Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the bacterial bond with a substratum and play important roles in bacterial adhesion, detachment and susceptibility to antimicrobials.

Nanoscale cell wall deformation impacts long-range bacterial adhesion forces on surfaces.

Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of different short- and long-range forces. Here we present a new atomic force microscopy (AFM)-based method to derive long-range bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading force, as applied during the use of AFM. The long-range adhesion forces of wild-type Staphylococcus aureus parent strains (0.5 and 0.8 nN) amounted to only one-third of these forces measured for their more deformable isogenic Δpbp4 mutants that were deficient in peptidoglycan cross-linking. The measured long-range Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed for the Δpbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak force-quantitative nanomechanical property mapping imaging mode confirmed a height reduction due to deformation in the Δpbp4 mutants of 100 to 200 nm. Across naturally occurring bacterial strains, long-range forces do not vary to the extent observed here for the Δpbp4 mutants. Importantly, however, extrapolating from the results of this study, it can be concluded that long-range bacterial adhesion forces are determined not only by the composition and structure of the bacterial cell surface but also by a hitherto neglected, small deformation of the bacterial cell wall, facilitating an increase in contact area and, therewith, in adhesion force.

Viscous nature of the bond between adhering bacteria and substratum surfaces probed by atomic force microscopy.

Here we report on the viscous nature of the bond between adhering bacteria and a substratum surface. A tailor-made script was written for an atomic force microscope, that enabled a constant loading force of 1 or 5 nN to act for 30 s upon a bacterium compressed between a cantilever and a glass surface, while measuring its deformation. Time-dependent deformation was fitted to a one element Kelvin-Voigt analogue of the bond to yield a characteristic relaxation time and viscosity of the bond. Viscosities of streptococcal bonds were smaller (<20 kPa s) than those of staphylococcal bonds (>31 kPa s). Since staphylococci are relatively rich in extracellular polymeric substances, it can be inferred that the presence of extracellular polymeric substances yields the major contribution to the viscous response. The viscous nature of the bond between adhering bacteria and substratum surfaces provides the bacteria with more time to respond and protect themselves against external stresses.

Formation and characterization of light-responsive TEMPO-oxidized konjac glucomannan microspheres.

A light-responsive delivery system has been developed. It consists of gelly microspheres made of TEMPO-oxidized Konjac glucomannan (OKGM) polymers where the carboxyl (COO(-)) groups are cross-linked via ferric ions (Fe(3+)) and in which functional ingredients may be incorporated. By irradiation with (simulated) sunlight, the microspheres degrade, thereby releasing the encapsulated component(s). The degree of oxidation (DO) of the OKGM polymers could be well-controlled between 15 and 80%, as confirmed by proton titrations and FT-IR spectroscopy. OKGM of DO 80% was selected to prepare the microspheres because the high COO(-) content leads to a high density of cross-links, yielding a strong gel. The electrokinetic potential of the OKGM particles increases with increasing pH and decreasing salt concentration. Mössbauer and FT-IR spectroscopy revealed that the cross-links are formed through two modes of COO(-)-Fe(3+) coordination, that is, 68.4% by bridging and 31.6% by unidentate binding. Thus, the unique properties of the OKGM microspheres make them potentially applicable as light-controlled biocompatible delivery systems.

Antiadhesive polymer brush coating functionalized with antimicrobial and RGD peptides to reduce biofilm formation and enhance tissue integration.

This paper describes the synthesis and characterization of polymer-peptide conjugates to be used as infection-resistant coating for biomaterial implants and devices. Antiadhesive polymer brushes composed of block copolymer Pluronic F-127 (PF127) were functionalized with antimicrobial peptides (AMP), able to kill bacteria on contact, and arginine-glycine-aspartate (RGD) peptides to promote the adhesion and spreading of host tissue cells. The antiadhesive and antibacterial properties of the coating were investigated with three bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa. The ability of the coating to support mammalian cell growth was determined using human fibroblast cells. Coatings composed of the appropriate ratio of the functional components: PF127, PF127 modified with AMP, and PF127 modified with RGD showed good antiadhesive and bactericidal properties without hampering tissue compatibility.

Residence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using surface-enhanced-fluorescence.

Bacterial adhesion to surfaces is accompanied by cell wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Nanoscale cell wall deformation upon adhesion is difficult to measure, except for Δpbp4 mutants, deficient in peptidoglycan cross-linking. This work explores surface enhanced fluorescence to measure the cell wall deformation of Staphylococci adhering on gold surfaces. Adhesion-related fluorescence enhancement depends on the distance of the bacteria from the surface and the residence-time of the adhering bacteria. A model is forwarded based on the adhesion-related fluorescence enhancement of green-fluorescent microspheres, through which the distance to the surface and cell wall deformation of adhering bacteria can be calculated from their residence-time dependent adhesion-related fluorescence enhancement. The distances between adhering bacteria and a surface, including compression of their extracellular polymeric substance (EPS)-layer, decrease up to 60 min after adhesion, followed by cell wall deformation. Cell wall deformation is independent of the integrity of the EPS-layer and proceeds fastest for a Δpbp4 strain.

Distribution of biomolecules in porous nitrocellulose membrane pads using confocal laser scanning microscopy and high-speed cameras.

The main focus of our research was to study the distribution of inkjet printed biomolecules in porous nitrocellulose membrane pads of different brands. We produced microarrays of fluorophore-labeled IgG and bovine serum albumin (BSA) on FAST, Unisart, and Oncyte-Avid slides and compared the spot morphology of the inkjet printed biomolecules. The distribution of these biomolecules within the spot embedded in the nitrocellulose membrane was analyzed by confocal laser scanning microscopy in the "Z" stack mode. By applying a "concentric ring" format, the distribution profile of the fluorescence intensity in each horizontal slice was measured and represented in a graphical color-coded way. Furthermore, a one-step diagnostic antibody assay was performed with a primary antibody, double-labeled amplicons, and fluorophore-labeled streptavidin in order to study the functionality and distribution of the immune complex in the nitrocellulose membrane slides. Under the conditions applied, the spot morphology and distribution of the primary labeled biomolecules was nonhomogenous and doughnut-like on the FAST and Unisart nitrocellulose slides, whereas a better spot morphology with more homogeneously distributed biomolecules was observed on the Oncyte-Avid slide. Similar morphologies and distribution patterns were observed when the diagnostic one-step nucleic acid microarray immunoassay was performed on these nitrocellulose slides. We also investigated possible reasons for the differences in the observed spot morphology by monitoring the dynamic behavior of a liquid droplet on and in these nitrocellulose slides. Using high speed cameras, we analyzed the wettability and fluid flow dynamics of a droplet on the various nitrocellulose substrates. The spreading of the liquid droplet was comparable for the FAST and Unisart slides but different, i.e., slower, for the Oncyte-Avid slide. The results of the spreading of the droplet and the penetration behavior of the liquid in the nitrocellulose membrane may (partly) explain the distribution of the biomolecules in the different slides. To our knowledge, this is the first time that fluid dynamics in diagnostic membranes have been analyzed by the use of high-speed cameras.

(Quasi-) 2D aggregation of polystyrene-b-dextran at the air-water interface.

Polystyrene-b-dextran (PS-b-Dextran) copolymers can be used to prepare dextran brushes at solid surfaces, applying Langmuir-Blodgett deposition. When recording the interfacial pressure versus area isotherms of a PS-b-Dextran monolayer, time-dependent hysteresis was observed upon compression and expansion. We argue that this is due to (quasi-) 2D aggregation of the copolymer at the air-water surface, with three contributions. First, at large area per molecule, a zero surface pressure is measured; we ascribe this to self-assembly of block copolymers into surface micelles. At intermediate area we identify a second regime ("desorption regime") where aggregation into large patches occurs due to van der Waals attraction between PS blocks. At high surface pressure ("brush regime") we observe hysteretic behavior attributed to H-bonding between dextran chains. When compared to hysteresis of other amphiphilic diblock copolymers (also containing PS, e.g., polystyrene-b-poly(ethylene oxide)) a general criterion can be formulated concerning the extent of hysteresis: when the hydrophobic (PS) block is of equal size as (or bigger than) the hydrophilic block, the hysteresis is maximal. The (quasi-) 2D aggregation of PS-b-Dextran has significant implications for the preparation of dextran brushes at solid surfaces using Langmuir-Blodgett deposition. For each grafting density the monolayer needs to relax, up to several hours, prior to transfer.

Spot morphology of non-contact printed protein molecules on non-porous substrates with a range of hydrophobicities.

Non-contact inkjet printing technology is one of the most promising tools for producing microarrays. The quality of the microarray depends on the type of the substrate used for printing biomolecules. Various porous and non-porous substrates have been used in the past, but due to low production cost and easy availability, non-porous substrates like glass and plastic are preferred over porous substrates. On these non-porous substrates, obtaining spot uniformity and a high signal to noise ratio is a big challenge. In our research work, we have modified pristine glass slides using various silanes to produce a range of hydrophobic glass substrates. The hydrophobicities of the slides expressed in the contact angle (θ) of a sessile drop of water were 49°, 61°, 75°, 88° and 103°. Using a non-contact inkjet printer, microarrays of biotinylated biomolecules (BSA and IgG) were produced on these modified glass substrates, pristine (untreated) glass and also on HTA polystyrene slides. The uniformity of the spots, reflecting the distribution of the biomolecules in the spots, was analyzed and compared using confocal laser scanning microscopy (CLSM). The quality of the spots was superior on the glass slide with a contact angle of ∼75°. We also investigated the influence of the hydrophobicity of the substrate on a two-step, real diagnostic antibody assay. This nucleic acid microarray immunoassay (NAMIA) for the detection of Staphylococcus aureus showed that on highly hydrophilic (θ < 10°) and hydrophobic substrates (θ > 100°) the assay signal was low, whereas an excellent signal was obtained on the substrates with intermediate contact angles, θ ∼ 61° and θ ∼ 75°, respectively.

Influence of soil humic and fulvic acid on the activity and stability of lysozyme and urease.

Humic substances (HS), including humic acids (HA) and fulvic acids (FA), are important components of soil systems. HS form strong complexes with oppositely charged proteins, which will lead to changes in the enzyme activity. The effect of soil HS on the activity and stability of two enzymes was investigated as a function of pH, ionic strength, and mass ratio HS/enzyme. Humic acid (JGHA) and fulvic acid (JGFA) are negatively charged, lysozyme is net positive at pH values below 10.4, and urease is net positive below pH 5.2 or net negative above pH 5.2. The enzyme activities in the HS-enzyme complexes were suppressed when the enzymes were oppositely charged to the HS. The largest activity suppression was observed around the mass ratio HS/enzyme where the HS-protein complex was at its isoelectric point (IEP). At the IEP strong aggregation of the complexes led to encapsulation of the enzyme. The ionic strength was important; an increase decreased complex formation, but increased aggregation. Due to the larger hydrophobicity of JGHA than JGFA, the reduction in enzyme activity was stronger for JGHA. The enzyme stability also decreased maximally at mass ratio around the IEP of the complex when HS and protein were oppositely charged. When urease and HS were both negatively charged no complexes were formed, but the presence of JGHA or JGFA improved the activity and stability of the enzyme.

Rapid mastitis detection assay on porous nitrocellulose membrane slides.

We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.

Bacterial adhesion forces with substratum surfaces and the susceptibility of biofilms to antibiotics.

Biofilms causing biomaterial-associated infection resist antibiotic treatment and usually necessitate the replacement of infected implants. Here we relate bacterial adhesion forces and the antibiotic susceptibility of biofilms on uncoated and polymer brush-coated silicone rubber. Nine strains of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa adhered more weakly to brush-coated silicone rubber (-0.05 ± 0.03 to -0.51 ± 0.62 nN) than to uncoated silicone rubber (-1.05 ± 0.46 to -5.1 ± 1.3 nN). Biofilms of weakly adhering organisms on polymer brush coatings remained in a planktonic state, susceptible to gentamicin, unlike biofilms formed on uncoated silicone rubber.

Stabilization of protein-loaded starch microgel by polyelectrolytes.

The interaction of biocompatible polyelectrolytes (chargeable poly(amino acids)) with oxidized starch microgel particles has been studied. The aim was to form a polyelectrolyte complex layer around the outer shell of microgel particles filled with functional ingredients to slow down the release of the ingredients from the gel and make this process less sensitive to salt. First, the distribution of positively charged poly(l-lysine) (PLL) of two different molecular weights ("small", 15-30 kDa, and "large", 30-70 kDa) in the negatively charged gel particles was measured. The small PLL distributes homogeneously throughout the gel particles, but the large PLL forms a shell; i.e., its concentration at the outer layer of the particles was found to be much higher than in their core. This shell formation does not occur at a relatively high salt concentration (0.07 M). The large PLL was selected for further study. It was found that upon addition of PLL to lysozyme-loaded gel particles the protein is exchanged by PLL. The exchange rate increases with increasing pH, in line with the increasing electrostatic attraction between the gel and the polyelectrolyte. Therefore, it was decided to use also a negatively charged poly(amino acid), poly(L-glutamic acid) (PGA), to form together with PLL a stable polyelectrolyte complex shell around the gel particles. This approach turned out to be successful, and the PLL/PGA complex layer effectively slows down the release of lysozyme from the microgel particles at 0.05 M salt. In addition, it was found that the PLL/PGA layer protects the gel particle from degradation by α-amylase.

Nanopatterned submicron pores as a shield for nonspecific binding in surface plasmon resonance-based sensing.

We present a novel approach to tackle the most common drawback of using surface plasmon resonance for analyte screening in complex biological matrices--the nonspecific binding to the sensor chip surface. By using a perforated membrane supported by a polymeric gel structure at the evanescent wave penetration depth, we have fabricated a non-fouling sieve above the sensing region. The sieve shields the evanescent wave from nonspecific interactions which interfere with SPR sensing by minimizing the fouled area of the polymeric gel and preventing the translocation of large particles, e.g. micelles or aggregates. The nanopatterned macropores were fabricated by means of colloidal lithography and plasma enhanced chemical vapor deposition of a polyethylene oxide-like film on top of a polymeric gel matrix commonly used in surface plasmon resonance analysis. The sieve was characterized using surface plasmon resonance imaging, contact angle, atomic force microscopy and scanning electron microscopy. The performance of the sieve was studied using an immunoassay for detection of antibiotic residues in full fat milk and porcine serum. The non-fouling membrane presented pores in the 92-138 nm range organized in a hexagonal crystal lattice with a clearance of about 5% of the total surface. Functionally, the membrane with the nanopatterned macropores showed significant improvements in immunoassay robustness and sensitivity in untreated complex samples. The utilization of the sensor built-in sieve for measurements in complex matrices offers reduction in pre-analytical sample preparation steps and thus shortens the total analysis time.

Statistical analysis of long- and short-range forces involved in bacterial adhesion to substratum surfaces as measured using atomic force microscopy.

Surface thermodynamic analyses of microbial adhesion using measured contact angles on solid substrata and microbial cell surfaces are widely employed to determine the nature of the adhesion forces, i.e., the interplay between Lifshitz-van der Waals and acid-base forces. While surface thermodynamic analyses are often viewed critically, atomic force microscopy (AFM) can also provide information on the nature of the adhesion forces by means of Poisson analysis of the measured forces. This review first presents a description of Poisson analysis and its underlying assumptions. The data available from the literature for different combinations of bacterial strains and substrata are then summarized, leading to the conclusion that bacterial adhesion to surfaces is generally dominated by short-range, attractive acid-base interactions, in combination with long-range, weaker Lifshitz-van der Waals forces. This is in line with the findings of surface thermodynamic analyses of bacterial adhesion. Comparison with single-molecule ligand-receptor forces from the literature suggests that the short-range-force contribution from Poisson analysis involves a discrete adhesive bacterial cell surface site rather than a single molecular force. The adhesion force arising from these cell surface sites and the number of sites available may differ from strain to strain. Force spectroscopy, however, involves the tedious task of identifying the minor peaks in the AFM retraction force-distance curve. This step can be avoided by carrying out Poisson analysis on the work of adhesion, which can also be derived from retraction force-distance curves. This newly proposed way of performing Poisson analysis confirms that multiple molecular bonds, rather than a single molecular bond, contribute to a discrete adhesive bacterial cell surface site.

Chemodynamic features of nanoparticles: Application to understanding the dynamic life cycle of SARS-CoV-2 in aerosols and aqueous biointerfacial zones.

We review concepts involved in describing the chemodynamic features of nanoparticles and apply the framework to gain physicochemical insights into interactions between SARS-CoV-2 virions and airborne particulate matter (PM). Our analysis is highly pertinent given that the World Health Organisation acknowledges that SARS-CoV-2 may be transmitted by respiratory droplets, and the US Center for Disease Control and Prevention recognises that airborne transmission of SARS-CoV-2 can occur. In our theoretical treatment, the virion is assimilated to a core-shell nanoparticle, and contributions of various interaction energies to the virion-PM association (electrostatic, hydrophobic, London-van der Waals, etc.) are generically included. We review the limited available literature on the physicochemical features of the SARS-CoV-2 virion and identify knowledge gaps. Despite the lack of quantitative data, our conceptual framework qualitatively predicts that virion-PM entities are largely able to maintain equilibrium on the timescale of their diffusion towards the host cell surface. Comparison of the relevant mass transport coefficients reveals that virion biointernalization demand by alveolar host cells may be greater than the diffusive supply. Under such conditions both the free and PM-sorbed virions may contribute to the transmitted dose. This result points to the potential for PM to serve as a shuttle for delivery of virions to host cell targets. Thus, our critical review reveals that the chemodynamics of virion-PM interactions may play a crucial role in the transmission of COVID-19, and provides a sound basis for explaining reported correlations between episodes of air pollution and outbreaks of COVID-19.

Food allergens profiling with an imaging surface plasmon resonance-based biosensor.

Food allergy is a growing health concern, which currently affects approximately 4% of adults and 8% of infants. For consumer protection purposes, food producers are required by law to disclose on the product label whether a major allergen is used during the production process. The commonly employed monitoring methods are highly laborious, time-consuming, and often expensive when screening for multiple allergens. Here, we utilize imaging surface plasmon resonance (iSPR) in combination with antibody array for rapid, quantitative, and multianalyte food allergens detection. We demonstrate how the use of this technology provides a complete allergen profile within short measurement time and with adequate sensitivity. The successful applicability of this approach is demonstrated by analyzing cookies and dark chocolate products from different manufacturers. Hazelnut content of the tested food products is also determined by enzyme linked immunosorbent assay and is found to correlate well with the hazelnut content determined by iSPR. This newly developed method opens the door to automated and high-throughput allergen analysis, ultimately aiming at providing the consumer with safer food.

Effects of polyelectrolyte complex micelles and their components on the enzymatic activity of lipase.

The enzymatic activity of Hl-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP(41)-PEO(205)) and poly(acrylic acid)(PAA(139)) is studied as a function of the PAA(139) + P2MVP(41)-PEO(205) complex composition. The measurements revealed that there are several factors that influence the enzymatic activity. When incorporated in micelles, the activity of lipase is increased, which suggests that the micelles favor the active state. The activity may further increase because the substrate tends to accumulate to the micelles. It is found that the presence of PAA(139) alone also increases the enzymatic activity somewhat. Increasing of the ionic strength decreases the enzymatic activity in all systems. However, at ionic strengths where the micelles are disintegrated (>0.5 M), the activity of lipase in the presence of both polyelectrolytes is still higher than the activity of free lipase. At 0.7 M NaCl it was found that lipase in the presence of (just) P2MVP(41)-PEO(205) is more active than lipase without this additive.

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with ß-lactoglobulin.

The complex formation between ß-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between ß-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the ß-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on ß-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.

Lysozyme uptake by oxidized starch polymer microgels.

With the aim of determining suitable conditions for uptake and release of globular proteins on microgels, we studied the interaction between phosphated, highly cross-linked, negatively charged oxidized potato starch polymer (OPSP) microgel particles and lysozyme from hen eggs. Our microgel shows a typical protein-induced deswelling behavior for charged microgels. The protein distributes rather homogenously through the microgel. We found that at low salt concentration the saturation protein uptake Gammasat increases with increasing pH. This is because the binding capacity is mainly determined by charge compensation: with increasing pH, the (positive) charge on the lysozyme molecules decreases, while the (negative) charge of the microgel particles increases. Therefore, more protein molecules are needed to compensate for the charge on the gel and the binding capacity increases. The protein binding affinity, however, decreases sharply with increasing pH, presumably because this affinity is mainly sensitive to the lysozyme charge density. At high pH the binding affinity is relatively low, and by adding salt, the protein can easily be released from the gel. This leads to a maximum in the curves of Gammasat versus pH, and this maximum shifts to lower pH values with increasing ionic strength. We conclude that, for protein uptake and release applications, the present system works best around pH 5 due to a sufficiently high binding affinity and a sufficiently high binding capacity.