As expected, the inactivated methoxy-capped regions (MeO, nonfluorescent, black) exhibit little chemical reactivity or non-specific binding as previously reported.30, 36 Open in a separate window Figure 2 (a, b) Scanned fluorescence images for (a) patterned protein A-TAMRA and (b) patterned fluorescein-PEG hapten on NHS/MeO patterned commercial polymer microarray slide surfaces; (c-e) fluorescence images of surface-bound Alexa647-labeled (c) 4-4-20 antibody, (d) non-specific murine Fab, and (e) murine Fc polyclonal fragment captured on protein A-patterned surfaces; (f-h) fluorescence images for bound Alexa647-labeled (f) 4-4-20 antibody, (g) non-specific murine Fab, and (h) murine Fc polyclonal fragments on fluorescein-patterned surfaces. 2.1.2 Capture of anti-fluorescein 4-4-20 (Ab-647) antibody by protein A- and fluorescein-patterned surfaces Anti-fluorescein 4-4-20 antibody (Ab-647) was affinity-immobilized onto patterned protein A (PM) and fluorescein (FM) single-ligand/methoxy-capped co-patterned samples. on the two different co-patterned surfaces generated side-by-side full antibody heads-up and tails-up oriented surface patterns. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis, sensitive to chemical information from the top 2-3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in heads-up regions, and ions from amino acids enriched in Fc domains for antibodies oriented in tails-up regions. Principal component analysis (PCA) improved the unique ToF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each heads-up versus tails-up patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain name- and structure- specific compositional differences by ToF-SIMS analysis. Since antibody immobilization Nelarabine (Arranon) and orientation are crucial to many Nelarabine (Arranon) technologies, orientation characterization using ToF-SIMS could be very useful Nelarabine (Arranon) and convenient for immobilization quality control and understanding methods for improving the overall performance of antibody-based surface capture assays. (black in Physique 1a)(white in Physique 1a)by adding iodine in portions until the reaction answer achieved a yellow to brown color. Iodine surplus was treated DGKH with 20% sodium pyrosulfite aqueous answer. After removal of 1 1,4-dioxane by rotary evaporation, the creamy suspension was filtered to yield the product 11,11-dithio-bis(undecanoic acid). Recrystallization from ethyl acetate/tetrahydrofuran (THF) afforded 8.0 g of product (yield 89%): ‘H NMR (400 MHz, CDCl3): 2.68 (t, 2H), 2.34 (t, 2H), 1.69-1.56 (m, 4H), and 1.40-1.29 (m, 12H). 1.9 Synthesis of 11,11- dithio-bis(succinimidylundecanoate) (DSU) 43 NHS (0.14 g, 1.2mmol) was added to 50 mL THF containing 11,11-dithio-bis(undecanoic acid) (0.26 g, 0.6 mmol), and 0.24 g DCC (1.2 mmol) and reacted at 0C for 3 hours. The reaction combination was warmed to RT and stirred for 36 hours at RT; the dicyclohexylurea was filtered off. Removal of solvent under reduced pressure, and recrystallization from acetone/hexane provided DSU as a white solid. Final purification was achieved by medium pressure liquid chromatography using silica gel and a 2:1 mixture of ethyl acetate and hexane, affording 0.29 g, (yield 75%): ‘H NMR (400 MHz, CDCI3): 5 2.83 (s, 4H), 2.68 (t, 2H, J = 7.3 Hz), 2.60 (t, 2H, J = 7.5 Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs, 20 keV): MALDI MS M+ m/z Calcd. for C26H44NO5S2 +:514.27, C30H48N2O8S2 628.29. Found: 514.24 and 628.28. 1.10 Formation of DSU monolayers on gold substrates Gold-coated silicon substrates were cleaned immediately before use under oxygen plasma (5 min, 80-90W, 0.15 mbar). These treated platinum substrates were immersed immediately into 1mM DSU ethanol answer for 16 hours, then removed from answer and rinsed extensively with ethanol and distilled water to remove physisorbed materials, and dried. 1.11 Covalent immobilization of protein A and fluorescein on platinum surfaces Nelarabine (Arranon) 1.11.1 Protein A immobilization on platinum Protein A (1.67 mg/mL) was first dissolved in PBS and then the DSU monolayer substrates were immersed into this protein Nelarabine (Arranon) solution for 48 hours at 4C. After rinsing with PBS and distilled water, the substrates were dried under a stream of nitrogen, and immediately utilized for capture of 4-4-20 antibody from answer as explained above. 1.11.2 Fluorescein hapten immobilization on platinum Carboxylic acid-terminated tetra(ethylene glycol) undecanethiol was dissolved in 20 mL ethanol (0.83 mg/mL). Freshly plasma-treated platinum substrates were immersed in this answer for 24 hours at RT. After rinsing with ethanol and distilled water, these substrates were dried under a stream of nitrogen and immediately immersed into a mixture of EDC (14 mg/mL) and NHS (3 mg/mL) aqueous answer for 2 hours at RT. Thereafter, the sample was rinsed with ethanol and distilled water and dried under a stream of nitrogen, and immediately utilized for reaction with fluoresceinamine. The NHS-adlayer-terminated gold substrates were immersed in fluoresceinamine isomer.
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