Preparation of Ferrocene-Labeled High Molecular Weight NAD Derivative

Supplementary Materials

Preparation of ferrocene-labeled high molecular weight NAD derivative

Ferrocene was firstly tethered to the polymer (PEI). FcCHO (90 mg) was dissolved in 15 mL ethanol and added within 1 h to 30 mL of ethanol solution containing 400 mg PEI. The mixture was stirred for 45 min at room temperature, then NaBH4 was carefully added in portions at 0ºC, and stirred continually for 90 min. Finally, the mixture was dried under vacuum condition and the residue was extracted with distilled water. The aqueous solution was purified by membrane dialysis against water for 12 h and dried. The polymer obtained was referred as PEI-Fc and used for further experiments.

PEI-Fc-NAD was further prepared. NAD+ (200 mg) was dissolved in 40 mL dimethylsulphoxide containing 8 g succinic anhydride. After 24 h at room temperature, NAD+ components (mixture of unreacted NAD+ and succinyl-NAD) were precipitated with 60 mL acetone. The precipitate was washed 3 times with acetone and recovered by centrifugation.After centrifugation, 20 mL water containing 700 mg EDC was added, and pH was adjusted to 4.7 for activating the carboxylic group of succinyl-NAD for 1 h. After activation, 300 mg PEI-Fc was added and reacted for another 12 h at 4ºC. The reaction mixture was dialyzed against 50 mM phosphate buffer (pH 7.0) for 12 h at 4ºC, followed by freeze drying.

Determination of the coenzyme activity of PEI-Fc-NAD

The coenzyme activity of PEI-Fc-NAD was determined spectrophotometrically. The assay mixture containing 2.9 mL of 0.1 M phosphate buffer (pH 7.0), 13.2 mol ethanol and appropriate amount of PEI-Fc-NAD was incubated at 30ºC for 5 min. A volume of 0.1 mL ADH solution (0.3 mgmL-1) was added to start the reaction. The absorbance change at 340 nm was measured for calculating the amount of NADH produced, with a molar absorption coefficient of 6.22×103 Lmol-1cm-1 for NADH.

Pretreatment of gold electrode

Commercially available polycrystalline Au disk electrodes (diameter: 2mm) were finely polished and electrochemically cleaned by cyclic voltammetry between –0.2 to +1.7 V vs. Ag/AgCl in 1 M H2SO4 at a scan rate of 100 mVs-1 until a reproducible plot was obtained. The surface roughness was calculated at 1.6.

Amperometric response of PEI-Fc-NAD/ADH/PVA bio-composite film modified electrode to ethanol

The amperometric response of PEI-Fc-NAD/ADH/PVA bio-composite film modified Au electrode was measured. The working potential was set at +0.45 V vs. Ag/AgCl in order to avoid the direct oxidation of produced NADH. Fig. S1 shows the calibration graph of the amperometric signal for ethanol.

Fig.S1 Calibration plot of PEI-Fc-NAD/ADH/PVA bio-composite film modified Au electrode for ethanol at a working potential of +0.45 V vs. Ag/AgCl.

Cyclic voltammograms of Au electrode modified withLBL multilayer film of PEI-Fc-NAD and ADH

The Au electrodes modified with PEI-Fc-NAD/ADH multilayer film were prepared, and Fig. S2 shows the cyclic voltammograms of the Au electrodes modified with different bilayer number of PEI-Fc-NAD/ADH.

Fig. S2Cyclic voltammograms of Au electrode modified with one, two, three, four, five and six PEI-Fc-NAD/ADH bilayers (from inner to outer) in 50 mM phosphate buffer (pH 7.0) at a scan rate of 50 mVs-1.

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