Controlling Mixed-Protein Adsorption Layers on Colloidal Alumina Particles by Tailoring Carboxyl and Hydroxyl Surface Group Densities Fabian Meder, Supreet Kaur, Laura Treccani, and Kurosch Rezwan* Faculty of Production Engineering, Advanced Ceramics, University of Bremen, D-28359 Bremen, Germany * S Supporting Information ABSTRACT: We show that different ratios of bovine serum albumin (BSA) and lysozyme (LSZ) can be achieved in a mixed protein adsorption layer by tailoring the amounts of carboxyl (-COOH) and aluminum hydroxyl (AlOH) groups on colloidal alumina particles (d 50 ≈ 180 nm). The particles are surface- functionalized with -COOH groups, and the resultant surface chemistry, including the remaining AlOH groups, is characterized and quantified using elemental analysis, ζ potential measurements, acid-base titration, IR spectroscopy, electron microscopy, nitrogen adsorption, and dynamic light scattering. BSA and LSZ are subsequently added to the particle suspensions, and protein adsorption is monitored by in situ ζ potential measurements while being quantified by UV spectroscopy and gel electrophoresis. A comparison of single-component and sequential protein adsorption reveals that BSA and LSZ have specific adsorption sites: BSA adsorbs primarily via AlOH groups, whereas LSZ adsorbs only via -COOH groups (1-2 -COOH groups on the particle surface is enough to bind one LSZ molecule). Tailoring such groups on the particle surface allows control of the composition of a mixed BSA and LSZ adsorption layer. The results provide further insight into how particle surface chemistry affects the composition of protein adsorption layers on colloidal particles and is valuable for the design of such particles for biotechnological and biomedical applications. ■ INTRODUCTION The deposition of specific protein layers with tailored compositions on colloidal particles is a crucial modification thereof for biotechnological and biomedical applications, e.g., when used as carriers for immunoassays, as biosensors, for cell targeting, or as enzyme carriers. 1-4 An interplay of the particle surface chemistry and protein properties governs which proteins preferentially associate with the particle surface. 5 Currently, it is unclear how particle surface chemistry selectively influences protein adsorption, particularly for linker-free, physisorption-based protein deposition, and how this surface chemistry can be exploited to deposit protein layers with tailored compositions. 6 This fundamental relationship is futhermore essential to understand the general phenomena of spontaneous and nonspecific protein adsorption onto colloidal particles that are exposed to the biological environment. 7,8 The composition of this protein layer determines the particles’ performance, selectivity, toxicity, and biocompatibility in such applications as protein purification and separation, imaging, and drug delivery. 7,9 Particle surface functionalization is a tool that influences protein-particle adsorption. Charged functional groups ex- posed on the particle surface direct the adsorption of oppositely charged proteins, and hydrophilic/hydrophobic groups guide protein-particle adsorption by interacting with water mole- cules. 10-12 However, protein adsorption behavior often differs from predictions that consider only the net charges or overall hydrophobicity. 13-16 Here, the actual molecular composition of the particle surface plays a crucial role. For instance, particles with a multifunctional surface chemistry may contain diverse functional groups with distinguishable affinities for certain proteins. 17,18 In addition, even small variations in the concentration of a specific particle functional surface group are shown to drastically change the adsorption of particular proteins. 14,19,20 Certain surface chemistries even feature a highly specific protein adsorption comparable to antigen- antibody interactions. 7,21 Although the interrelationships are complex and not completely clear, such effects might be intentionally employed. By modifying the particle surface with different functional groups for which specific proteins have an affinity, the composition of a protein layer might be controlled. A tailored particle surface functionalization and its detailed characterization are therefore mandatory. Colloidal alumina (Al 2 O 3 ) particles are an excellent model material to investigate protein-particle interactions as a function of surface chemistry. These particles can be easily surface-functionalized, and Al 2 O 3 is hydrolytically stable and bioinert. 12,22 Furthermore, Al 2 O 3 is an important, promising material in biotechnology and biomedicine as a substrate for the production of biomedical devices, protein separation and Received: June 1, 2013 Revised: July 21, 2013 Published: July 23, 2013 Article pubs.acs.org/Langmuir © 2013 American Chemical Society 12502 dx.doi.org/10.1021/la402093j | Langmuir 2013, 29, 12502-12510