*2.2. Enzymes*

Flavocytochrome *b*2 (Fc*b*2) was isolated from the yeas<sup>t</sup> *Ogataea (Hansenula) polymorpha 356* and purified, as described earlier [48,49]. The Fc*b*2 (20 <sup>U</sup>·mg<sup>−</sup>1) was stored at −10 ◦C in a suspension of 70% ammonium sulfate, prepared with 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA and 0.1 mM dithiothreitol. To prepare a fresh solution, the enzyme was precipitated from the suspension by centrifugation (10,000 rpm, 10 min, 4 ◦C) and dissolved in 50 mM phosphate buffer, pH 7.5, up to 50 U·mL−1. An assay of Fc*b*2 activity in solution was performed as described earlier [48,49]. One unit of the enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol of L-lactate in 1 min under standard assay conditions (20 ◦C; 30 mM phosphate buffer, pH 7.5; 0.33 M L-lactate; 0.83 mM K3Fe(CN)6; 1 mM EDTA).

A commercial lyophilized horseradish peroxidase (PO or HRP, EC 1.11.1.7) from *Armoracia rusticana* (Aster, Lviv, Ukraine) with 600 <sup>U</sup>·mg<sup>−</sup><sup>1</sup> activity was dissolved in 20 mM phosphate buffer, pH 6.0, up to 400 U·mL−1.

A commercial lyophilized glucose oxidase (GO, EC 1.1.3.4) from *Asperigillus niger* (Sigma, St. Louis, MO, USA) with an activity of 100,000 <sup>U</sup>·g<sup>−</sup><sup>1</sup> in a solid form was dissolved in 20 mM phosphate buffer, pH 6.0, up to a concentration of 0.1 mg·mL−1. GO activity was assayed in a reaction mixture containing 0.16 mM *o*-dianisidine, 1.61% ( *w*/*v*) glucose and 2 U mL−<sup>1</sup> of PO in 50 mM sodium acetate buffer (NaOAc), pH 5.0, as described earlier [50].

#### *2.3. Synthesis of Hexacyanoferrates*

Synthesis of gHCF was carried out according to the scheme presented in Figure 1 [40]. A reaction mixture containing 6 mM K3[Fe(CN)6], 20 mM sodium lactate, 0.03–0.15 U mL−<sup>1</sup> Fc*b*2 in 50 mM phosphate buffer, pH 8.0, was prepared and incubated at 37 ◦C for 30 min. Formation of gHCF was initiated by the addition of salt to a final concentration of 10–100 mM.


**Figure 1.** Scheme of green hexacyanoferrate synthesis using flavocytochrome *b*2 (Fc*b*2) in enzymatic (**1**) and chemical (**2**) reactions; M—metal.

To obtain chemically synthesized HCFs (chHCFs), a solution of 6 mM K3Fe(CN)6 and 60 mM transition metal salt in 50 mM phosphate buffer, pH 8.0, was mixed with H2O2, added dropwise up to 100 mM. After 0.5–10 min incubation, the resulting mixture was fractionated by centrifugation at 13,000 rpm for 1 min, and the precipitate was resuspended in water. The centrifugation–redispersion procedure was repeated 2–4 times. The obtained HCFs were resuspended in water and kept at +4 ◦C until used.

#### *2.4. Characterization of the Synthesized HCFs*

### 2.4.1. Optical Properties

The optical properties of the synthesized HCFs, their concentrations and PO-like activities were characterized using a Shimadzu UV1650 PC spectrophotometer (Kyoto, Japan).

#### 2.4.2. Scanning Electron Microscopy (SEM)

Morphological analyses of the samples were performed using a SEM microanalyzer REMMA-102-02 (Sumy, Ukraine). The samples of different dilutions (2 μL) were dropped onto the surface of a silicon wafer and dried at room temperature. The distance from the last lens of the microscope to the sample (WD) ranged from 17.1 to 21.7 mm. The accelerator voltage was in the range of 20 to 40 eV.
