Adsorption Of Human Serum Albumin Research Articles

Adsorption of Human Serum Albumin (HSA) on a mixed-mode adsorbent: equilibrium and kinetics

The adsorption equilibrium and kinetics of Human Serum albumin (HSA) onto a novel high particle density multimodal adsorbent were studied by batch adsorption experiments. MabDirect MM, from Upfront Chromatography A/S, is an agarose-tungsten carbide composite adsorbent with an anionic mixed mode ligand. The effects of ionic strength (by addition of salt) and of pH are assessed. Langmuir isotherms parameters are obtained along with pore diffusivity values by fitting the batch experiments using a pore diffusion model. Under the studied conditions for pH 5.0, without salt, it was obtained the largest adsorption capacity (36.0 ± 3.5 mg·gadsorbent,dry −1) a typical behavior for a mixed mode adsorbent. Effective pore diffusivity (Dpe) was estimated as (2.2 ± 0.1) × 10−6 cm2·min−1 for this condition. Additionally, the lowest adsorption capacity (8.6 ± 2.1 mg·gadsorbent,dry −1) and an estimation of Dpe equal to (2.4 ± 0.6) × 10−6 cm2·min−1 were obtained for pH 7.0 without salt. An alternative method of Dpe estimation is used and validate the results obtained by simulations. Furthermore, several experiments were carried out in a fixed bed column with the aim to understand the kinetics and hydrodynamics, and to validate the batch adsorption results. For a feed concentration of ±0.92 g·L−1, pH 5.0, without salt, for cleaned and fresh adsorbent, fixed bed dynamic binding capacities of 13.84 and 14.37 mg·gadsorbent,dry −1 were obtained, respectively, representing near 50% of saturation capacity.

Preparation and evaluation of mixed-mode resins with tryptophan analogues as functional ligands for human serum albumin separation

Abstract Five tryptophan analogues with a hydrophobic indole ring and an amino group on each molecule were used as functional ligands of mixed-mode resins for human serum albumin (HSA) purification. Their adsorption performance was evaluated and the effects of pH and salt addition on HSA adsorption were studied. The resins prepared showed typical pH-dependent adsorption and the highest adsorption capacity and affinity were found at pH 5.0 for all the resins tested. The saturated adsorption capacity was 138.02 mg·g− 1 with the tryptamine-functionalized resin, which significantly decreased at pH below 4.0 due to electrostatic repulsion between ligands and HSA. Moreover, the addition of NaCl or (NH4)2SO4 in media reduced HSA adsorption capacity, although the two salts showed different affecting profiles. The tryptamine-functionalized resin showed the best salt-tolerant performance, and its high adsorption capacity was maintained under high salt concentrations. In addition, the five resins prepared showed good adsorption selectivity for recombinant HSA from Pichia pastoris broth. Molecular docking results between tryptamine and HSA indicated that tryptamine was favorable to bind on Site II (indole-binding site) of HSA.

Monolayers of immunoglobulin G on polystyrene microparticles and their interactions with human serum albumin

The adsorption of mouse monoclonal immunoglobulin G (IgG) on negatively charged polystyrene microparticles was studied by the laser Doppler velocimetry (LDV) electrophoretic mobility measurements. The dependence of the electrophoretic mobility of microparticles on the IgG concentration in the suspension was determined for different ionic strengths and pHs (3.5, 7.4). The increase in the electrophoretic mobility was quantitatively interpreted in terms of the 3D electrokinetic model. The maximum coverage of IgG on latex was determined by the depletion method aided by AFM imaging. It was shown by monitoring the electrophoretic mobility and hydrodynamic dimeter of IgG covered microparticles over prolonged time periods that IgG adsorption was irreversible. The acid-base properties of the IgG monolayers were also determined in pH cycling experiments. It was also confirmed that the adsorption of human serum albumin (HSA) on saturated IgG monolayers, often referred to as blocking, was negligible at pH 7.4.

Monolayers of Poly(styrene/α-tert-butoxy-ω-vinylbenzyl-polyglycidol) Microparticles Formed by Controlled Self-Assembly with Potential Application as Protein-Repelling Substrates.

The kinetics of the self-assembly of poly(styrene/α-tert-butoxy-ω-vinylbenzyl-polyglycidol) microparticles on poly(allylamine hydrochloride)-derivatized silicon/silica substrate was determined by direct AFM imaging and streaming potential (SP) measurements. The kinetic runs acquired under diffusion-controlled transport were quantitatively interpreted in terms of the extended random sequential adsorption (RSA) model. This allowed confirmation of a core/shell morphology of the microparticles. The polyglycidol-rich shell of thickness equal to 25 nm exhibited a fuzzy structure that enabled penetration of particles into each other resulting in high coverage inaccessible for ordinary microparticles. The SP measurements interpreted by using the 3D electrokinetic model confirmed this microparticle structure. Additionally, the acid-base characteristics of the microparticle monolayers were determined for a broad pH range. By using the streaming potential measurements, human serum albumin (HSA) adsorption on the microparticle monolayers was investigated under in situ conditions. It was confirmed that the protein adsorption was considerably lower than for the reference case of bare silicon/silica substrate under the same physicochemical conditions. This effect was attributed to the presence of the shell diminishing the protein/microparticle physical interactions.

Controlling the Integration of Polyvinylpyrrolidone onto Substrate by Quartz Crystal Microbalance with Dissipation To Achieve Excellent Protein Resistance and Detoxification.

Blood purification systems, in which the adsorbent removes exogenous and endogenous toxins from the blood, are widely used in clinical practice. To improve the protein resistance of and detoxification by the adsorbent, researchers can modify the adsorbent with functional molecules, such as polyvinylpyrrolidone (PVP). However, achieving precise control of the functional molecular density, which is crucial to the activity of the adsorbent, remains a significant challenge. In the present study, we prepared a model system for blood purification adsorbents in which we controlled the integration density of PVP molecules of different molecular weights on an Au substrate by quartz crystal microbalance with dissipation (QCM-D). We characterized the samples with atomic force microscopy, X-ray photoelectron spectroscopy, and QCM-D and found that the molecular density and the chain length of the PVP molecules played important roles in determining the properties of the sample. At the optimal condition, the modified sample demonstrated strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg) by 92.5% and 79.2%, respectively. In addition, the modified sample exhibited excellent detoxification, and the adsorption of bilirubin increased 2.6-fold. Interestingly, subsequent atomistic molecular dynamics simulations indicated that the favorable interactions between PVP and bilirubin were dominated by hydrophobic interactions. An in vitro platelet adhesion assay showed that the adhesion of platelets on the sample decreased and that the platelets were maintained in an inactivated state. The CCK-8 assay indicated that the modified sample exhibited negligible cytotoxicity to L929 cells. These results demonstrated that our method holds great potential for the modification of adsorbents in blood purification systems.

Impact of mesoporous silica surface functionalization on human serum albumin interaction, cytotoxicity and antibacterial activity

Aiming to broaden our understanding on the interplay of silica nanomaterials with living biocells, we designed herein various surface-functionalized mesoporous SBA-15-type silicates, including native one SBA-OH, aminated SBA-NH2, thiolated SBA-SH and carboxy-terminated SBA-COOH. These high-surface area materials are then subjected to interactions with human serum albumin (HSA), in order to reproduce the real picture of what occurs within the body upon delivering active drugs. First, we examined circular dichroism measurements, fluorescence quenching, HSA adsorption and hemolysis in the presence and absence of HSA. Subsequently, we studied the toxicity of these materials toward eukaryotic and prokaryotic cells and the effect of the silica surface on the antibacterial activity. Irrespective to the functional groups, silica interacts with HSA resulting in the suppression of hemolysis. In terms of antibacterial activity, the pivotal parameter was found to be the amorphous state of the silica carrier rather than the nature of the functional groups. In the whole, this informative work provides an overall understanding of the role of the silica surface for bio-cells interaction and can serve as a guideline for the rational-design of blood-compatible nanomaterials.

Adsorption of human fibrinogen and albumin onto hydrophobic and hydrophilic Ti6Al4V powder

Adsorption of proteins on solid surfaces has been widely studied because of its importance in various biotechnological, medical and technical applications, such as medical implants or biosensors. One of the main problems is the adsorption-induced conformational changes because they often modify the biological activity of the proteins, which is believed to be a key factor on the subsequent cellular adhesion. The aim of this work is the study of the adsorption of human fibrinogen (Fg) and human serum albumin (HSA) onto Ti6Al4V particles, commercially available on different size, that are used to elaborate scaffolds to provide structural support to cell proliferation, promoting tissue development and bone regeneration among others. The study was done through the analysis of the adsorption isotherms and the electrical characterization of surfaces after adsorption in terms of the zeta potential (ζ). From this analysis it seems that Fg adsorbs preferentially vertically oriented (end-on) and HSA moves sequentially over the surface of the Ti6Al4V particles through dimmer formation, allowing adsorption progress over this initial bilayer. The zeta potential values of both proteins remain constant when the monolayer is formed. The study also extends the analysis of both adsorption behaviour and ζ potential characterization factors to the influence of the substrate hydrophobicity as this property can be modified for the Ti6Al4V by irradiating it with ultraviolet light (UV-C) without changes on its chemical composition [1,2]. Differences at low protein concentrations were found for both isotherms and zeta-potential values.

An antifouling electrode based on electrode–organic layer–nanoparticle constructs: Electrodeposited organic layers versus self-assembled monolayers

Electrodes modified with organic layers and nanoparticles are promising tools for analytical devices. A major challenge however is how to make electrodes that resist the nonspecific protein adsorption from biological media? Herein, we report gold electrode–organic layer-gold nanoparticle constructs as a platform for electrochemical biosensing. This study focuses on the fabrication of antifouling electrodes with the organic layers formed by two different approaches; self-assembled monolayer (SAM) procedure and electrochemical reduction of aryl diazonium salts, followed by the attachment of gold nanoparticle (AuNP) to allow the electrochemistry to proceed. The results show that the electrodes modified with ethylene oxide-based SAM enable a better antifouling behaviour with less adsorption of human serum albumin (HSA) as compared to the electrodes modified with ethylene oxide-based aryl diazonium-derived layer. Furthermore, findings suggest such distinction could be due to the surface packing and/or conformation of organic film on Au electrodes.

Zwitterionic surface coating of quantum dots reduces protein adsorption and cellular uptake.

We have studied the effect of the zwitterionic surface coating of quantum dots (QDs) on their interaction with a serum supplemented cell medium and their internalization by human cervical carcinoma (HeLa) cells. Zwitterionic QDs showed negligible adsorption of human serum albumin (HSA) selected as a model serum protein, in contrast to similar but negatively charged QDs. The incorporation of zwitterionic QDs by HeLa cells was found to be lower than for negatively charged QDs and for positively charged QDs, for which the uptake yield was largest. Our results suggest that the suppression of protein adsorption, here accomplished by zwitterionic QD surfaces, offers a strategy that allows for reducing the cellular uptake of nanoparticles.

A mixed-mode resin with tryptamine ligand for human serum albumin separation

Mixed-mode chromatography (MMC) is a new technology that uses specially-designed ligands to improve the adsorption selectivity with multimodal protein–ligand interactions for protein separation. A new MMC resin TA-B-6FF with tryptamine as the functional ligand was prepared and used for human serum albumin (HSA) separation. Adsorption equilibria of plasma-derived HSA (pHSA) were investigated and compared with a commercial tryptophan-based resin (MX-Trp-650m), and the influence of pH and salt addition was studied. The results showed that weak acidic conditions (pH 5.0–7.0) were favorable for HSA adsorption. The maximum adsorption capacity of TA-B-6FF was 141.33mg/g at pH 5.0, which was two times higher than that of MX-Trp-650m. TA-B-6FF also showed better salt-tolerance than MX-Trp-650m. Moreover, TA-B-6FF was used to separate recombinant HSA (rHSA) from Pichia pastoris culture broth. The results indicated that rHSA could be directly captured by TA-B-6FF without dilution or pH adjustment. High purity (87.75%) of rHSA monomer could be obtained with a recovery of 98.53% through two-step elution process. Total content of rHSA monomer and degraded fragment was 99.75%, the removal of host cell proteins reached about 90%. The results demonstrate that new TA-B-6FF resin has a great potential for rHSA purification directly from the complex fermentation broth.

Application of a fluorescent biosensor based-on magneto-γ-Fe2 O3 -methyldopa nanoparticles for adsorption of human serum albumin.

Understanding and controlling the interaction between the polymer methyldopa (2-amino-3-(3,4-dihydroxyphenyl)-2-methyl-propanoic acid) (PMDP)-γ-Fe2 O3 nanoparticles and biological fluids is important if the potential of nanoparticles (NPs) in biomedicine is to be realized. Physicochemical studies on the interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of the NP surface, interactions between human serum albumin (HSA) and PMDP-γ-Fe2 O3 NPs were investigated. Here, the adsorption of HSA onto small (10-30 nm diameter) PMDP-γ-Fe2 O3 NPs was quantitatively analyzed using spectroscopic methods. The fluorescence quenching data were checked for the inner-filter effect, the main confounding factor in the observed quenching. The binding constants, Ka , were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ∆H, ∆S and ∆G were given. The obtained thermodynamic signature suggests that hydrophobic interactions at least are present. This result indicates that the structure of the protein turns from a structureless denatured state at pH 3 into an ordered biologically active native state on addition of PMDP-γ-Fe2 O3 NPs. Copyright © 2015 John Wiley & Sons, Ltd.

Single and competitive protein adsorption on polymeric surfaces

The biological response to material surfaces is often influenced by protein layers formed at the bio‐interface. Understanding this protein layer is of paramount importance for biomedical materials and cell culture devices, which often require protein coating for optimal cell growth. An insert system fitting exactly into standard tissue culture plates was developed and can be used for biological investigations without the influence of the cell culture material. Inserts prepared from polystyrene (PS), polycarbonate (PC), poly(styrene‐co‐acrylonitrile) (PSAN) and poly(ether imide) (PEI) exhibit a similar surface roughness and wettability so that only the chemistry is varied. Previously, stem cell adhesion responses were found to be different for these inserts, possibly because of their different protein adsorption profiles. This work investigated if the surface functional groups of these inserts influence their protein binding ability. Single and competitive adsorption of two most abundant blood proteins, human serum albumin (HSA) and immunoglobulin G (IgG) on these polymers was investigated by labeling both proteins with different near‐infrared (IR) dyes. PEI showed the highest protein adsorption propensity in single and competitive adsorption of IgG and HSA while PS exhibited the least adsorption capability, whereas PSAN and PC showed an intermediate protein adsorption profile. Chemical inertness of PS could be the reason for this low protein binding ability and limited cell growth. Conclusively, a novel method to efficiently detect protein adsorption on polymer surfaces was established, and using this method the high relevance of the chemical composition of polymeric substrates on their protein adsorption profile could be proven. Copyright © 2015 John Wiley & Sons, Ltd.

Comprehensive Multispectroscopic Analysis on the Interaction and Corona Formation of Human Serum Albumin with Gold/Silver Alloy Nanoparticles.

In the present investigation, we have systematically studied the binding mechanism of model protein human serum albumin (HSA) with gold/silver alloy nanoparticles (Au/Ag NPs) using multiple spectroscopic techniques. Absorption spectral studies of Au/Ag NPs in the presence of increasing concentrations of HSA resulted in a slight red shift of the surface plasmon resonance band (SPR) of Au/Ag NPs, suggesting changes in the refractive index around the nanoparticle surface owing to the adsorption of HSA. The results from high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta potential analysis substantiated the formation of a dense layer of HSA on the surface of Au/Ag NPs. The formation of a ground-state complex between HSA and Au/Ag NPs was evident from the outcome of the steady-state emission titration experiments of the HSA-Au/Ag NPs system. The binding parameters computed from corrected emission quenching data revealed that HSA exhibited a significant binding affinity toward Au/Ag NPs. The identical fluorescence lifetime values of HSA and HSA-Au/Ag NPs from time-resolved fluorescence spectroscopic analysis further authenticated the findings of steady-state emission measurements. The formation of HSA corona on the Au/Ag NPs surface was established on the basis of experimental quenching data and theoretical values. The occurrence of partial unfolding of HSA upon its interaction with the Au/Ag NPs surface was established by using an extrinsic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS). Absorption, Fourier transform infrared (FT-IR), Raman, circular dichroism (CD), and excitation-emission matrix (3D) spectral studies were also carried out to explore Au/Ag NPs-induced tertiary and secondary conformational changes of HSA. The influence of Au/Ag NPs on the esterase-like activity of HSA was established by probing the hydrolysis of p-nitrophenyl acetate.

On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range.In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution.Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6nm) and the dissociation rate constant (0.055s−1). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake.

Here we have investigated the effect of enshrouding polymer-coated nanoparticles (NPs) with polyethylene glycol (PEG) on the adsorption of proteins and uptake by cultured cells. PEG was covalently linked to the polymer surface to the maximal grafting density achievable under our experimental conditions. Changes in the effective hydrodynamic radius of the NPs upon adsorption of human serum albumin (HSA) and fibrinogen (FIB) were measured in situ using fluorescence correlation spectroscopy. For NPs without a PEG shell, a thickness increase of around 3 nm, corresponding to HSA monolayer adsorption, was measured at high HSA concentration. Only 50% of this value was found for NPs with PEGylated surfaces. While the size increase clearly reveals formation of a protein corona also for PEGylated NPs, fluorescence lifetime measurements and quenching experiments suggest that the adsorbed HSA molecules are buried within the PEG shell. For FIB adsorption onto PEGylated NPs, even less change in NP diameter was observed. In vitro uptake of the NPs by 3T3 fibroblasts was reduced to around 10% upon PEGylation with PEG chains of 10 kDa. Thus, even though the PEG coatings did not completely prevent protein adsorption, the PEGylated NPs still displayed a pronounced reduction of cellular uptake with respect to bare NPs, which is to be expected if the adsorbed proteins are not exposed on the NP surface.

Surface Acoustic Wave (SAW) Resonators for Monitoring Conditioning Film Formation.

We propose surface acoustic wave (SAW) resonators as a complementary tool for conditioning film monitoring. Conditioning films are formed by adsorption of inorganic and organic substances on a substrate the moment this substrate comes into contact with a liquid phase. In the case of implant insertion, for instance, initial protein adsorption is required to start wound healing, but it will also trigger immune reactions leading to inflammatory responses. The control of the initial protein adsorption would allow to promote the healing process and to suppress adverse immune reactions. Methods to investigate these adsorption processes are available, but it remains difficult to translate measurement results into actual protein binding events. Biosensor transducers allow user-friendly investigation of protein adsorption on different surfaces. The combination of several transduction principles leads to complementary results, allowing a more comprehensive characterization of the adsorbing layer. We introduce SAW resonators as a novel complementary tool for time-resolved conditioning film monitoring. SAW resonators were coated with polymers. The adsorption of the plasma proteins human serum albumin (HSA) and fibrinogen onto the polymer-coated surfaces were monitored. Frequency results were compared with quartz crystal microbalance (QCM) sensor measurements, which confirmed the suitability of the SAW resonators for this application.

Gold Nanoparticle-Based Facile Detection of Human Serum Albumin and Its Application as an INHIBIT Logic Gate.

In this work, a facile colorimetric method is developed for quantitative detection of human serum albumin (HSA) based on the antiaggregation effect of gold nanoparticles (Au NPs) in the presence of HSA. The citrate-capped Au NPs undergo a color change from red to blue when melamine is added as a cross-linker to induce the aggregation of the NPs. Such an aggregation is efficiently suppressed upon the adsorption of HSA on the particle surface. This method provides the advantages of simplicity and cost-efficiency for quantitative detection of HSA with a detection limit of ∼1.4 nM by monitoring the colorimetric changes of the Au NPs with UV-vis spectroscopy. In addition, this approach shows good selectivity for HSA over various amino acids, peptides, and proteins and is qualified for detection of HSA in a biological sample. Such an antiaggregation effect can be further extended to fabricate an INHIBIT logic gate by using HSA and melamine as inputs and the color changes of Au NPs as outputs, which may have application potentials in point-of-care medical diagnosis.

Detecting human serum albumin using screen-printed carbon electrode by cyclic voltammetry

Purpose: Electrochemical analysis is a powerful tool for analyzing interfacial reactions as well as composition of analytes. In this study, we made a lowcost, portable, and easy-to-use instrument consists of hardware and software for electrochemical analysis. The cost of our design is 1/1000 fold to compare with commercial systems, and the weight of this device is light for 120 grams. Finally, this study used this portable device to detect human serum albumin (HSA) by cyclic voltammetry analysis employing screenprinted carbon electrode (SPCE). Methods: In this study, we made a portable device which consists of hardware and software in it. The hardware part consists of PCB and electrical elements on the PCB. We used ATXmega32A4 as the microcontroller, and TLC2264ID as OP amplifier. The software part was written by C language and compiled with AVR studio. We used homemade instruments to detect HSA with CV with SPCE. All the solutions were prepared at room temperature(25 2 C)and DI water. The concentration of HSA is 0e450 mg/L. Results: Figure shows the CV response of the detection of HSA. With adsorption of HSA on the electrode, we can observe that the peak current decreases when the concentration of HSA increases. This is due to the adsorption of HSA hinders transfer of electrons between electrode and the solution.

Characteristic of silicon doped diamond like carbon thin films on surface properties and human serum albumin adsorption

Diamond-like carbon (DLC) coatings are useful for creating biocompatible surfaces for medical implants. DLC and silicon doped DLC have been synthesised using plasma enhanced chemical vapour deposition (PECVD). The effects of surface morphology on the interaction of human serum albumin (HSA) with doped and undoped DLC films have been investigated using a range of surface analysis techniques using Raman spectroscopy and atomic force microscopy (AFM). Raman spectra of doped DLC show that silicon doped DLC reduces the growth range of the ID/IG ratio, with a significant red-shift of the G peak position. Following exposure to protein, for undoped DLC the peaks at 1664cm−1 and around 1241cm−1 can be attributed to amide I and III, respectively, with an increase in the surface morphology of the surfaces giving some indication of the protein structure on the surfaces. Results indicate that HSA exhibit the majority of β-sheet during the adsorption on the surfaces. The results showed that the silicon incorporation DLC tends to increase of surface roughness and the adsorbed level of HSA is higher with higher levels of silicon doping of the DLC. Therefore, doping DLC may provide a method of controlling the adsorption of protein.

Molecular Dynamics Simulations of a Binary Protein Mixture Adsorption onto Ion-Exchange Adsorbent

Molecular dynamics (MD) simulations with a coarse-grained model are applied to investigate the binary adsorption of proteins onto an ion-exchange chromatographic medium. In particular, the adsorption of human serum albumin (HSA) and bovine hemoglobin (bHb) on the anion exchanger Q Sepharose FF is studied. Simulations with different initial orientations of the proteins and different protein–protein distances are carried out over a 2000 ns time scale. The protein–ligand potential energies and protein–ligand minimum distances are analyzed, and stable adsorption of HSA but no stable adsorption of bHb is observed. Interactions between HSA and bHb as well as a coupled movement of these two proteins are observed in all simulations. Because of the interaction between these two proteins, their rotations and adjustments to reach a favorable adsorption configuration are hindered. The competition is further indicated by changing adsorption sites on the HSA surface. The interaction and aggregation of the two proteins ...