thin hydrogel film attached to the silicon oxide surface of the other wafer was obtained.
28.PlasmaTherm 72 reactive ion etcher at Cornell NanoScale Science and Technology Facility (CNF) (electrode area ) was used to perform the oxygen RIE. oxygen flow rate and rf power were employed. chamber pressure was used for anisotropic etch and for isotropic etch parameters.
29.The thermal silicon oxide surface was treated twice for with 3-methacryloxypropyl(trichlorosilane) at the chamber pressure of (MVD-100, Applied Microstructures, Inc., San Jose, CA).
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