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4 protocols using tris 2 2 bipyridyl ruthenium 2 chloride hexahydrate

1

Ruthenium Complexes Preparation Protocol

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Tris(2,2′-bipyridyl)ruthenium(ii) chloride hexahydrate (Ru(bpy)3(H2O)6), potassium hexacyanoruthenate(ii) hydrate (K4[Ru(CN)6]xH2O), hexaammineruthenium(iii) chloride ([Ru(NH3)6]Cl3), pentaamminechlororuthenium(iii) chloride ([Ru(NH3)5Cl]Cl2), and cis-bis(2,2′-bipyridine)-dichlororuthenium(ii) hydrate, ([Ru(bpy)2Cl2]xH2O)) were purchased from Sigma Aldrich and used without further purification. The five complexes were dissolved in water yielding 50 mM ([RuII(bpy)3]2+), 100 mM ([RuII(CN)6]4−), 100 mM ([RuIII(NH3)6]3+), 10 mM ([RuIII(NH3)5Cl]2+) and 10 mM ([RuII(bpy)2Cl2]) solutions.
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2

Synthesis and Characterization of GO-CONHTPP Hybrid

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Graphene oxide (GO-powder < 35 mesh. C/O atomic ratio = 2.5–2.6) was purchased from LayerOne. Anhydrous DMF and tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate were purchased from Sigma Aldrich. Reagents for porphyrin synthesis were obtained from commercial sources and were used as received. For solution preparation ultrapure water (18 MΩ cm) was used. The synthesis of TPPNH2 followed a literature procedure22 (link) and is described in the Supporting Information along with 1H NMR and MALDI spectra (Figs. S1S3). For all experiments GO or GO-CONHTPP hybrid suspensions were prepared by dispersing GO or hybrid powder in DMF or DMF-H2O (1:2, v/v) followed by ultrasonication for 30 min.
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3

Photocrosslinked A2YA2-A48-SAv Hydrogels

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Lyophilized A2YA2-A48-SAv proteins were dissolved in 20 mM tris buffer while incubating on a rocker at 4°C for approximately 1 hr. Ammonium persulfate and tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 20 mM tris and added to the protein solution for a final concentration of 15 or 80 mM and 31 or 250 μM, respectively, with 10% w/v A2YA2-A48-SAv. Hydrogels were prepared in a 4°C cold room by pipetting the mixture into a 14 mm diameter x 2 mm height mold and crosslinking 10 inches under a 24 W, 14 × 14 LED array emitting 460 nm blue light for 10 min. During photo-cross-linking, tris(2,2’-bipyridyl)ruthenium(II) ([Ru(II)bpy3]2+) oxidation of the tyrosyl phenyl groups form a spontaneous chemical bond, consuming a persulfate anion. Once completed, hydrogels were cut to an 8 mm diameter to match the rheometer geometry.
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4

Electrochemical Detection of Glucose Oxidase

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All chemicals were of analytical grade unless noted otherwise and were used as received. Ferricyanide (99.98%), ferrocyanide (≥98.5%), ferrocene methanol (97%), ferrocene (98%), tris(2 2′-bipyridyl)-ruthenium(II) chloride hexahydrate (98%), methylene blue, hydrochloric acid (37 wt %), chitosan (from shrimp shells, ≥75% (deacetylated)), lithium perchlorate, tetrabutylammonium perchlorate (TBAP) (≥99%), methanol (≥99.9%), and 5% Nafion were obtained from Sigma-Aldrich. Potassium chloride (USP/FCC/crystalline), potassium nitrate (certified ACS, crystalline), and dextrose (d-glucose) (certified ACS, granular powder) were obtained from Fischer Bioreagents. Glucose oxidase (GOx) from Aspergillus niger was obtained from MP Biomedicals. Dulbecco’s phosphate-buffered saline (DPBS) (1×) with calcium and magnesium (pH = 6.8) was purchased from Corning. All stock solutions were made with ultrapure water and kept in the dark throughout use.
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