# Polymer-Stabilized Silver (Gold)-Zinc Oxide Nanoheterodimer Structures as Antimicrobials
## 2 Materials and Methods
### Methods  
#### 2.3.1 Preparation of Polymer-Stabilized AgNPs, AuNPs, and Heterodimers  
Preparation of VM/Ag\({}^{0}\), EM/Ag\({}^{0}\), and SM/Ag\({}^{0}\)  
Synthesis of nanosized silver was described elsewhere [78]. Briefly: To the solution of copolymer of maleic acid (0.01 M, here the molar concentration of copolymer refers to the maleic acid units) in deionized (DI) water at pH 7 (titration with NaOH solution, 5%), a freshly prepared solution of AgNO\({}_{3}\) (0.1 M) (for the ratio of reagents 1:1 (mol.)) was added with vigorous stirring. Then, after about 5 min, a freshly prepared solution ofNaBH\({}_{4}\) (0.1 M, 2-fold molar excess with respect to silver ions) in DI water was added to the reaction solution with vigorous stirring. The reaction mixture was then stirred for 24 h at room temperature. The samples of polymer stabilized silver nanoparticles were dried using lyophilization (0.05 mbar, \(-\)55 \({}^{\circ}\)C) after ultrafiltration (YM5 membrane (\"DIAFLO\", AMICON CORPORATION, Palo Alto, CA, USA).  
Preparation of VM/Au\({}^{0}\)  
Nano-sized gold was synthesized by the reduction of gold ions at \(-\)20 \({}^{\circ}\)C with NaBH\({}_{4}\) in the presence of copolymer of maleic acid following the procedure [83]. Briefly: To the solution of the VM (0.007 M) in DI water at pH 6 (titration with water solution of NaOH, 5%), the freshly prepared solution of HAuCl\({}_{4}\cdot\)3H\({}_{2}\)O (0.01 M) (for the ratio 1:5 of reagents, respectively) was added with vigorous stirring. Then, after about ten minutes, a freshly prepared solution of NaBH\({}_{4}\) in DI water (0.1 M, in 3-fold molar excess with respect to metal ions) was added to the reaction solution with vigorous stirring. The reaction mixture was then stirred for 24 h at room temperature. The dried sample was obtained using lyophilization (0.05 mbar, \(-\)55 \({}^{\circ}\)C) after ultrafiltration (or dialysis) of the reaction solution.  
Synthesis of VM/Au\({}^{0}\)/ZnO  
A measure of 20 mL of VM solution (0.007 M, pH 8), 0.322 mL ZnSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.1 M), 2.1 mL HAuCl\({}_{4}\cdot\)3H\({}_{2}\)O (0.01 M) in DI water, and 0.4 mL NaOH (1 M) were mixed in glass vial with a screw-caps tightly closed. The pH of the reaction was 12. Then, the test vial was kept in a boiling water bath for 3 h. The molar ratio of units of maleic acid residues of copolymer to gold cations and zinc cations was 1/0.15/0.23. After cooling to room temperature, the solution was left overnight at this temperature and then was dialyzed against DI water and lyophilized (0.05 mbar, \(-\)55 \({}^{\circ}\)C).  
Synthesis of VM (EM, SM)/Ag\({}^{0}\)/ZnO  
The solutions of ZnSO\({}_{4}\cdot\)7H\({}_{2}\)O (0.262 mL, 0.1 M), VM (25 mL, 0.007 M), AgNO\({}_{3}\) (0.52 mL, 0.1 M), all in DI water, and NaOH (0.325 mL, 1 M) were mixed in glass vial with a screw-caps tightly closed. After shaking (the pH of solution was 12), the test vial was kept in a boiling water bath for 6 h. The molar ratio of units of maleic acid residues of copolymer to silver cations and zinc cations was 1/0.3/0.15. The samples EM/Ag\({}^{0}\)/ZnO and SM/Ag\({}^{0}\)/ZnO were synthesized at the same temperature and at the same molar ratio of reagents. The reaction time for these preparations was 7 h. Samples containing zinc ions were prepared with the addition of a zinc salt to the copolymer- or heterodimer-contained copolymer. The samples were dialyzed against DI water or ultrafiltered (YM5 membrane, \"DIAFLO\", Amicon Corporation, Palo Alto, CA, USA) and then lyophilized (-55 \({}^{\circ}\)C, 0.05 mbar).