Bovine Serum Albumin Monomer Research Articles

Integrated system for temperature-controlled fast protein liquid chromatography. IV. Continuous ‘one-column’ ‘low-salt’ hydrophobic interaction chromatography

Systematic development of a temperature-controlled isocratic process for one-column low-salt hydrophobic interaction chromatography (HIC) of proteins employing a travelling cooling zone reactor (TCZR) system, is described. Batch binding and confocal scanning microscopy were employed to define process conditions for temperature-reversible binding of bovine serum albumin (BSA) which were validated in pulse-response temperature switching HIC experiments, before transferring to TCZR-HIC. A thin-walled stainless-steel column mounted with a movable assembly of copper blocks and Peltier elements (travelling cooling zone, TCZ) was used for TCZR-HIC. In pulse-response TCZR-HIC, 12 TCZ movements along the column desorbed 86.3% of the applied BSA monomers in 95.3% purity depleted >6-fold in 2–4 mers and nearly 260-fold in higher molecular weight (HMW) species. For continuous TCZR-HIC, the TCZ was moved 49–58 times during uninterrupted loading of BSA feeds at 0.25, 0.5 or 1 mg·mL-1. Each TCZ movement generated a sharp symmetrical elution peak. In the best case, (condition 1: 0.25 mg·mL-1 BSA; >17 mg BSA applied per mL of bed) the height of TCZ elution peaks approached pseudo-steady midway through the loading phase with no rise in baseline UV280 signal between peaks. Peak composition remained constant averaging 94.4% monomer, 5.6% 2–4 mers and <0.05% HMW. Monomers were recovered in quantitative yield depleted >3.1 fold in 2–4 mers and 92-fold in HMW species cf. the feed (63.6% monomers, 21.8% 2–4 mers, 14.6% HMW). However, increasing the BSA concentration to 1 mg·mL-1 (condition 2) or employing a fouled HIC column with 0.5 mg·mL-1 BSA (condition 3) compromised monomer purification performance.

Multiphase reactions of proteins in the air: Oligomerization, nitration and degradation of bovine serum albumin upon ambient exposure

Proteins in atmospheric aerosol can react with atmospheric pollutants such as ozone (O3) and nitrogen dioxide (NO2) in the atmosphere via the reactions of oxidation, nitration, and cross-linking etc. Currently, the reactions have been more thoroughly studied in the laboratory but rarely investigated in the ambient environment. In this study, we used bovine serum albumin (BSA) as the model protein to conduct the exposure experiment in the ambient environment in southern China, an area with increasing oxidative capacity, to investigate the reactions of proteins in the atmosphere. We observed the occurrence of oligomerization, nitration and degradation of BSA upon exposure. The mass fraction of BSA monomer decreased by 5.86 ± 1.61% after exposure and those of dimers, trimers and higher oligomers increased by 1.04 ± 0.49%, 1.37 ± 0.74% and 3.40 ± 1.06%, respectively. Simultaneously, the nitration degrees of monomers, dimers, trimers and higher oligomers increased by 0.42 ± 0.15%, 0.53 ± 0.15%, 0.55 ± 0.28% and 2.15 ± 1.01%, respectively. The results show that oligomerization was significantly affected by O3 and temperature and nitration was jointly affected by O3, temperature and relative humidity, indicating the important role of atmospheric oxidants in the atmospheric reactions of protein. Atmospheric degradation of BSA was observed with the release of free amino acids (FAAs) such as glycine, alanine, serine and methionine. Glycine was the dominant FAA with a molar yield ranging from ∼8% to 33% for BSA. The estimated stoichiometric coefficient (α) of glycine is 10−7–10−6 for the degradation of BSA upon O3. Our observation suggests the occurrence of protein reactions in the oxidative ambient environment, leading to the production of nitrated products, oligomers and low molecular weight products such as peptides and FAAs. This study may deepen the current understanding of the atmospheric reaction mechanisms and reveal the influence of environmental factors in the atmosphere.

The effect of fatty acids, ionic strength, and electric fields on the microscopic dynamics of BSA aggregates

This paper presents the microscopic dynamics of the concentrated suspensions of bovine serum albumin (BSA) proteins and their aggregates by dynamic light scattering (DLS) experiments. The effects of fatty acids binding to BSA, as well as the ionic strength and weak electric field, are discussed for affecting the stability of BSA suspensions against calcium-induced aggregation. By variation of the ionic strength, in the absence of an external electric field, DLS experiments show that monomer–BSA interactions (in the essentially fatty acid-free case) are overall repulsive but that, nevertheless, aggregation occurs to some extent. Also, the diffusive properties of different types of BSA are explored under an applied low-AC electric field by means of in situ electric small-angle depolarized DLS experiments, which reveal a significant decrease of the translational BSA–monomer diffusion coefficient with increasing frequency, while the aggregates indicate orientational motion via rotation on applying an electric field. These observations are interpreted in terms of (localized) orientation interactions obtained as oscillations in the intermediate scattering correlation function, as well as the anomalous slower relaxations as resulting in effective (collective) dynamics between monomeric BSA and their protein aggregates.

Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part II. Mechanisms of protein aggregation on the chromatographic surface

AbstractBACKGROUNDProteins on chromatographic surfaces can undergo conformational changes, leading to aggregate formation. Such effects have been reported previously for hydrophobic interaction and cation exchange chromatography. In Part I of this work, we examined protein adsorption behavior on monomodal (Nuvia HP‐Q) and multimodal anion exchangers (Nuvia aPrime and Capto Adhere), observing that aggregates are also formed on these multimodal resins. In this work we examine the underlying mechanisms and the conditions that affect aggregate formation.RESULTSIncubating the multimodal anion exchangers with pure bovine serum albumin monomer at low ionic strength results in the formation of dimer and higher‐order oligomers (up to hexamers) to an extent that depends on the incubation time. The percentage of total protein initially bound that is desorbed decreased from &gt;85% with a 5 min incubation to &lt;40% with 24 h incubation at pH 7.0, indicating that additional irreversibly bound species are formed on the resin surface. Load buffers leading to stronger binding (higher pH and/or lower salt) and higher temperatures (32 °C) result in more extensive aggregation. Minimal aggregation occurs, instead, for Nuvia HP‐Q.CONCLUSIONBSA oligomers are formed directly on the surface of the multimodal resins studied and are eluted as discrete species at increasing salt concentrations. Aggregate formation is promoted by the ligand hydrophobicity and affects the separation of monomer–dimer mixtures by frontal chromatography, as the ability of the dimer in the feed to displace the adsorbed protein is impaired by gradual conversion of the monomer to stronger bound species. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry (SCI).

Albumin/Thiacalix[4]arene Nanoparticles as Potential Therapeutic Systems: Role of the Macrocycle for Stabilization of Monomeric Protein and Self-Assembly with Ciprofloxacin.

The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization of monomeric bovine serum albumin and preventing aggregation. Spectral methods (UV-vis, CD, fluorescent spectroscopy, and dynamic light scattering) established the influence of the synthesized compounds on the content of monomeric and aggregated forms of BSA even without the formation of stable thiacalixarene/protein associates. The effect of thiacalixarenes on the efficiency of protein binding with the antibiotic ciprofloxacin was shown by fluorescence spectroscopy. The binding constant increases in the presence of the macrocycles, likely due to the stabilization of monomeric forms of BSA. Our study clearly shows the potential of this macrocycle design as a platform for the development of the fundamentally new approaches for preventing aggregation.

Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer-dimer mixture in situ.

Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g., by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules.

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography

Thermodynamic studies on protein adsorption onto chromatographic surfaces mainly focus on the molecular level interaction between proteins and ligands. Yet, not much attention is given to the study of polymer grafted ligand architecture effect on thermodynamic parameters, nor to the relation between chromatographic parameters and the directly obtained thermodynamic parameters. These relations are needed in order to confer meaning and to ease future data interpretation of thermodynamic studies of protein adsorption. In this study, the adsorption of bovine serum albumin monomer (BSAm) onto chromatographic surfaces with grafted ligands was studied from a thermodynamic point of view together with chromatographic data. Isothermal titration calorimetry (ITC) results showed that BSAm adsorption is exothermic (ΔH¯ads < 0) when adsorbs onto Toyopearl GigaCapQ 650 M, Toyopearl Q600AR, and Q Sepharose XL, but endothermic (ΔH¯ads > 0) when adsorbs onto Toyopearl SuperQ and a conventional resin (Q Sepharose Fast Flow), showing clear differences in the driving forces of adsorption caused by different ligand architectures. In addition, we found a new relation between the salt required for protein elution and the change in adsorption enthalpy (ΔH¯ads) directly measured with ITC, intrinsically connecting both adsorption and desorption mechanisms.

Comparing Protein Adsorption onto Alumina and Silica Nanomaterial Surfaces: Clues for Vaccine Adjuvant Development

Protein adsorption onto nanomaterial surfaces is important for various nanobiotechnology applications such as biosensors and drug delivery. Within this scope, there is growing interest to develop alumina- and silica-based nanomaterial vaccine adjuvants and an outstanding need to compare protein adsorption onto alumina- and silica-based nanomaterial surfaces. Herein, using alumina- and silica-coated arrays of silver nanodisks with plasmonic properties, we conducted localized surface plasmon resonance (LSPR) experiments to evaluate real-time adsorption of bovine serum albumin (BSA) protein onto alumina and silica surfaces. BSA monomers and oligomers were prepared in different water-ethanol mixtures and both adsorbing species consistently showed quicker adsorption kinetics and more extensive adsorption-related spreading on alumina surfaces as compared to on silica surfaces. We rationalized these experimental observations in terms of the electrostatic forces governing protein-surface interactions on the two nanomaterial surfaces and the results support that more rigidly attached BSA protein-based coatings can be formed on alumina-based nanomaterial surfaces. Collectively, the findings in this study provide fundamental insight into protein-surface interactions at nanomaterial interfaces and can help to guide the development of protein-based coatings for medical and biotechnology applications such as vaccines.

Measurement of the Translational Diffusion Coefficient and Hydrodynamic Radius of Proteins by Dynamic Light Scattering.

Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally, measurement of the diffusion coefficient and the sedimentation coefficient, and application of the Svedberg equation, yields the molecular weight, which is particularly useful in the characterization of very large macromolecules. Dynamic light scattering (DLS) is the most common method to measure the diffusion coefficient of macromolecules. We describe a procedure to perform DLS measurements on monomeric bovine serum albumin (BSA) purified by size-exclusion chromatography using the Zetasizer Nano S particle size analyzer. We compare several analytical methods in existing software programs to estimate the diffusion coefficient of BSA (extrapolated to water at 20°C at infinite dilution, ) and describe a statistical method to obtain 95% confidence limits of the precision of the estimates. We compare estimates to literature values obtained by diffusiometry, sedimentation velocity analytical ultracentrifugation, and other DLS instruments. The method of cumulant analysis in the program SEDFIT (www.analyticalultracentrifugation.com) produced the most precise estimate, 6.06 ± 0.07 F (1 F = 10-7 cm2 s-1), which was within the range of estimates obtained by diffusiometry or sedimentation velocity. This protocol is useful for DLS method validation and quality control.

Laccase-catalyzed cross-linking of BSA mediated by tyrosine

Tyrosine was explored as a cross-linking agent to form cross-linked bovine serum albumin (BSA) using laccase as a catalyst. Liquid chromatography-mass spectrometry (LC-MS) and fluorescence spectra indicated that tyrosine can be mainly oxidized to be dityrosine. Spectra analysis and molecular weight were used to characterize the BSA treated with tyrosine and laccase. Both SDS-PAGE and size exclusion chromatography confirmed the formation of cross-linked BSA, while most of the protein products existed as BSA–tyrosine conjugates. The MALDI-TOF analysis revealed that five tyrosine units were grafted on one BSA monomer, however one cross-linked BSA consists of two BSA monomers and 18 tyrosine. Furthermore, the content of the amino acid of BSA was identified using amino acid analysis, among those the percentage of lysine presented a visible decline from 12.36% to 11.43%, corresponding to 4–5 lysine residues. The pure and modified BSA were hydrolyzed by trypsin and the corresponding peptides were obtained. Different mass of five peptides from LC-MS spectra after hydrolysis indicated that tyrosine could react with Lys-136, Lys-204, Lys-224, Lys-322 and Lys-537 in BSA, promoting the formation of BSA–tyrosine conjugates and cross-linked BSA.

Reaction Rate Governs the Viscoelasticity and Nanostructure of Folded Protein Hydrogels.

Hydrogels constructed from folded protein domains are of increasing interest as resilient and responsive biomaterials, but their optimization for applications requires time-consuming and costly molecular design. Here, we explore a complementary approach to control their properties by examining the influence of crosslinking rate on the structure and viscoelastic response of a model hydrogel constructed from photochemically crosslinked bovine serum albumin (BSA). Gelation is observed to follow a heterogeneous nucleation pathway in which BSA monomers crosslink into compact nuclei that grow into fractal percolated networks. Both the viscoelastic response probed by shear rheology and the nanostructure probed by small-angle X-ray scattering (SAXS) are shown to depend on the photochemical crosslinking reaction rate, with increased reaction rates corresponding to higher viscoelastic moduli, lower fractal dimension, and higher fractal cluster size. Reaction rate-dependent changes are shown to be consistent with a transition between diffusion- and rate-limited assembly, and the corresponding changes to viscoelastic response are proposed to arise from the presence of nonfractal depletion regions, as confirmed by SAXS. This controllable nanostructure and viscoelasticity constitute a potential route for the precise control of hydrogel properties, without the need for molecular modification.

Effects of molecule size and resin structure on protein adsorption on multimodal anion exchange chromatography media.

The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 μm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 μm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors.

Following Structural Changes by Thermal Denaturation Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T ∼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H]12+ to [M + 16H]16+ at ∼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H]46+ at ∼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature Tm ∼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 μM), this method can detect nonspecific BSA dimers and trimers which dissociate early (Td ∼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).

Aggregation features of partially unfolded bovine serum albumin modulated by hydrogenated and fluorinated surfactants: Molecular dynamics insights and experimental approaches

Protein aggregation plays important roles in life science as, for instance, those associated to neurodegenerative diseases. Although extensive efforts have been done to elucidate all the possible variables related to the aggregation process, much has yet to be done to unveil the main pathways governing protein assembling. In the current work, we induce bovine serum albumin (BSA) association, at pH 3.7, by adding sodium dodecyl sulfate (SDS) and sodium perfluorooctanoate (SPFO) surfactants to BSA solution as promoters of protein aggregation. Firstly, we combine molecular dynamic simulations (MD) to obtain a partially unfolded state of BSA’s monomer at the acid pH and small angle X-ray scattering (SAXS) to validate the model. Interestingly, we found by SAXS that at pH 3.7 BSA monomers coexist with dimers in surfactant-free solution. Upon SDS and SPFO addition, the partial unfolded BSA may evolve to large aggregates depending on surfactant concentration. The threshold occurs at 30:1 and 45:1 SDS:BSA and SPFO:BSA molar ratio, respectively, according to turbidity, Thioflavin (ThT) fluorescence, synchrotron radiation circular dichroism (SRCD), SAXS and scanning electron microscopy (SEM) experiments. BSA aggregates are larger in the presence of SDS and structurally more defined upon SPFO binding. Isothermal titration calorimetry (ITC) results give support to infer that both surfactants initially bind to the BSA macromolecule forming a complex. Then, these complexes self-associate towards supramolecular aggregates. Taking into account the physicochemical characteristics of both surfactants and also MD simulations we may suggest that the higher rigidity of the fluorinated chains in respect to hydrogenated ones is crucial to induce more ordered and smaller BSA’s aggregates. Our results thus evidence that the ligand structural flexibility might be of a key importance in the pathway of protein aggregation and may pave the way to better understand the early steps of neurodegenerative disorders.

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique. Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Encapsulation of zinc phthalocyanine into bovine serum albumin aggregates

Artificial hybridization of molecules with proteins is beneficial for biomedical applications. Herein, we describe a novel method for encapsulation of zinc phthalocyanine (ZnPc) aggregates into bovine serum albumin (BSA) aggregates, but not monomeric BSA. This property is potentially useful for not only photodynamic or photothermal therapy but also development of a novel delivery system for hydrophobic drug molecules.

Application of native polyacrylamide gel electrophoresis for protein analysis: Bovine serum albumin as a model protein

Native polyacrylamide gel electrophoresis (N-PAGE) is a simple qualitative technology to determine heterogeneity of proteins. Here, we have applied N-PAGE to examine heat-induced aggregation and oligomerization of bovine serum albumin (BSA) and the effects of temperature, caprylic acid, N-acetyl-tryptophan, DNA, arginine and gallic acid. Arginine showed marginal protection of BSA against heat-induced aggregation and oligomerization, while other compounds showed varying degree of protections. It is interesting to point out that new bands were formed in the presence of some of these compounds upon heating. Among the compounds tested, gallic acid showed protection of monomeric BSA (observed in N-PAGE as a prominent band) and increased the mobility of native BSA. The increased mobility indicates binding of gallic acid to the native BSA.

Temperature-Induced Denaturation of BSA Protein Molecules for Improved Surface Passivation Coatings.

Bovine serum albumin (BSA) is the most widely used protein for surface passivation applications, although it has relatively weak, nonsticky interactions with hydrophilic surfaces such as silica-based materials. Herein, we report a simple and versatile method to increase the stickiness of BSA protein molecules adsorbing onto silica surfaces, resulting in up to a 10-fold improvement in blocking efficiency against serum biofouling. Circular dichroism spectroscopy, dynamic light scattering, and nanoparticle tracking analysis showed that temperature-induced denaturation of BSA proteins in bulk solution resulted in irreversible unfolding and protein oligomerization, thereby converting weakly adhesive protein monomers into a more adhesive oligomeric form. The heat-treated, denatured BSA oligomers remained stable after cooling. Room-temperature quartz crystal microbalance-dissipation and localized surface plasmon resonance experiments revealed that denatured BSA oligomers adsorbed more quickly and in larger mass quantities onto silica surfaces than native BSA monomers. We also determined that the larger surface contact area of denatured BSA oligomers is an important factor contributing to their more adhesive character. Importantly, denatured BSA oligomers were a superior passivating agent to inhibit biofouling on silica surfaces and also improved Western blot application performance. Taken together, the findings demonstrate how temperature-induced denaturation of BSA protein molecules can lead to improved protein-based coatings for surface passivation applications.

Indirect Nanoplasmonic Sensing Platform for Monitoring Temperature-Dependent Protein Adsorption.

The development of highly surface-sensitive measurement approaches to monitor protein adsorption across different temperatures would advance understanding of how thermally activated processes contribute to the denaturation of adsorbed proteins. Herein, we established an indirect nanoplasmonic sensing approach to monitor the temperature-dependent adsorption and denaturation of bovine serum albumin (BSA) protein onto a silica-coated array of plasmonic gold nanodisks. A theoretical model was developed to explain how the denaturation of an individual, adsorbed protein molecule influences the localized surface plasmon resonance (LSPR) measurement response and provided an analytical framework to estimate the effect of temperature-dependent protein denaturation on the corresponding adsorption kinetics. The sensing performance of this measurement platform was also characterized across the tested range of temperatures. With increasing temperature (up to 50 °C), it was observed that adsorbed proteins undergo greater denaturation. Circular dichroism spectroscopy and dynamic light scattering experiments verified that individual BSA monomers in bulk solution had increasingly lower conformational stability at higher temperatures within this range, which correlated with the extent of denaturation in the adsorbed state. At higher temperatures, distinct kinetic profiles arising from multilayer/aggregate formation on the sensor surface were also detected. Taken together, our findings identify that the high surface sensitivity and temperature stability of LSPR sensors make them broadly useful analytical tools for monitoring thermally activated biomacromolecular interaction processes.

Protein exclusion is preserved by temperature sensitive PEG brushes

Poly(ethylene glycol) (PEG) is widely used in biotechnology-related applications yet its temperature-dependent functionality is not well understood. Here, we use bovine serum albumin (BSA) monomers and cross-linked dimers to directly probe the height of strongly stretched PEG brushes using surface plasmon resonance (SPR) in aqueous solution. Our results show that PEG brush height follows a smooth decrease as a function of increasing temperature commencing near room temperature. Measurements obtained by BSA monomers and dimers are comparable and suggest that BSA effectively probes the leading edge of the brush with minimal penetration into its interior being supported by SPR reflectivity calculations. Further, the BSA-PEG interaction remains largely inert over the entire temperature range. Overall, PEG brushes undergo a smooth conformational transition while fully preserving its protein excluding properties far from the lower critical solution temperature.