The role of ligands on protein retention in adsorption chromatography: a surface energetics approach.

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650-C, and Toyopearl Butyl 650-C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.

Adsorption and orientation of the physiological extracellular peptide glutathione disulfide on surface functionalized colloidal alumina particles.

Understanding the interrelation between surface chemistry of colloidal particles and surface adsorption of biomolecules is a crucial prerequisite for the design of materials for biotechnological and nanomedical applications. Here, we elucidate how tailoring the surface chemistry of colloidal alumina particles (d50 = 180 nm) with amino (-NH2), carboxylate (-COOH), phosphate (-PO3H2) or sulfonate (-SO3H) groups affects adsorption and orientation of the model peptide glutathione disulfide (GSSG). GSSG adsorbed on native, -NH2-functionalized, and -SO3H-functionalized alumina but not on -COOH- and -PO3H2-functionalized particles. When adsorption occurred, the process was rapid (≤5 min), reversible by application of salts, and followed a Langmuir adsorption isotherm dependent on the particle surface functionalization and ζ potential. The orientation of particle bound GSSG was assessed by the release of glutathione after reducing the GSSG disulfide bond and by ζ potential measurements. GSSG is likely to bind via the carboxylate groups of one of its two glutathionyl (GS) moieties onto native and -NH2-modified alumina, whereas GSSG is suggested to bind to -SO3H-modified alumina via the primary amino groups of both GS moieties. Thus, GSSG adsorption and orientation can be tailored by varying the molecular composition of the particle surface, demonstrating a step toward guiding interactions of biomolecules with colloidal particles.

Controlling mixed-protein adsorption layers on colloidal alumina particles by tailoring carboxyl and hydroxyl surface group densities.

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 (d50 ≈ 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.

Effective bacterial inactivation and removal of copper by porous ceramics with high surface area.

In this study, we present porous ceramics combining the antibacterial effect of copper with an integrated copper removal adsorbent. After preparing and characterizing the antibacterial copper-doped microbeads and monoliths (CuBs and CuMs), their antibacterial efficiency is probed against different nonpathogenic and pathogenic bacteria (Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa). An antibacterial efficiency of 100% is reached within 15 min to 3 h for all tested strains under static conditions. Dynamic tests with B. subtilis and E. coli showed high antibacterial efficiency up to 99.93% even at continuous flux. To avoid any adverse effects on the environment, continuous removal of released copper-ions is accomplished with porous, high surface area monolithic adsorbents (MAds). MAds are prepared similarly to the CuMs but without adding copper during the manufacturing process. MAds reduce the amount of copper released from the CuMs ≥ 99% during the first 15 min, ≥90% up to 2 h, and after 22 h of continuous filtration up to 56% of the released copper is removed.

Highly efficient enzyme-functionalized porous zirconia microtubes for bacteria filtration.

In contrast to polymer membranes, ceramic membranes offer considerable advantages for safe drinking water provision due to their excellent chemical, thermal, and mechanical endurance. In this study, porous ceramic microtubes made of yttria stabilized zirconia (YSZ) are presented, which are conditioned for bacteria filtration by immobilizing lysozyme as an antibacterial enzyme. In accordance with determined membrane pore sizes of the nonfunctionalized microtube of ≤200 nm, log reduction values (LRV) of nearly 3 (i.e., bacterial retention of 99.9%) were obtained for bacterial retention studies using gram-positive model bacterium Micrococcus luteus. Immobilization studies of lysozyme on the membrane surface reveal an up to six times higher lysozyme loading for the covalent immobilization route as compared to unspecific immobilization. Antibacterial activity of lysozyme-functionalized microtubes was assessed by qualitative agar plate test using Micrococcus luteus as substrate showing that both the unspecific and the covalent lysozyme immobilization enhance the microtubes' antibacterial properties. Quantification of the enzyme activity at flow conditions by photometric assays reveals that the enzyme activities of lysozyme-functionalized microtubes depend strongly on applied flow rates. Intracapillary feeding of bacteria solution and higher flow rates lead to reduced enzyme activities. In consideration of different applied flow rates in the range of 0.2-0.5 mL/min, the total lysozyme activity increases by a factor of 2 for the covalent immobilization route as compared to the unspecific binding. Lysozyme leaching experiments at flow conditions for 1 h show a significant higher amount of washed-out lysozyme (factor 1.7-3.4) for the unspecific immobilization route when compared to the covalent route where the initial level of antibacterial effectiveness could be achieved by reimmobilization with lysozyme. The presented platform is highly promising for sustainable bacteria filtration.

Adsorption and reduction of glutathione disulfide on α-Al2O3 nanoparticles: experiments and modeling.

Glutathione disulfide (GSSG; γ-GluCysGly disulfide) was used as a physiologically relevant model molecule to investigate the fundamental adsorption mechanisms of polypeptides onto α-alumina nanoparticles. Its adsorption/desorption behavior was studied by enzymatic quantification of the bound GSSG combined with zeta potential measurements of the particles. The adsorption of GSSG to alumina nanoparticles was rapid, was prevented by alkaline pH, was reversed by increasing ionic strength, and followed a nearly ideal Langmuir isotherm with a standard Gibbs adsorption energy of -34.7 kJ/mol. Molecular dynamics simulations suggest that only one of the two glutathionyl moieties contained in GSSG binds stably to the nanoparticle surface. This was confirmed experimentally by the release of GSH from the bound GSSG upon reducing its disulfide bond with dithiothreitol. Our data indicate that electrostatic interactions via the carboxylate groups of one of the two glutathionyl moieties of GSSG are predominantly responsible for the binding of GSSG to the alumina surface. The results and conclusions presented here can provide a base for further experimental and modeling studies on the interactions of biomolecules with ceramic materials.

Anchoring of Iron Oxyhydroxide Clusters at H and L Ferritin Subunits.

Ferritin (Fn) proteins or their isolated subunits can be used as biomolecular templates for the selectively heterogeneous nucleation and growth of nanoparticles, in particular of iron oxyhydroxides. To shed light on the atomistic mechanisms of ferritin-promoted mineralization, in this study we perform molecular dynamics simulations to investigate the anchoring sites for Fe(III) clusters on Fn subunit assemblies using models of goethite and ferrihydrite nanoparticles. For this aim, we develop and parametrize a classical force field for Fe(III) oxyhydroxides based on reference density functional theory calculations. We then reveal that stable Fn-nanoparticle contacts are formed not only via negatively charged amino acid residues (glutamic and aspartic acid) but also, in a similar amount, via positively charged (lysine and arginine) and neutral (histidine) residues. A large majority of the anchoring sites are situated at the inner side of protein cages, consistent with the natural iron storage function of ferritin in many organisms. A slightly different distribution of anchoring sites is observed on heavy (H) and light (L) Fn subunits, with the former offering a larger amount of negative and neutral sites than the latter. This finding is exploited to develop a Fn mineralization protocol in which immobilized Fn subunits are first loaded with Fe2+ ions in a long "activation" step before starting their oxidation to Fe3+. This leads to the formation of very dense and uniform iron oxide films, especially when H subunits are employed.

A New Porous Hybrid Material Derived From Silica Fume and Alginate for Sustainable Pollutants Reduction.

In this work a new mesoporous adsorbent material obtained from a natural, high abundant raw material and a high volume industrial by-product is presented. The material is consolidated by the gelling properties of alginate and by decomposition of sodium-bicarbonate controlled porosity at low temperatures (70-80°C) at different scale lengths. The structural, thermal, and morphological characterization shows that the material is a mesoporous organic-inorganic hybrid. The material is tested as adsorbent, showing high performances. Methylene blue, used as model pollutant, can be adsorbed and removed from aqueous solutions even at a high concentration with efficiency up to 94%. By coating the material with a 100 nm thin film of titania, good photodegradation performance (more than 20%) can be imparted. Based on embodied energy and carbon footprint of its primary production, the sustainability of the new obtained material is evaluated and quantified in respect to activated carbon as well. It is shown that the new proposed material has an embodied energy lower than one order of magnitude in respect to the one of activated carbon, which represents the gold standards. The versatility of the new material is also demonstrated in terms of its design and manufacturing possibilities In addition, this material can be printed in 3D. Finally, preliminary results about its ability to capture diesel exhaust particulate matter are reported. The sample exposed to diesel contains a large amount of carbon in its surface. At the best of our knowledge, this is the first time that hybrid porous materials are proposed as a new class of sustainable materials, produced to reduce pollutants in the wastewaters and in the atmosphere.

Antibacterial active open-porous hydroxyapatite/lysozyme scaffolds suitable as bone graft and depot for localised drug delivery.

An engineered synthetic scaffold for bone regeneration should provide temporary structural support and a medium for controlled and localised release of bioavailable medical drugs. In this work, a method is proposed to incorporate biologically active agents without impairing agent activity into open-porous resorbable hydroxyapatite scaffolds. Scaffolds are obtained by a one-pot freeze gelation process and loaded with different amounts of lysozyme, a model macromolecular drug with antibacterial activity. The antibacterial activity is tested by submerging hydroxyapatite scaffolds with 0.5 to 2.5 wt.% lysozyme into two different bacteria stock solutions. A complete dieback of M. luteus bacteria when in contact with the scaffolds is observed. Higher lysozyme amount in the scaffold leads to faster dieback. In contact with scaffolds containing 2.5 wt.% lysozyme after 30 min, no viable bacteria can be observed. An amount of 0.5 wt.% lysozyme in the scaffolds is sufficient to kill all bacteria after a contact time of 24 h. For L. innocua, a bacteriostatic effect is observed. The scaffolds have spongiosa-like stability and are suitable bone implant substitutes. As agents are released from the scaffolds by degrees over a time period of at least 9 days, they are particularly attractive as depot for localised drug delivery of bioactive macromolecular drugs.

Co-delivery of cisplatin and doxorubicin from calcium phosphate beads/matrix scaffolds for osteosarcoma therapy.

Bone substitute materials with a controlled drug release ability can fill cavities caused by the resection of bone tumours and thereby combat any leftover bone cancer cells. The combined release of different cytostatics seems to enhance their toxicity. In this study, calcium phosphate beads and matrix scaffolds are combined for a long-term co-delivery of cis-diamminedichloroplatinum (cisplatin, CDDP) and doxorubicin hydrochloride (DOX) as clinical relevant model drugs. Tricalcium phosphate/alginate beads as additional drug carrier are produced by droplet extrusion with ionotropic gelation and incorporated in scaffold matrix by freeze gelation without sintering. CDDP shows a short burst release while DOX has a continuous release measurable over the entire study period of 40days. Drug release from matrix is decreased by ~30% compared to release from beads. Nevertheless, all formulations follow the Korsmeyer-Peppas release kinetic model and show Fickian diffusion. Cytotoxic activity was conducted on MG-63 osteosarcoma cells after 1, 4, and 7days with WST-1 cell viability assay. Co-loaded composites enhance activity towards MG-63 cells up to ~75% toxicity while reducing the released drug quantity. The results suggest that co-loaded beads/matrix scaffolds are highly promising for osteosarcoma therapy due to synergistic effects over a long period of more than a month.

Multi-loaded ceramic beads/matrix scaffolds obtained by combining ionotropic and freeze gelation for sustained and tuneable vancomycin release.

For a targeted release against bacteria-associated bone diseases (osteomyelitis) ceramic beads with a high drug loading capacity, loaded with vancomycin as model antibiotic, are synthesized as drug carrier and successfully incorporated in an open porous hydroxyapatite matrix scaffold via freeze gelation to prevent bead migration at the implantation site and to extend drug release. We demonstrate that the quantity of loaded drug by the hydroxyapatite and ß-tricalcium phosphate beads, produced by ionotropic gelation, as well as drug release can be tuned and controlled by the selected calcium phosphate powder, sintering temperature, and high initial vancomycin concentrations (100mg/ml) used for loading. Bead pore volume up to 68mm(3)/g, with sufficiently large open pores (pore size of up to 650nm with open porosity of 72%) and high surface area (91m(2)/g) account likewise for a maximum drug loading of 236mg/g beads or 26mg/sample. Multi-drug loading of the beads/matrix composite can further increase the maximum loadable amount of vancomycin to 37mg/sample and prolong release and antibacterial activity on Bacillus subtilis up to 5days. The results confirmed that our approach to incorporate ceramic beads as drug carrier for highly increased drug load in freeze-gelated matrix scaffolds is feasible and may lead to a sustained drug release and antibacterial activity.

Physisorption of α-chymotrypsin on SiO2 and TiO2: A comparative study via experiments and molecular dynamics simulations.

In order to understand fundamental interactions at the interface between immobilized enzymes and ceramic supports, the authors compare the adsorption features of chymotrypsin on SiO2 and TiO2 colloidal particles by means of a combination of adsorption experiments and molecular dynamics simulations. While the dependency of the adsorption amount on pH is consistent with the trend predicted the Derjaguin-Landau-Verwey-Overbeek theory, other effects can only be rationalized if the atomic-scale details of the water-mediated protein-surface interactions are considered. On both surfaces, a clear driving force for the formation of a double monolayer at the saturation coverage is found. Although nearly equal free energies of adsorption are estimated on the two materials via a Langmuir adsorption analysis, about 50% more proteins per unit of surface can be accommodated on TiO2 than on SiO2. This is probably due to the lower surface diffusion mobility of the adsorbed protein in the latter case. Surface anchoring is realized by a combination of direct ionic interactions between charged proteins and surface sites (more pronounced for SiO2) and distinct structuring of the surface hydration layers in which the contact residues are embedded (more pronounced for TiO2). Finally, normalization of the data with respect to particle surface areas accessible to the proteins, rather than determined by means of the Brunauer-Emmett-Teller nitrogen adsorption isotherm, is crucial for a correct interpretation of the results.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials.

Utilizing the protein corona around silica nanoparticles for dual drug loading and release.

A protein corona forms spontaneously around silica nanoparticles (SNPs) in serum-containing media. To test whether this protein corona can be utilized for the loading and release of anticancer drugs we incorporated the hydrophilic doxorubicin, the hydrophobic meloxicam as well as their combination in the corona around SNPs. The application of corona-covered SNPs to osteosarcoma cells revealed that drug-free particles did not affect the cell viability. In contrast, SNPs carrying a protein corona with doxorubicin or meloxicam lowered the cell proliferation in a concentration-dependent manner. In addition, these particles had an even greater antiproliferative potential than the respective concentrations of free drugs. The best antiproliferative effects were observed for SNPs containing both doxorubicin and meloxicam in their corona. Co-localization studies revealed the presence of doxorubicin fluorescence in the nucleus and lysosomes of cells exposed to doxorubicin-containing coated SNPs, suggesting that endocytotic uptake of the SNPs facilitates the cellular accumulation of the drug. Our data demonstrate that the protein corona, which spontaneously forms around nanoparticles, can be efficiently exploited for loading the particles with multiple drugs for therapeutic purposes. As drugs are efficiently released from such particles they may have a great potential for nanomedical applications.

Effect of silica on porosity, strength, and toughness of pressureless sintered calcium phosphate-zirconia bioceramics.

The preparation of dense, high-strength calcium phosphate-zirconia (CaP-ZrO2) composed bioceramics is realized via versatile pressureless sintering by adding silica nanoparticles. Two different weight ratios of HAp:ZrO2, 9:1 and 1:1, are used with varying silica contents from 5 to 20 wt%. After sintering at 1200 °C, the phase composition, microstructure, porosity, biaxial bending strength, and fracture toughness as well as SBF in vitro bioactivity are characterized. We show that the addition of silica altered the crystal phase composition, inhibiting the formation of non-favourable cubic ZrO2. Furthermore, SiO2 addition leads to an increase of the biaxial bending strength, and the fracture toughness of CaP-ZrO2-containing materials. With the addition of 20 wt% silica we find the highest characteristic strength (268 MPa) and toughness (2.3 ± 0.1 MPam(0.5)) at <1% porosity. Both mechanical properties are 2 times higher than those of pure hydroxyapatite. At the same time we observe for the very same composition similar bioactivity to that of pure hydroxyapatite.

Dual fluorophore doped silica nanoparticles for cellular localization studies in multiple stained cells.

Fluorescently labeled nanoparticles (NPs) are used in a wide range of biomedical and nanotoxicological studies to elucidate their interactions with cellular components and their intracellular localization. As commonly used fluorescence microscopes are usually limited in their performance to a few channels which detect the emitted fluorescence light in the red, green and blue color range, the simultaneous colocalization of accumulated fluorescent NPs with cellular markers is often difficult and remains a challenge due to spectral overlay of NP fluorescence and fluorescence of stained cellular components. To overcome this problem we have synthesized three different photostable dual-labeled fluorescent core/shell silica NPs with high fluorescence intensity and well-defined shape, size and surface chemistry. The synthesis route of dual fluorophore doped silica (DFDS) NPs was based on a water-in-oil microemulsion method and includes the separate incorporation of two fluorophores in the core or shell. The suitability of DFDS for colocalization studies was assessed and successfully demonstrated with human osteoblast cells. Parallel visualization of DFDS NPs with two separate microscope channels allowed cellular NP uptake and discrimination from fluorescently stained cellular components, even in triple stained cells that show fluorescence for the cytoskeleton protein actin (green), the nucleus (blue) and collagen (red). Our results demonstrate the feasibility and straightforwardness of the approach for colocalization studies at a single-cell level to discern clearly the accumulation of NPs from triple-stained cellular components. Such NPs with multiple fluorescence characteristics have a great potential to replace single fluorescent NPs for in vitro studies, when multiple staining of cellular components is required.

Modulation of Silica Nanoparticle Uptake into Human Osteoblast Cells by Variation of the Ratio of Amino and Sulfonate Surface Groups: Effects of Serum.

To study the importance of the surface charge for cellular uptake of silica nanoparticles (NPs), we synthesized five different single- or multifunctionalized fluorescent silica NPs (FFSNPs) by introducing various ratios of amino and sulfonate groups into their surface. The zeta potential values of these FFSNPs were customized from highly positive to highly negative, while other physicochemical properties remained almost constant. Irrespective of the original surface charge, serum proteins adsorbed onto the surface, neutralized the zeta potential values, and prevented the aggregation of the tailor-made FFSNPs. Depending on the surface charge and on the absence or presence of serum, two opposite trends were found concerning the cellular uptake of FFSNPs. In the absence of serum, positively charged NPs were more strongly accumulated by human osteoblast (HOB) cells than negatively charged NPs. In contrast, in serum-containing medium, anionic FFSNPs were internalized by HOB cells more strongly, despite the similar size and surface charge of all types of protein-covered FFSNPs. Thus, at physiological condition, when the presence of proteins is inevitable, sulfonate-functionalized silica NPs are the favorite choice to achieve a desired high rate of NP internalization.

Enhancing Cellular Uptake and Doxorubicin Delivery of Mesoporous Silica Nanoparticles via Surface Functionalization: Effects of Serum.

In this study, we demonstrate how functional groups on the surface of mesoporous silica nanoparticles (MSNPs) can influence the encapsulation and release of the anticancer drug doxorubicin, as well as cancer cell response in the absence or presence of serum proteins. To this end, we synthesized four differently functionalized MSNPs with amine, sulfonate, polyethylene glycol, or polyethylene imine functional surface groups, as well as one type of antibody-conjugated MSNP for specific cellular targeting, and we characterized these MSNPs regarding their physicochemical properties, colloidal stability in physiological media, and uptake and release of doxorubicin in vitro. Then, the MSNPs were investigated for their cytotoxic potential on cancer cells. Cationic MSNPs could not be loaded with doxorubicin and did therefore not show any cytotoxic and antiproliferative potential on osteosarcoma cells, although they were efficiently taken up into the cells in the presence or absence of serum. In contrast, substantial amounts of doxorubicin were loaded into negatively charged and unfunctionalized MSNPs. Especially, sulfonate-functionalized doxorubicin-loaded MSNPs were efficiently taken up into the cells in the presence of serum and showed an accelerated toxic and antiproliferative potential compared to unfunctionalized MSNPs, antibody-conjugated MSNPs, and even free doxorubicin. These findings stress the high importance of the surface charge as well as of the protein corona for designing and applying nanoparticles for targeted drug delivery.

Enzyme-assisted calcium phosphate biomineralization on an inert alumina surface.

In this study a bioinspired approach to induce self-mineralization of bone-like material on alumina surfaces is presented. The mineralizing enzyme alkaline phosphatase (ALP) is covalently immobilized by a carbodiimide-mediated chemoligation method. The enzymatic activity of immobilized ALP and its mineralization capability are investigated under acellular conditions as well as in the presence of human bone cells. Analytical, biochemical and immunohistochemical characterization show that ALP is efficiently immobilized, retains its activity and can trigger calcium phosphate mineralization on alumina at acellular conditions. In vitro cell tests demonstrate that ALP-functionalized alumina clearly boosts and enhances bone cell mineralization. Our results underpin the great potential of ALP-functionalized alumina for the development of bioactive surfaces for applications such as orthopaedic and dental implants, enabling a fast and firm implant osseointegration.

Physisorption of enzymatically active chymotrypsin on titania colloidal particles.

In this study we use a straightforward experimental method to probe the presence and activity of the proteolytic enzyme α-chymotrypsin adsorbed on titania colloidal particles. We show that the adsorption of α-chymotrypsin on the particles is irreversible and pH-dependent. At pH 8 the amount of adsorbed chymotrypsin is threefold higher compared to the adsorption at pH 5. However, we observe that the adsorption is accompanied by a substantial loss of enzymatic activity, and only around 6-9% of the initial enzyme activity is retained. A Michaelis-Menten kinetics analysis of both unbound and TiO2-bound chymotrypsin shows that the K(M) value is increased from ∼10 µM for free chymotrypsin to ∼40 µM for the particle bound enzyme. Such activity decrease could be related by the hindered accessibility of substrate to the active site of adsorbed chymotrypsin, or by adsorption-induced structural changes. Our simple experimental method does not require any complex technical equipment, can be applied to a broad range of hydrolytic enzymes and to various types of colloidal materials. Our approach allows an easy, fast and reliable determination of particle surface-bound enzyme activity and has high potential for development of future enzyme-based biotechnological and industrial processes.