Life science research has largely benefited from the use of nanoparticles (NPs), yet fundamental issues such as colloidal stability and control over NP size and shape affect NP properties and functions in biomedical applications. Here we show that including capping agents directly into zinc oxide (ZnO) NP synthesis can lead to better control of these properties and their enhanced functionality. A systematic study of the influence capping agents has on the physicochemical and antibacterial properties of ZnO NP synthesized using the microwave (MW)-assisted polyol method is presented. Primary NP size (10–20 nm) was controlled by zinc precursor concentration, and NP shape was influenced by capping agent type. Capping agents enabled control over the surface charge and water interaction properties of ZnO for further investigations involving bacteria. The superior antibacterial effect was observed using positively charged, hydrophilic HMTA-capped ZnO, yet negatively charged hydrophobic OA-capped ZnO still exhibited an antibacterial effect. These observations suggest different underlying mechanisms, and we discuss these differences with particular reference to the specific surface area of ZnO, and how this is key to bacteria-nanoparticle interactions. Appropriate selection of capping agents is crucial for the synthesis of potent ZnO NPs intended for antibacterial applications, specifically for combating resistance.European Commission, EC; TAČR, (TM03000033); US-UK Fulbright Commission, (2022-21-1, RP/CPS/2024-28/007); US-UK Fulbright Commission; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT, (CZ.02.01.01/00/22_008/0004596 - SenDiSo); Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT; České Vysoké Učení Technické v Praze, ČVUT, (SGS23/166/OHK4/3T/13); České Vysoké Učení Technické v Praze, ČVUT; Grantová Agentura České Republiky, GAČR, (24-10607J); Grantová Agentura České Republiky, GAČRTACR; European Union; Grant agency of the Czech Republic; CTU; Fulbright Commission in the Czech Republic; Ministry of Education, Youth and Sports of the Czech Republic; [TM03000033]; [CZ.02.01.01/00/22_008/0004596-SenDiSo]; [24-10607J]; [SGS23/166/OHK4/3T/13]; [2022-21-1]; [RP/CPS/2024-28/007
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