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Potential-Assisted Adsorption of Bovine Serum Albumin onto Optically Transparent Carbon Electrodes
... [7][8][9][10][11] On conducting substrates, an external applied potential can also be used to control the accumulation of the protein molecules at the polarized interface. [12][13][14][15][16][17][18] This phenomenon is particularly important because it could open the door for the possibility to control the efficiency, selectivity, and kinetics of the adsorption process. [19] Illustrating the intriguing complexity of the adsorption process, several authors have linked the effect of the potential applied to a surface with electrostatic interactions, [20][21][22][23] orientation, [14,24,25] as well as hydration of the protein. ...
... [34] Thus, alternating electric fields have the potential to interact with the dipoles in a protein and promote conformational changes, leading to polarization. [35][36][37][38] While previous results from our group support the capacity of DC signals to induce the formation of these dipoles, [16][17][18]30] it is reasonable to expect that an oscillating electrical signal with frequencies in the correct range could maximize this effect and further enhance the protein adsorption process. [37,39] It is also important to state that performing adsorption experiments under precise electrochemical conditions and frequencies [40] is not a trivial task and requires not only specific instrumentation but also training in multiple areas. ...
... Adsorption experiments were carried out using a variable angle spectroscopic ellipsometer (WVASE, J.A. Woollam Co.; Lincoln, NE), following a procedure described in previous papers. [16,17,30] Briefly, SE measures the change in the reflectance and phase difference between the parallel (RP) and perpendicular (RS) components of a polarized light beam upon reflection from a surface, which are related to the amplitude ratio (Ψ) and phase difference (Δ). [45,46] Experimental data were collected as a function of wavelength or time, and then modeled by using the WVASE software package (J.A. Woollam Co.; Lincoln, NE). ...
This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular‐wave signal with frequencies varying from 0.5 to 2 Hz. The basic experiment, which can be performed in less than 5 min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100 mV and superimposed to +800 mV (applied as a working potential) and varying the frequency in the 0.1–10000 Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1–1.0 Hz), results that are in line with the corresponding dielectric features of the protein.
... The surface electric potential is naturally considered to be one of the factors that affect protein adsorption behavior. In actual fact, protein adsorption can be altered by applying an external potential to the adsorptive surface, in which several electrode surfaces as adsorption surface have been used [2,[26][27][28][29][30][31][32]. The adsorption of albumin, cytochrome c, and soybean peroxidase to a Au surface was reported to increase as the result of imposing http://dx.doi.org/10.1016/j.colsurfb.2016.07.042 0927-7765/© 2016 Elsevier B.V. All rights reserved. ...
... both negative and positive external potentials [28][29][30], and a similar tendency was observed for the adsorption of fibrinogen to a platinum surface [26]. In the case for a carbon-based electrode as an adsorptive surface, increasing the surface potential from negative to positive resulted in a monotoneous decrease in the surface coverage by the adsorbed protein (bovine serum albumin and fibrinogen) while the adsorption rate showed a complicated dependence on the surface potential [26,31]. It was also reported that the rate and amount of protein adsorption on an optically transparent carbon electrode (OTCE) were increased when a positive external potential was imposed [26,31], which was more significant for a hard protein rather than for a soft one [32]. ...
... In the case for a carbon-based electrode as an adsorptive surface, increasing the surface potential from negative to positive resulted in a monotoneous decrease in the surface coverage by the adsorbed protein (bovine serum albumin and fibrinogen) while the adsorption rate showed a complicated dependence on the surface potential [26,31]. It was also reported that the rate and amount of protein adsorption on an optically transparent carbon electrode (OTCE) were increased when a positive external potential was imposed [26,31], which was more significant for a hard protein rather than for a soft one [32]. All these reports demonstrate that it is possible to control protein adsorption by adjusting the external electric potential [2,30]. ...
The impact of external electric potential on the adsorption of a protein to base metal surfaces was examined. Hen egg white lysozyme (LSZ) and six types of base metal plates (stainless steel SUS316L (St), Ti, Ta, Zr, Cr, or Ni) were used as the protein and adsorption surface, respectively. LSZ was allowed to adsorb on the surface under different conditions (surface potential, pH, electrolyte type and concentration, surface material), which was monitored using an ellipsometer. LSZ adsorption was minimized in the potential range above a certain threshold and, in the surface potential range below the threshold, decreasing the surface potential increased the amount of protein adsorbed. The threshold potential for LSZ adsorption was shifted toward a positive value with increasing pH and was lower for Ta and Zr than for the others. A divalent anion salt (K2SO4) as an electrolyte exhibited the adsorption of LSZ in the positive potential range while a monovalent salt (KCl) did not. A comprehensive consideration of the obtained results suggests that two modes of interactions, namely the electric force by an external electric field and electrostatic interactions with ionized surface hydroxyl groups, act on the LSZ molecules and determine the extent of suppression of LSZ adsorption. All these findings appear to support the view that a base metal surface can be controlled for the affinity to a protein by manipulating the surface electric potential as has been reported on some electrode materials.
... Aiming to address this gap in knowledge, the present study utilized interdisciplinary approaches, novel laboratory setups, cellular models, as well as biochemical assays to examine the electrochemical pre-adsorption of collagen type I (the predominant protein in the organic phase of bone) on optically transparent carbon films and its subsequent effects on adult human mesenchymal stem cell adhesion, the first and most important function of anchorage-dependent cells which is required for cell survival and subsequent functions pertinent to new tissue-formation related functions. The present research was also motivated by the following research contributions related to the effect of electric current stimulation on (1) cellular-and molecular-level functions of osteoblasts [6,7] and of adult human mesenchymal stem cells [8,9] pertinent to new bone tissue formation; and (2) adsorption of proteins onto nanostructured carbon films [10][11][12][13] by our respective laboratories. The combined research interests and approaches provided a unique opportunity to address aspects of the underlying mechanism(s) involved when stem cells interact with (and function on) substrates under electric stimulation. ...
... Silica wafers layered with thin, optically-transparent carbon films (Si/SiO 2 /OTCE) were prepared following the procedure described in previous publications [10,14]. Briefly, standard <111> silicon wafers (Si/SiO 2 , Sumco; Phoenix, AZ) were first scored using a computercontrolled engraver (Gravograph IS400, Gravotech; Duluth, GA). ...
... MSE values lower than 15 were considered acceptable and were in agreement with published reports [16,17]. The optical model used in this study to interpret the raw ellipsometric data was developed in the Garcia Lab and was introduced in earlier publications [10]. [14,20] were used to define the ellipsometric response of the OTCE (d = 19.6 ± 0.7 nm). ...
The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon films (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential (OCP; control), +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and cyclic voltammetry (CV). Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (pre-coated with the collagen type I films) under standard cell culture conditions for 2 hours was affected by the extent of collagen pre-adsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential > +800 mV) however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion.
... 8−13 In this regard, our group recently demonstrated that increases in the interfacial potential could yield to significant enhancements in the adsorption of bovine serum albumin (BSA) onto optically transparent carbon electrodes (OTCE). 14 This phenomenon, which was attributed to the induction of dipoles (polarization) within the structure of the incoming protein molecules when placed in the vicinity of the electrode surface, showed a significant dependence on the potential applied, the solution pH, the concentrations of protein, and the ionic strength. 14 Furthermore, subsequent studies demonstrated that the adsorption of glucose oxidase (GOx, a model protein for the development of biosensors) was also susceptible to the potential applied to the sorbent surface and that significant increases in the catalytic activity of the substrates can be achieved by this method. ...
... 14 This phenomenon, which was attributed to the induction of dipoles (polarization) within the structure of the incoming protein molecules when placed in the vicinity of the electrode surface, showed a significant dependence on the potential applied, the solution pH, the concentrations of protein, and the ionic strength. 14 Furthermore, subsequent studies demonstrated that the adsorption of glucose oxidase (GOx, a model protein for the development of biosensors) was also susceptible to the potential applied to the sorbent surface and that significant increases in the catalytic activity of the substrates can be achieved by this method. 15 While very interesting from the catalytic point of view, the use of proteins like BSA and GOx does not provide enough information to identify the key variables involved in the effect of electric potential on the adsorption of proteins with different properties. ...
... In agreement with previous reports, MSE < 15 were considered acceptable. 30,31 The ellipsometric measurements were interpreted using a previously developed optical model 13,14 allowing the description of the substrates in terms of the refractive index (n), extinction coefficient (k), and thickness (d). Consequently, five uniaxial layers with optical axes parallel to the surface substrate were considered in this optical model. ...
The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins, whereas myoglobin (Mb), α-
lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800 mV to the sorbent surface induced the formation of multiple layers of protein. These results suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.
... 4 In addition, several groups have developed approaches to influence the adsorption process (amount, orientation, kinetics, etc.) of proteins based on the natural heterogeneous distribution of charges, 5,6 by performing chemical modifications on either the -NH 2 or the -COOH terminal groups, 7,8 or by directed mutations. 9 Alternatively, and although it can only be applied to conductive materials, the adsorption process can also be influenced by the application of an external electric field, [10][11][12][13][14] potentially affecting the orientation. [15][16][17][18][19] Given the importance of the orientation on the resulting bioactivity, the adsorption process has also be investigated with several computational models, ranging from highly detailed (but slow), molecular dynamics, 20,21 to more phenomenological (yet faster), coarse-grained approaches. ...
... We can now use Eqs. (13), (17), and (18) to generate a linear system for every index j, as ...
Under the most common experimental conditions, the adsorption of proteins to solid surfaces is an spontaneous process that leads to a rather compact layer of randomly oriented molecules. Due to the importance of this process for the development of catalytic surfaces, a number of existing computational and experimental approaches try to predict and control the orientation of such molecules. However, and despite their own advantages, these tend to be either too expensive computationally, or oversimplified, undermining their ability to predict the most appropriate experimental conditions to maximize the catalytic activity of adsorbed proteins. To address this current need, we present an efficient computational approach to model the behavior of proteins near surfaces in the presence of an external electric field, based on continuum electrostatics. Our model can not only estimate the overall affinity of the protein with the surface, but also their most likely orientation as a function of the potential applied. In this way, a rational selection of the potential can be performed to maximize the accessibility of the protein's active site to the solvent. The model relies on the Poisson-Boltzmann equation and was implemented in an extension of the code PyGBe that includes an external electric field, and renders the electrostatic component of the solvation free energy. To demonstrate the feasibility of this technique, we investigate the adsorption of trypsin onto a carbon electrode under potentiostatic conditions both numerically and experimentally. We found that even though the adsorption process is largely dominated by hydrophobic effects, the orientation of trypsin can be controlled through an external potential, influencing the position of the active sites, and resulting in an important change in the catalytic activity of the surface.
... Based on the findings that protein-surface electrostatic interactions are crucial for such an adsorption, as described above, it is possible that protein adsorption might be controlled by electrochemically controlling the electric potential of the adsorbent surface. Previous studies have reported on attempts to control (reduce or enhance) protein adsorption by applying an electric potential to the adsorbent metal surface [21][22][23][24][25]. In our previous study [26], we also reported in the influence of an external electric field on the adsorption of a protein to a base metal oxide surface, using lysozyme (LSZ) and six types of base metals (St, Ti, Ta, Zr, Cr, and Ni) as model substrates. ...
... When the applied surface potential was increased to +0.4 V vs Ag/AgCl, the slow adsorption process continued over the time period for the test (∼7000 s). Such a consecutive adsorption of a protein under certain external electric fields has been frequently reported in the literature [24,25]. The attained amounts of adsorbed ß-Lg on a stainless steel surface in the presence of an external electric field were determined at different pHs (4.0, 5.8 and 7.0) and are shown as a function of the applied surface potential in Fig. 2. The amount adsorbed at 7000 s is plotted in Fig. 2 in the case where the amount adsorbed continued to increase (closed keys in Fig. 2). ...
The effect of the properties of a protein on its adsorption to a metal surface in the presence of external electric potential was investigated. Protein adsorption processes at different surface potentials were measured for fifteen types of proteins using an in-situ ellipsometry. The tested proteins were classified into three groups, based on the amount of protein that was adsorbed as a function of the surface potential: In First group of proteins, an increasing trend for the amount adsorbed with a more positive surface potential was found; The amount adsorbed of α-chymotrypsinogen A and ribonuclease A (Second group) were roughly constant and independent of the applied surface electric potentials; In Third group, the amount adsorbed decreased with increasing surface potential. This protein classification was correlated with the isoelectric points of the proteins (First group: ≤9.3; Second group: 9.3-10; Third group: >10). Increasing the pH positively and negatively shifted the surface potentials, allowing ß-lactoglobulin (First group) and lysozyme (Third) to become adsorbed, respectively. The surface potential range for protein adsorption was also markedly shifted depending on the metal substrate type. These findings were interpreted based on the electrostatic interactions among the protein, surface hydroxyl groups, and the applied external electric field.
... Experiments dealing with the adsorption of proteins to nanomaterials can be performed in either static (batch experiments) [16,108] or dynamic fashion. The latter methodology allows calculating not only the adsorbed amount but also the adsorption rate, which could be then used to calculate the probability of attachment for a protein under specific experimental conditions [120][121][122]. Among other techniques [123][124][125][126] that have been applied, adsorption kinetics of proteins can be investigated using quartz crystal microbalance (QCM) [92,[127][128][129][130] or ellipsometry [131,132]. ...
... BSA has also been adsorbed to the surface of PMMA nanoparticles to enhance the subsequent attachment of GOx by electrostatic interactions, retaining most of the activity of the free enzyme [308,309]. It is also important to mention that Benavidez et al. [121] recently reported the possibility to induce the accumulation of multiple layers of BSA by the application of an external potential, a phenomenon that is compatible with the polarization of the protein described by other authors [310]. ...
... Jedoch galt dies vornehmlich für Elektrosorption der SRNOM und nur in sehr geringem Maße für jene des BB-Moleküls[152]. Dies kann damit begründet werden, dass insbesondere die Ladung von NOM-Molekülen stark abhängig von der Ionenstärke ist[153]. Mit zunehmender Ionenstärke verlieren die NOM-Substanzen aufgrund ihres molekularen Aufbaus an Ladung[35,154], was jedoch nicht für das BB-Molekül gilt.Neben der Ionenstärke hatte auch der pH-Wert einen Einfluss auf die Elektrosorption von SRNOM (Abbildung 7.7-D), aber nicht auf die Elektrosorption des BB-Farbstoffes (Abbildung 7.7-C). ...
Humanity is facing a water crisis due growing water demand of an increasing population, economic development, water pollution, and climate change. Ultrafiltration (UF) is a promising technology for countering the global water crisis due to its high removal potential for pathogens and turbidity at high recovery and low demand of chemicals. However, it suffers from membrane fouling and low removal performance for organic water constitutes such as natural organic matter (NOM). The selectivity for NOM increases when the pore size of the UF membrane is decreased but this also leads to reduction of permeability and energy efficiency. This problem is called the selectivity-permeability trade-off. Electrically conductive UF membranes are a new approach which might offer a solution to these problems. Most NOM are negatively charged. By application of a negative or positive electrical potential to the membrane surface, a repulsive or attractive force is induced on the charged substances in the feed water, respectively, which influences the rejection and fouling behavior of these membranes.
In this work, an ultra-thin gold coating was applied on the active and support layer of flat-sheet polymer membranes to achieve electrical conductivity. Due to the coating of both sides of the membrane, no additional counter electrode was necessary to apply an external potential. An intrinsically negatively charged polyethersulfone (PES, UP150) and an intrinsically positively charged polyamide (M5) membrane were used for electro-repulsive and electro-sorptive filtration experiments, respectively.
In the first part of this thesis, the membranes were characterized before and after the gold-coating regarding its filtration and electrochemical properties. Sputter coating only slightly changed the filtration properties of the membranes. The molecular weight cut-off was almost not affected by the gold coating. However, the pure-water permeability was reduced by 15 % and 40 % for the M5 and UP150 membrane, respectively.
In the second part of this thesis, electro-repulsive filtration was conducted with model NOM solutions and natural lake water with the UP150 membrane. At filtration of Hohloh lake water the permeability decreased by 49 % (± 2 %) when no external potential was applied (0 V). However, when negative potential was applied the permeability only decreased by 17 % (± 3 %) (at -2.5 V). The application of negative potential to the membrane active layer led to less fouling and an increased NOM rejection at cross-flow mode. The molecular weight cut-off was shifted from 150 kDa at no applied potential to 5 kDa at -2.5 V (cell potential). Therefore, it could be seen that the duplex-coated membrane configuration was almost as effective as conventional counter electrode configuration in fouling mitigating and rejection enhancement.
In the third part, electro-sorptive dead-end filtration experiments showed that the application of a positive potential led to adsorption of NOM and negatively charged organic dye molecules. When the potential was reversed to negative potential, the previously adsorbed substances could be desorbed. The process of electrosorptive UF worked with the intrinsically positively charged M5 membrane but not with the intrinsically negatively charged UP150. The molecular weight cut-off of the M5 membrane was shifted from approx. 1000 kDa at no applied potential to approx. 0.7 kDa at +2.5 V (cell potential). Therefore, the electrosorptive UF achieved a NOM rejection performance in the range of commercially available nanofiltration membranes. At the same time, the positively charged M5 membrane showed permeability in the range of loose UF membranes and fouling was not observed to be problematic. The additional energy consumption for the application of the external potential was low with 0.03 kWh/m³ of permeate.
Overall, electro-repulsive and electro-sorptive UF membranes, both, broke the selectivity-permeability trade-off of the UF process. Whereas, the electrosorptive enhancement of NOM removal was more pronounced than the electro-repulsive.
... Increasing ionic strength has a substantial impact on the sorption capacity of NOM but only minor impact on BB dye (Xing et al., 2012) electrosorption (Fig. 13A&B). At an ionic strength of more than 10 mmol/L, sorption capacity of NOM decreases significantly (Benavidez & Garcia, 2013). This is because the charge of NOM is more sensitive to the presence of ions than the charge of BB dye. ...
Negatively charged electrically conductive ultrafiltration (UF) membranes have been intensively investigated for fouling mitigation and rejection enhancement in recent years. This study reports the novel approach of applying positive charge (+2.5 V cell potential) to a conductive membrane to induce electrosorption of negatively charged substances onto the membrane. Subsequently, desorption of negatively charged substances is achieved by changing the potential periodically (e.g., after 30 min) to negative charge (-2.5 V cell potential). For this purpose, sputter deposition of ultra-thin gold layers (40 nm) is used to generate electrically conductive gold-polymer-gold flat sheet membranes by coating the active and the support layer of two commercial polymer UF membranes (polyethersulfone UP150, polyamide M5). When M5 membrane was charged positively during filtration (+2.5 V), Suwannee River NOM, Hohloh lake NOM, humic acid and Brilliant Blue ionic dye showed removal rates of 70 %, 75% and 93% and 99%, respectively. Whereas, when no potential was applied (0 V) removal rates were only 1 – 5 %. When a positive potential was applied to the active membrane layer and a negative potential was applied to the support layer (cell potential 2.5 V), a significant increase of flux with 25 L/(m² h) was observed due to the induction of electro-osmosis. Electrosorption was only observed for M5 membrane (ζ: +13 mV, pH 7) and not with UP150 membrane (ζ: -29 mV, pH 7). Due to a low current density of 1.1 A/m² at a flux of 100 L/(m² h), the additional energy consumption of electrosorption and desorption process was low with 0.03 kWh per m³ of permeate. This study delivered the proof of concept for the novel process of electrosorptive UF with energy consumption between microfiltration and ultrafiltration but NOM removal rates of nanofiltration membranes.
... This behaviour of BSA at positively charged surfaces was corroborated by contact angle measurements at anodic potential (Fig. 7C) and also in the literature [69,70]. Other research groups investigated the electrosorption and desorption of BSA and other proteins onto different electrode materials [71,72] with the result that BSA is electro-adsorbed easily at low positive potentials. Furthermore, it is reported [73] that the application of positive potential leads to severe adsorption of negative charged proteins on gold electrodes which -in contrast to other negatively charged colloids -cannot be desorbed by reversing the potential. ...
Recent studies showed that the application of an electrical potential onto an ultrafiltration membrane surface exhibit several advantages with respect to the fouling and rejection behaviour. Sputter deposition of ultra-thin metal layers onto commercial flat sheet membranes proved to be a simple way of producing conductive metal-polymer-composite membranes. Adopting the novel approach of duplex-coating the active and support layer of a flat sheet membrane eliminates the need for an additional counter electrode and significantly simplifies module design for conductive flat sheet systems. Cross-flow filtration experiments were conducted with organic model foulants such as sodium alginate, bovine serum albumin (BSA), humic acids as well as with natural organic matter (NOM) from Hohloh lake water. The duplex-coating induced an additional electric field through the membrane itself, resulting in enhanced performance not only for cathodic but also for anodic charging of the active membrane layer. Hohloh lake water fouling experiments showed decreased permeability of 47% for the uncharged membrane to 16% and 38% for the cathodic and anodic charging, respectively. Moreover, a significant increase in NOM-rejection was observed with 27% for the uncharged membrane and 72% and 49% for the cathodic and anodic potentials, respectively. Finally, the effectiveness of externally charged duplex-coated membranes was demonstrated by comparing rejection and fouling rates with conventional membrane-electrode vs. counter electrode configuration, showing an almost similar enhancement in performance.
... The first insight into biofilm development was achieved by following the change in the OCP previous to any other electrochemical test. The OCP measurement is a recurrent practice in electrochemistry because this parameter could be related to biofilm stability (bacteria attachment to FTO), the redox environment and the charge interactions between the FTO, the biofilm and the electrolyte [39,40]. Fig. 1, shows the OCP value of FTO electrodes with and without biofilm at different incubation times. ...
Geobacter sulfurreducens is a model organism for understanding the role of bacterial structures in extracellular
electron transfer mechanism (EET). This kind of bacteria relies on different structures such as type IV pili and
over 100 c-type cytochromes to perform EET towards soluble and insoluble electron acceptors, including
electrodes. To our knowledge, this work is the first electrochemical study comparing a G. sulfurreducens PilRdeficient mutant and wild type biofilms developed on fluorine-doped tin oxide (FTO) electrodes. Open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), were used to evaluate
the electroactive properties of biofilms grown without externally imposed potential. Parallel studies of Confocal
Laser Scanning Microscopy (CLSM) correlated with the electrochemical results. PilR is a transcriptional regulator
involved in the expression of a wide variety of genes, including pilA (pilus structural protein) relevant c-type cytochromes and some other genes involved in biofilm formation and EET processes. Our findings suggest that PilRdeficient mutant forms a thinner (CLSM analysis) and less conductive biofilm (EIS analysis) than wild type,
exhibiting different and irreversible redox processes at the interface (CV analysis). Additionally, this work reinforces some of the remarkable features described in previous reports about this G. sulfurreducens mutant.
... It has been immobilized onto different types of membranes via covalent grafting 5 , and adsorption onto transparent electrodes. 6 However, surface leaching, low wettability, biofouling of nonspecific proteins, bacteria adhesion and adsorption of hydrophobic analytes are the major problems associated with protein based substrates. 7 These flaws further result in lower sensitivity and short stability of biomedical devices and implants. ...
The present study was aimed to investigate the direct electrooxidation of bovine serum albumin (BSA) without stabilizer and adsorbent to design antifouling screen printed carbon interface. Herein, a simple strategy revealed good immobilization support for biological molecules, better conducting transducer interface, and above all improved antifouling properties which are typically contradictory in one material. BSA was electro grafted onto the carbon surface of screen printed electrode in presence of NBu4BF4. Furthermore, to inhibit non-specific adsorption diethanolamine (DEA) was used as a blocking agent followed by adsorption studies. The comprehensive study of various BSA protein concentrations, pH buffer solutions and interaction of different analyte molecules such as DNA and toxin molecules was conducted for the adopted procedure as model analytes. The BSA grafting protocol was also validated by comparing the analyte adsorptions with simply drop casted modifications. The obtained results not only demonstrated the antifouling characteristics of the transducer surface but also proved the obtained transducer surface as a conductive immobilization support. Most of the medical devices face complications and failures due to nonspecific biofouling of microorganisms, cells and proteins. Towards imparting these devices with nonfouling capabilities, this stabilizer free SBA grafting may result a highly promising bio-surface for biomedical and engineering applications.
... To demonstrate the possible application of carbon electrodes fabricated from pyrolyzed paper, uric acid biosensors were developed by adsorbing uricase onto the substrates [66,67]. After adsorbing the enzyme, each electrode was transferred to the electrochemical cell containing 35 mL phosphate buffer solution (10 mmol L −1 , pH = 8.5) and the electrochemical response was recorded by chronoamperometry. ...
Electrostatic and electrochemical separations offer significant potential in the deionization of water as either stand‐alone processes or pre‐/posttreatment processes for achieving targeted water quality. This article critically reviews these electrically driven methods for the desalination of water. We begin by chronicling the historical development of electrostatic and electrochemical separation processes in both research and commercial applications. Further, we review the fundamental transport and reaction phenomenon critical to process performance and define metrics for assessing this performance. The subsequent two sections provide a deeper review of CDI (electrostatic) and electrosorption (electrochemical ion removal), and electromembrane separation systems, such as electrodialysis (ED), electrodialysis reversal (EDR), and electrodeionization (EDI), as well as membrane‐assisted electrosorption systems. For each of these electrostatic or electrochemical separation methods, we discuss the process fundamentals, key challenges associated with these technologies as well as recent innovations in materials and process design that are overcoming historical limits. Finally, we conclude with a discussion of practical applications of these methods and summarize opportunities to advance electrochemical processes for materially and energy‐efficient ionic separations.
Water stress must be addressed by developing cost-effective wastewater treatment technologies. Reactive electrochemical membranes (REM) have gained popularity in this scenario due to the benefits of both electrochemical advanced oxidation processes (EAOP) and membrane filtration processes (MFP) in reducing mass transport limitations and fouling issues. In this context, this review aims to consolidate recent advances in REM, focusing on sub-stoichiometric titanium oxide-based REM (TiSO-REM), mainly Ti4O7, the most promising ceramic material due to its high conductivity and ability to generate •OH. The primary fabrication methods for TiSO porous anodes are discussed, resulting in a significant reminder of their electrocatalytic, porosimetric and physicochemical intrinsic properties. In addition to the real advantages of using this material in flow-through configurations, which allow convection-enhanced mass transport and provide a high electrochemical surface area. The latest progresses in TiSO-REM are presented, with specific emphasis on understanding the main operating parameters in performance efficiency: the determinative key role among pollutant nature, matrix effect and anode in removal mechanisms, and where the anode is in relation to the cathode in the reactor design. These points are intended to identify process limitations and challenges to be overcome when implementing various reactor configurations until the scaling-up. All these aspects are reviewed critically in order to provide future research directions for the industrial advancement of this technique, which has proven to be more energy-efficient and therefore economically feasible than alternative EAOP.
In vivo sensing based on implantable microelectrodes has been widely used to monitor neurochemicals due to its high spatial and temporal resolution and engineering interface designability, which has become a powerful drive to decode the mysteries of degenerative diseases and regulate neural activity. Over the past few decades, with the development of a variety of advanced materials and technologies, encouraging progress has been made in quantifying various neurochemical transients. However, because of the complex chemical atmosphere including thousands of small and large biomolecules and the inherent low mechanical property of brain tissue, the design of a compatible microelectrode for the in vivo electrochemical tracking of neurochemicals with high selectivity and stability still faces great challenges. This Perspective presents a brief account of recent representative progress in the rational regulation of the microelectrode interface to resolve the questions of selectivity and sensitive decrease resulting from antiprotein adsorption, and how to decrease the mechanical mismatch of an implanted electrode with that of brain tissue. Possible future research directions on further addressing the above key issues and a more biocompatible microelectrode for in vivo long-time electrochemical analysis are also discussed.
ConspectusThe development of in vivo analytical tools and methods for recording electrical signals and accurately quantifying chemical signals is a key issue for a comprehensive understanding of brain events. The electrophysiological microelectrode was invented to monitor electrical signals in free-moving brains. On the other hand, electrochemical assays with excellent spatiotemporal resolution provide an effect way to monitor chemical signals in vivo. Unfortunately, the in vivo electrochemical biosensors still have three limitations. First, many biological species such as reactive oxygen species (ROS) and neurotransmitters demonstrate large overpotentials at conventional electrodes. Thus, it is hard to convert the chemical/electrochemical signals of these molecules into electric signals. Second, the interfacial properties of the recognition molecules assembled onto the electrode surfaces have a great influence on the transmission of electric charge through the interface and the stability of the modified recognition molecules. Meanwhile, the surface of biosensors implanted in the brain is easily absorbed by many proteins present in the brain, resulting in the loss of signals. Finally, activities in the brain including neuron discharges and electrophysiological signals may be affected by electrochemical measurements due to the application of extra potentials and/or currents.This Account presents a deep view of the fundamental design principles and solutions in response to the above challenges for developing in vivo biosensors with high performance while meeting the growing requirements, including high selectivity, long-time stability, and simultaneously monitoring electrical and chemical signals. We aim to highlight the basic criteria based on a double-recognition strategy for the selective biosensing of ROS, H2S, and HnS through the rational design of specific recognition molecules followed by electrochemical oxidation or reduction. Recent developments in designing functionalized surfaces through a systematic investigation of self-assembly with Au-S bonds, Au-Se bonds, and Au≡C bonds for facilitating electrochemical properties as well as improving the stability are summarized. More importantly, this Account highlights the novel methodologies for simultaneously monitoring electrical and chemical signals ascribed to the dynamic changes in K+, Na+, and Ca2+ and pH values in vivo. Additionally, SERS-based photophysiological microarray probes have been developed for quantitatively tracking chemical changes in the live brain together with recording electrophysiological signals.The design principles and novel strategies presented in this Account can be extended to the real-time tracking of electrical signals and the accurate quantification of more chemical signals such as amino acids, neurotransmitters, and proteins to understand the brain events. The final part also outlines potential future directions in constructing high-density microarrays, eventually enabling the large-scale dynamic recording of the chemical expression of multineuronal signals across the whole brain. There is still room to develop a multifiber microarray which can be coupled with photometric methods to record chemical signals both inside and outside neurons in the live brains of freely moving animals to understand physiological processes and screen drugs.
Under the most common experimental conditions, the adsorption of proteins to solid surfaces is a spontaneous process that leads to a rather compact layer of randomly oriented molecules. However, controlling such orientation is critically important for the development of catalytic surfaces. In this regard, the use of electric fields is one of the most promising alternatives. Our work is motivated by experimental observations that show important differences in catalytic activity of a trypsin-covered surface, which depended on the applied potential during the adsorption. Even though adsorption results from the combination of several processes, we were able to determine that (under the selected conditions) mean-field electrostatics play a dominant role, determining the orientation and yielding a difference in catalytic activity. We simulated the electrostatic potential numerically, using an implicit-solvent model based on the linearized Poisson-Boltzmann equation. This was implemented in an extension of the code PyGBe that included an external electric field, and rendered the electrostatic component of the solvation free energy. Our model (extensions available at the Github repository) allowed estimating the overall affinity of the protein with the surface, and their most likely orientation as a function of the potential applied. Our results show that the active sites of trypsin are, on average, more exposed when the electric field is negative, which agrees with the experimental results of catalytic activity, and confirm the premise that electrostatic interactions can be used to control the orientation of adsorbed proteins.
Understanding the phenomenon of protein adsorption on electrified conducting polymer‐based electrodes is an important issue in organic bioelectronics. To investigate both specific and nonspecific protein adsorption on electrified electrodes, C‐reactive protein (CRP) binding behavior is measured in situ and is compared with the nonspecific binding of bovine serum albumin and lysozyme on electrified phosphorylcholine‐functionalized poly(3,4‐ethylenedioxythiophene) (poly(EDOT‐PC)) by using an electrochemical quartz crystal microbalance with dissipation (EQCM‐D) and an electrochemical atomic force microscopy (EC‐AFM). According to the result of EQCM‐D, an unexpected enhancement is observed in the CRP binding on the electrified poly(EDOT‐PC) when both −0.5 and 0.5 V (versus Ag/AgCl) potentials are applied to poly(EDOT‐PC). Furthermore, an EC‐AFM is used to in situ map the surface topography and modulus of poly(EDOT‐PC) under the application of surface potentials. Together with EQCM‐D measurements, the large enhancement of CRP binding can be visualized as the formation of a loose and thick CRP multilayer of low surface modulus, high root‐mean‐square roughness, and high dissipation when a potential of 0.5 V is subjected to poly(EDOT‐PC). It is concluded that the enhancement in CRP binding is mainly attributed to the synergistic effect of specific protein recognition and electrostatic interaction between the CRP and poly(EDOT‐PC). In this study, an electrochemical quartz crystal microbalance with dissipation and an electrochemical atomic force microscopy is utilized to investigate the recognition and nonspecific binding of proteins on electrified zwitterionic conducting polymers. An unusual protein binding behavior is attributed to the synergistic effect of specific protein recognition and electrostatic interaction between proteins and zwitterionic conducting polymers.
We investigated an alternate technique to coat the surface with a protein having no surface affinity, without the use of any exotic chemical agents. An external electric field was utilized to prepare the protein coating on a metal substrate. Stainless steel (St) substrate and lysozyme (LSZ) were used as the surface to be coated and the model non-adsorptive protein, respectively. Dynamics of the adsorption of LSZ on the St surface in the presence and absence of an external electric potential (EEP) were monitored by in-situ ellipsometry. Applying negative surface potential (-0.4 V vs Ag/AgCl) forced the adsorption of LSZ onto the St surface where LSZ did not adsorb without applying any EEP. The repetition of the EEP-application and -cut-off indicated the controllability of the LSZ coating amount depending on the total duration of the EEP-application. The coated LSZ largely remained bound to the surface even by the cut-off of the external electric field, the ratio of which to the detached amount was roughly constant (approximately 7:3). Furthermore, the LSZ coated surface on the St substrate was found to be reversibly switched between being affinitive and non-affinitive to a typical model protein adsorbate (bovine serum albumin) by the EEP-application and cut-off.
A method for detection of causative agent of tularemia in aqueous solutions by surface plasmon resonance spectrometry was proposed. The kinetics of immobilization of antibodies to Francisella tularensis on the surface of gold-plated glass chips was studied. Based on a quantitative analysis of the results, it was concluded that the antibody molecules were predominantly laterally oriented on the surface. This conclusion was confirmed by analysis of sensorgrams of the subsequent antigen–antibody interaction. It is shown that the proposed method can reliably determine the presence of the causative agent of tularemia in aqueous solutions with a sensitivity of 1.0 × 10² cell/mL for several minutes. Various options for improving this method for detection of the tularemia causative agent to increase its sensitivity have been proposed.
This work presents a detailed investigation of interface interactions between natural rubber (NR) particles and solid surfaces in aqueous medium at high ionic strength (0.1 M) using AFM in fast force spectroscopy mode. In this study, an original method for fixing the NR on the substrate was developed. This avoided the usual perturbations common in standard immobilization techniques. We proved that the adhesion process of the NR is monitored by slight changes in the surface charge state of the contacting solid surfaces made of silicon oxide or silicon nitride. The results were interpreted using Dynamic Force Spectroscopy theory, with the introduction of a supplementary term describing the electrostatic energy. Furthermore, these experiments revealed that adhesion between NR and tip was time dependent in a cumulative process. In addition, an increase of the adhesion between NR and AFM tip with the size of the rubber particles was measured. This was related to the higher concentration in lipids versus proteins for larger NR particles. These results are of great importance both for practical applications in solution-based industrial processes and to the fundamental knowledge of adhesion process involved for biopolymers or living cells.
Enzymatic cleaning is a potentially useful method for removing proteinaceous fouling from solid surfaces under mild conditions. Herein, the influence of an external electric field on the enzymatic cleaning of a metal surface fouled with a protein was investigated. The model fouling protein (BSA; lysozyme) was prepared on a stainless steel (St) surface, and the resulting surface subjected to enzymatic cleaning with an electric potential being applied to the St plate. Trypsin, α-chymotrypsin, and thermolysin were used as model proteases. The amounts of protein remaining on the plate before and during the cleaning process were measured by means of a reflection absorption technique using Fourier transform infrared spectroscopy. In the case for BSA fouling, the cleaning efficiency of the protease tended to increase at more negative applied potentials. Whereas, there was an optimum applied potential for removing the lysozyme fouling. Atomic force microscopy analyses indicated that applying an adequate range of electric potential enhanced the enzymatic removal of protein fouling inside scratches on the St plate surface. These findings suggest the existence of two modes of electrostatic interactions for the external electric field, one with protease molecules and the other with digested fragments of the fouling protein.
As a viable alternative with respect to carbon-based materials prepared by vapor deposition, the pyrolysis of non-volatile organic precursors has allowed the development of substrates with advantageous properties towards the development of sensors. Considering the importance and versatility of these materials, this review provides a summary of representative articles describing the procedures and most important considerations linked to the fabrication of these films, their characterization (structure, thickness, topography, contact angle, as well as optical and electrochemical properties). The review focuses on analytical applications (electroanalysis, biosensors, dielectrophoresis, and solid phases for separations) published in the last five years but additional contributions outside this period have been included to provide readers background information to link the chemical functionality of the films with the corresponding performance. Without aiming to make a prediction, a series of potential directions for the future of the field are also described.
A model system based on bovine serum albumin (BSA)-laden drop is selected. AC ElectroWetting-On-Dielectrics (EWOD), increasingly used in digital microfluidics for medical assays, is implemented in coplanar configuration. The moderate frequency required by EWOD actuation enables shape oscillations of the BSA-laden sessile drop and promotes internal micro-streaming. The non reversible impact of EWOD-induced micro-streaming upon surface pressures is investigated. In contrast to the liquid-gas drop surface where thermodynamical equilibrium is instantaneously achieved, the adsorption of BSA molecules at the lubricated solid substrate can be characterized by a time-dependent solid-liquid surface pressure. As demonstrated from dynamic contact angle (DCA) imaging, adsorption of BSA is promoted under AC EWOD. A significant dependence of the solid-liquid surface pressure on the resonant capillary frequencies is observed: for a large enough actuation frequency corresponding to an oscillating mode, n = 6, numerical simulations are performed which show that corner eddies located in the Stuart boundary layer, near the oscillating contact line, yield an efficient molecular renewal. In this way, oscillating EWOD could be used purposely as a tool to speed up label-free detection from DCA measurements.
The objective of this work is to investigate the rate, extent, and structure of amphoteric proteins with charged solid surfaces over a range of applied potentials and surface charges. We use Electrochemical Quartz Crystal Microbalance with Dissipation Monitoring (E-QCM-D) to investigate the adsorption of amphoteric Bovine Serum Albumin (BSA) to a gold electrode while systematically varying the surface charge on the adsorbate and adsorbent by manipulating pH and applied potential, respectively. We also perform cyclic voltammetry-E-QCM-D on an adsorbed layer of BSA to elucidate conformational changes in response to varied applied potentials. We confirm previous results demonstrating that increasing magnitude of applied potential on the gold electrode is positively correlated with increasing mass adsorption when the protein and the surface are oppositely charged. On the other hand, we find that the rate of BSA adsorption is not governed by simple electrostatics, but instead depends on solution pH, an observation not well documented in the literature. Cyclic voltammetry with simultaneous E-QCM-D measurements suggest that BSA protein undergoes a conformational change as the surface potential varies.
During the last few decades, electrochemistry and electrode modification have seen a tremendous fall off in creativity with the emergence of the nanoarchitectonic-based layer-by-layer (LbL) film deposition technique. An unprecedented variety of building blocks can be immobilized on surfaces, leading to progress in several fields including sensing, electrochromic, electro-responsive and energy devices. This review describes the state of the art of electrochemical devices based on LbL assemblies, with a focus on supercapacitors, biosensors, and electroresponsive LbL such as electrodissolution/electroswelling of coatings. Recently, electrochemistry has also been used as an “active trigger” to induce the formation of films by covalent coupling, leading to new nanoarchitectonic approaches beyond the LbL strategy. These emerging electro-coupling reactions, including electroclick and carbazole chemistry, open new perspectives toward architecture and patterning of functional films and are extensively reviewed.
The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800mV to the sorbent surface induced the formation of multiple layers of protein. These results also suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.
This paper describes a simple and inexpensive procedure to produce thin-films of poly(dimethylsiloxane). Such films were characterized by a variety of techniques (ellipsometry, nuclear magnetic resonance, atomic force microscopy, and goniometry) and used to investigate the adsorption kinetics of three model proteins (fibrinogen, collagen type-I, and bovine serum albumin) under different conditions. The information collected from the protein adsorption studies was then used to investigate the adhesion of human dermal microvascular endothelial cells. The results of these studies suggest that these films can be used to model the surface properties of microdevices fabricated with commercial PDMS. Moreover, the paper provides guidelines to efficiently attach cells in BioMEMS devices.
Atomic Force Microscope images and other experiments show us that very small stable bubbles, known as nanobubbles, can be present on surfaces despite well founded theoretical considerations that predict otherwise. Nanobubbles are thought to play a role in the rupture of thin films during froth flotation, hydrodynamic slip over surfaces, interaction forces between hydrophobic surfaces and influence the electroplating and electrolysis processes. Here we describe what is known of nanobubbles and discuss the challenges in understanding nanobubble morphology and stability.
Using neutron reflectivity together with an appropriate electrochemical cell,
we have studied the effects of transverse electric field on the Bovine Serum
Albumin (BSA) monolayer initially adsorbed at the interface of the aqueous
solution and a conductive doped-silicon wafer. Depending on the sign of the
initial potential, a second layer is adsorbed on top of the first whereas a
subsequent reversal of potential has no effect. We show that this behavior
reveals the slow and remanent electric polarization of the first BSA layer and
suggest an analogy with spin glasses based on the dipolar structure of this
protein.
The points of zero charge/potential of proteins depend not only on pH but also on how they are measured. They depend also on background salt solution type and concentration. The protein isoelectric point (IEP) is determined by electrokinetical measurements, whereas the isoionic point (IIP) is determined by potentiometric titrations. Here we use potentiometric titration and zeta potential (ζ) measurements at different NaCl concentrations to study systematically the effect of ionic strength on the IEP and IIP of bovine serum albumin (BSA) aqueous solutions. It is found that high ionic strengths produce a shift of both points toward lower (IEP) and higher (IIP) pH values. This result was already reported more than 60 years ago. At that time, the only available theory was the purely electrostatic Debye-Hückel theory. It was not able to predict the opposite trends of IIP and IEP with ionic strength increase. Here, we extend that theory to admit both electrostatic and nonelectrostatic (NES) dispersion interactions. The use of a modified Poisson-Boltzmann equation for a simple model system (a charge regulated spherical colloidal particle in NaCl salt solutions), that includes these ion specific interactions, allows us to explain the opposite trends observed for isoelectric point (zero zeta potential) and isoionic point (zero protein charge) of BSA. At higher concentrations, an excess of the anion (with stronger NES interactions than the cation) is adsorbed at the surface due to an attractive ionic NES potential. This makes the potential relatively more negative. Consequently, the IEP is pushed toward lower pH. But the charge regulation condition means that the surface charge becomes relatively more positive as the surface potential becomes more negative. Consequently, the IIP (measuring charge) shifts toward higher pH as concentration increases, in the opposite direction from the IEP (measuring potential).
This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the "inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions.
Using constant current chronopotentiometry we showed that in 50 mM sodium phosphate (pH 7) bovine serum albumin and some other proteins were not significantly denatured at a bare mercury electrode while at higher phosphate concentrations they underwent electric field-driven denaturation on the electrode surface.
Modification and conjugation techniques are dependent on two interrelated chemical reactions: the reactive functional groups present on the various cross-linking or derivatizing reagents and the functional groups present on the target macromolecules to be modified. Without both these types of functional groups being available and chemically compatible, the process of derivatization would be unfeasible. The reactive functional groups on cross-linking reagents, tags, and probes provide the means to label specifically certain target groups on ligands, peptides, proteins, carbohydrates, lipids, synthetic polymers, nucleic acids, and oligonucleotides. Knowledge of the main mechanisms by which the reactive groups couple with target functional groups provides the ways to design a modification or conjugation strategy. The basis for successful chemical modification is choosing the correct reagent systems that can react with the chemical groups available on target molecules. The principal reactive functional groups commonly encountered on bioconjugate reagents are now present on scores of commercially obtainable compounds. This chapter describes the major targets for these reagent systems. The functional groups discussed are found on virtually every conceivable biological molecule, including amino acids, peptides, proteins, sugars, carbohydrates, polysaccharides, nucleic acids, oligonucleotides, lipids, and complex organic compounds.
Albumin is clearly an extraordinary molecule of manifold functions and applications. Although the exact function of albumin has been debated, much of the present data support the notion that the principal role of albumin in the circulatory system is to aid in the transport, metabolism, and distribution of exogenous and endogenous ligands. The ability of albumin to act as an important extracellular antioxidant (Halliwell, 1988) or impart protection from free radicals, and other harmful chemical agents (Emerson, 1989) agrees well with the increased susceptibility of analbuminemic rats to cancer (Kakizoe and Sugimura, 1988). The expression and delivery of albumin to the circulatory system by the liver therefore seem appropriate. An overview of the prolific ligand-binding properties of albumin is summarized in Fig. 21. The positions of known binding sites for important pharmaceutical markers such as diazepam, ibuprofen, aspirin, and warfarin are illustrated. In addition, the important endogenous markers tryptophan, octanoate, and bilirubin are also shown. With the exception of the definitive positions of the long-chain fatty acids, most albumin-ligand chemistry can now be explained by the atomic coordinates derived from crystal structures. Knowledge of the atomic structure coupled with the current applications of genetic engineering, such as site-directed mutagenesis, promises to provide an even greater understanding of albumin chemistry. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficacy of many drugs can be altered based on their affinity to serum albumin. In addition, many promising new drugs are rendered ineffective because of their unusually high affinity for this abundant protein. Obviously, an understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design, placing albumin in its rightful place as the 'second step in rational drug design.' Application of albumin in other therapeutic approaches is widely known. Some studies have suggested that modified serum albumin may be used as a selective contrast agent for tumor detection and/or therapy (Sinn et al., 1990). Other studies have demonstrated that albumin may be used to deliver toxic compounds for elimination of Mycobacterium tuberculosis via receptor-mediated drug delivery (Majumdar and Basu, 1991). Recently, chimeric albumin molecules such as HSA-CD4 (Yeh et al., 1992) and HSA-Cu,Zn-superoxide dismutase (Mao and Poznansky, 1989) have been utilized to increase the half-life and distribution, and reduce the immunogenicity, of these potential protein therapeutics. Albumin has now been cloned and expressed in several bacterial and fungal systems. The primary motivation for many of these studies has been the potential of recombinant albumin to serve as a serum replacement product that is free from unwanted viral contaminants, e.g., hepatitis, herpes, and human immunodeficiency virus (HIV). The most successful production has been achieved by extracellular expression in yeast (Etcheverry et al., 1986; Hinchcliffe and Kenney, 1986; Kalman et al., 1990; Okabayashi et al., 1986; Quirk, et al., 1989; Sijmons et al., 1990; Sleep et al., 1991). Clearly, future scientific and therapeutic applications of albumin appear limitless. In conclusion, albumin may be unique among proteins in that so many scientists have spent the largest portion of their professional careers studying very specific aspects of this protein. New appreciation for the complexity and potential applications presented by the structure of albumin promises to consume the careers of many more scientists.
The controlled surface placement of protein molecules represents a crucial step toward many new biotechnological devices and processes. A promising means of directing the structure and formation rate of an adsorbed protein layer is through an applied electric potential difference. We present here a method for continuously measuring the protein adsorption under a direct current voltage using optical waveguide lightmode spectroscopy. An indium tin oxide-coated waveguiding sensor chip serves as the anode and adsorbing substrate, and a platinum counter electrode serves as the cathode in a parallel plate arrangement. For (negatively charged) human serum albumin in either pure water or N-[2-hydroxyethyl]piperazine-N'-ethanesulfonic acid (HEPES) buffer, we find the transport-limited and initial surface-limited rates of adsorption to significantly increase with the applied potential. For (positively charged) horse heart cytochrome c, we observe no influence of the voltage on the transport-limited adsorption rate in either solvent and a decrease with the voltage in the initial surface-limited rate in a HEPES (but not a pure water) solvent. Interestingly, we find the rate of adsorption at moderate to high surface density to greatly increase with the voltage for both proteins; this effect is more pronounced in water than in HEPES. We attribute this enhanced adsorption to contact between electrode and protein patches of complementary charge, leading to more oriented and efficiently packed adsorbed molecules and, in the case of high voltage, to multilayer formation.
Using the perturbation treatment developed by Aspnes and Rowe [Phys.
Rev. B 5, 4022 (1972)], an analytic expression for the third-order
nonlinear optical susceptibility
χ(3)(ω;0,0,ω) is computed and analyzed for
single walled zigzag carbon nanotubes. By improving their method, our
calculations based on a tight-binding model take into account the
transitions between all pairs of valence and conduction bands and
thereby the contributions to the third-order susceptibility associated
with different energy bands are investigated. With increasing radius of
the nanotube, a nonmonotonous increase of the quadratic electro-optic
effect has been demonstrated except for the fundamental peak. The
nonuniformity is a result of the overlap between two energy bands as
well as the reduced effective masses associated with each pair of
conduction and valence bands. A nonperturbative numerical calculation is
applied to obtain the high-field response as well as to assess the
applicability of the low-field perturbation expression.
Label-free electrochemical (EC) protein biosensors that derive electrical signal from redox-active amino acid (AA) residues can avoid disruption of delicate protein structures, and thus provide a great opportunity to reveal valid information about protein functions. However, the challenge is that such a signal is usually very limited due to the sluggish EC reaction of free AAs on most common electrodes and slow electron-transfer rates from the deeply-buried AA residues in a protein to the electrode. Signal enhancement therefore becomes crucial. We first survey recent progress in this area.
We present a signal-enhancing system that relies on the electrocatalytic oxidation of tyrosine mediated by osmium bipyridine or phenoxazine complexes. We describe several applications of label-free protein EC biosensors based on this detection principle for the analysis of protein functions, including the monitoring of protein-conformation change, study of ligand/protein binding, and detection of protein oxidative damage and protein phosphorylation.
We describe related works on protein-function analysis using other signal-enhancing methods. The results suggest that label-free EC protein biosensors are suitable for the rapid survey of protein functions due to their fast response, ease of integration, cost effectiveness and convenience. Proof-of-concept work on the application of our system is paving the way for bio-analytical detections and protein-function analysis in future work.
External electric field effects on state energy and photoexcitation dynamics have been examined for a mutant of UV-excited green fluorescent protein (GFPuv5) in a PVA film. The electrofluorescence spectrum of GFPuv5 is reproduced by a linear combination between the fluorescence spectrum and its second derivative spectrum, indicating the field-induced fluorescence quenching and the difference in electric dipole moment between the fluorescent state and the ground state. The direct measurements of the field-induced change in fluorescence decay show that the field-induced quenching results from the field-induced increase in the rate of the non-radiative process from the fluorescent state.
The present paper describes the results related to the optical and electrochemical characterization of thin carbon films fabricated by spin coating and pyrolysis of AZ P4330-RS photoresist. The goal of this paper is to provide comprehensive information allowing for the rational the selection of the conditions to fabricate optically-transparent carbon electrodes (OTCE) with specific electro-optical properties. According to our results, these electrodes could be appropriate choices as electrochemical transducers to monitor electrophoretic separations. At the core of this manuscript is the development and critical evaluation of a new optical model to calculate the thickness of the OTCE by variable angle spectroscopic ellipsometry (VASE). Such data was complemented with topography and roughness (obtained by AFM), electrochemical properties (obtained by cyclic voltammetry), electrical properties (obtained by electrochemical impedance spectroscopy), and structural composition (obtained by Raman spectroscopy). Although the described OTCE were used as substrates to investigate the effect of electrode potential on the real-time adsorption of proteins by ellipsometry, these results could enable the development of other biosensors that can be then integrated into various CE platforms. This article is protected by copyright. All rights reserved.
From the Publisher:This volume presents the results of approximately 15 years of work from researchers around the world on the use of fuzzy set theory to represent imprecision in databases. The maturity of the research in the discipline and the recent developments in commercial/industrial fuzzy databases provided an opportunity to produce this survey. Fuzzy Databases: Principles and Applications is self-contained providing background material on fuzzy sets and database theory. It is comprehensive covering all of the major approaches and models of fuzzy databases that have been developed including coverage of commercial/industrial systems and applications. Background and introductory material are provided in the first two chapters. The major approaches in fuzzy databases comprise the second part of the volume. This includes the use of similarity and proximity measures as the fuzzy techniques used to extend the relational data modeling and the use of possibility theory approaches in the relational model. Coverage includes extensions to the data model, querying approaches, functional dependencies and other topics including implementation issues, information measures, database security, alternative fuzzy data models, the IFO model, and the network data models. A number of object-oriented extensions are also discussed. The use of fuzzy data modeling in geographical information systems (GIS) and use of rough sets in rough and fuzzy rough relational data models are presented. Major emphasis has been given to applications and commercialization of fuzzy databases. Several specific industrial/commercial products and applications are described. These include approaches to developing fuzzy front-end systems and special-purpose systems incorporating fuzziness.
We present results for the enhancement or retardation of the attachment of the protein soybean peroxidase to a gold substrate using electric fields. We detect the influence of the electric fields on the proteins using the surface plasmon resonance phenomenon. The gold surface on which the surface plasmon resonance is generated also acts as one of the electrodes required to apply the electric field. The second electrode has a semi-insulating layer that limits current flow, and limits electrolysis effects. The results show that at pH 7, when the soybean peroxidase is negatively charged, a greatly enhanced deposition of protein is obtained when a positive potential is applied to the gold electrode. A negative potential can inhibit protein attachment, or reduce the amount of protein attached; however, after repeated applications of a positive potential, a negative potential has little effect. Results are,presented of experiments using colloidal silica that has negatively charged particles of similar size to soybean peroxidase, showing the attraction and repulsion of negatively charged particles by the gold surface. (c) 2007 Elsevier B.V. All rights reserved.
Spectroscopic ellipsometry was used to characterize the optical properties of thin (< 5 nm) films of nanostructured titanium dioxide (TiO2). These films were then used to investigate the dynamic adsorption of bovine serum albumin (BSA, a model protein), as a function of protein concentration, pH, and ionic strength. Experimental results were analyzed by an optical model and revealed that hydrophobic interactions were the main driving force behind the adsorption process, resulting in up to 3.5 mg/m2 of albumin adsorbed to nanostructured TiO2. The measured thickness of the adsorbed BSA layer (less than 4 nm) supports the possibility that spreading of the protein molecules on the material surface occurred. Conformational changes of adsorbed proteins are important because they may subsequently lead to either accessibility or inaccessibility of bioactive sites which are ligands for cell interaction and function relevant to physiology and pathology.
Rapid oxidation processes relevant to the degradation of [4Fe4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential-pulse voltammograms of native [4Fe4S] ferredoxin showed two well-defined oxidation peaks corresponding to apparent E-values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe4Se] derivative for which peak positions were shifted. Square-wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super-oxidised’ [4Fe4S]3+ oxidation level, undergoes rapid degradation (t < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (⪢100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FES(Se) substructure.
Direct oxidation peaks relating to proteins have been clearly observed by exploiting H-terminated boron-doped diamond (BDD) elec-trodes. Hydrogen generation by reduction treatment was enabled to provide the possibility of using BDD. The pH dependence of bovine serum albumin (BSA) oxidation suggested that the oxidation involved of at least three redox-active amino acid residues (cysteine, tryp-tophan and tyrosine). Furthermore, the electrochemical detection of not only BSA but also immunosuppressive acidic protein (IAP, a cancer marker) was demonstrated by using cyclic voltammetry and flow injection analysis. A linear dynamic in the concentration range of 5 and 3000 mg/dl (r 2 = 0.9) with a lower detection limit (LOD) of 5 mg/dl was achieved for BSA. In the case of IAP, the calibration curve was linear over the concentration range from 200 to 800 lg/ml, with an experimental LOD of 100 lg/dl. Excellent reproducibility was also demonstrated, suggested that direct electrochemical detection using BDD electrodes can be performed with advantages in terms of simplicity, sensitivity and stability.
This study is the first to focus on the potential use of carbon nanotube (CNT) scaffolds as enzyme immobilization substrates for analytical purposes. Besides all the well-known advantages of CNT, three-dimensional scaffolds can significantly increase the amount of enzymes adsorbed per unit area, preserve the catalytic activity of the adsorbed molecules, and allow effective exposure to substrates present in the adjacent medium. Additionally, our results indicate that the sensitivity of analytical probes based on enzyme-loaded CNT scaffolds is proportional to the thickness of the scaffold providing 3-fold sensitivity improvements with respect to the control surfaces.
Under most conditions proteins show a strong tendency to adsorb at interfaces. The general principles underlying the interaction between proteins and solid surfaces in an aqueous environment are discussed. These principles are illustrated by experimental results obtained with well-defined systems. The approach is mainly based on thermodynamic arguments.
Quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to characterize the adsorption of the model proteins, bovine serum albumin (BSA) and fibronectin (FN), to polypyrrole doped with dextran sulfate (PPy-DS) as a function of DS loading and surface roughness. BSA adsorption was greater on surfaces of increased roughness and was above what could be explained by the increase in surface area alone. Furthermore, the additional mass adsorbed on the rough films was concomitant with an increase in the rigidity of the protein layer. Analysis of the dynamic viscoelastic properties of the protein adlayer reveal BSA adsorption on the rough films occurs in two phases: (1) arrival and initial adsorption of protein to the polymer surface and (2) postadsorption molecular rearrangement to a more dehydrated and compact conformation that facilitates further recruitment of protein to the polymer interface, likely forming a multilayer. In contrast, FN adsorption was independent of surface roughness. However, films prepared from solutions containing the highest concentration of DS (20 mg/mL) demonstrated both an increase in adsorbed mass and adlayer viscoelasticity. This is attributed to the higher DS loading in the conducting polymer film resulting in presentation of a more hydrated molecular structure indicative of a more unfolded and bioactive conformation. Modulating the redox state of the PPy-DS polymers was shown to modify both the adsorbed mass and viscoelastic nature of FN adlayers. An oxidizing potential increased both the total adsorbed mass and the adlayer viscoelasticity. Our findings demonstrate that modification of polymer physicochemical and redox condition alters the nature of protein-polymer interaction, a process that may be exploited to tailor the bioactivity of protein through which interactions with cells and tissues may be controlled.
The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.
Adsorption on solid surface is of fundamental importance in the research and development of chromatographic science and technology. Due to the limitation of the currently available experimental approaches, molecular simulation, a research tool with sufficiently small scales in both time and space, has been used to explore the molecular insights into adsorption phenomena. This article offers an overview of the molecular simulation studies of adsorption on solid surfaces. First of all, various models of adsorbents used in different chromatographic modes are reviewed, including coarse-grained models and all-atom models, depending on the description precision required and the computational power provided. In the adsorbent models, the surface morphology is visualized using Monte Carlo simulation or molecular dynamics simulation. Then, studies on the adsorption and retention behaviors of small molecules by these models and methods are summarized. Finally, emphases are focused on the application of molecular simulation to protein adsorption, including protein–surface interaction, protein orientation and conformational transition on solid surfaces. In these studies, the effects of ligand parameters, including the ligand composition, ligand length, bonding density, ligand distribution have been examined. Meanwhile, chromatographic parameters, including the mobile phase composition and temperature, have also been investigated. Based on the successful applications reviewed herein, it is concluded that molecular simulation studies have contributed to the development of adsorption and chromatography in bioseparations. Moreover, it is suggested that molecular simulation combined with computational quantum chemistry and experiments would provide more comprehensive understanding of adsorption phenomena in the future.
Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the "protein-adsorption problem" to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations C(B). This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume V(I) by expulsion of either-or-both interphase water and initially adsorbed protein. Interphase protein concentration C(I) increases as V(I) decreases, resulting in slow reduction in interfacial energetics. Steady state is governed by a net partition coefficient P=(C(I)/C(B)). In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This "adsorption-dehydration" step is the significant free energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, adsorbent capacity monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ→65(°). Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ<65(°). For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption ΔG(ads)(o) is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein adsorption to any surface by competing with adsorbing protein molecules. This Leading Opinion ends by proposing several changes to the protein-adsorption paradigm that might advance answers to the three core questions that frame the "protein-adsorption problem" that is so fundamental to biomaterials surface science.
A sensitive electrochemical procedure based on bovine serum albumin (BSA)/poly-o-phenylenediamine (PoPD)/carbon-coated nickel (C-Ni) nanobiocomposite film modified glassy carbon electrode (BSA/PoPD/C-Ni/GCE) has been developed to explore the electrochemical detection of BSA damage induced by hydroxyl radical. It is the first time that the electrochemical method has been applied for the analysis of Fenton-mediated oxidative damage to proteins. The hydroxyl radical was generated by Fenton reaction (Fe(2+)/H(2)O(2)), which was also validated by ultraviolet-visible (UV-vis) spectroscopy. The decrease in intensity of the PoPD oxidation signals was used as an indicator for the detection of BSA damage. Damage to BSA was also validated by horizontal Attenuation Total Reflection Fourier Transform Infra-red (ATR-FTIR) spectroscopy and the change of protein carbonyl group content achieved by UV-vis spectroscopy. Effects of H(2)O(2) concentration, the ratio of Fe(2+) and H(2)O(2) and incubation time on BSA damage were examined. Protections of BSA from damage by antioxidants were also investigated. These conclusions demonstrated that the proposed electrochemical method is expected to the further application for protein damage studies.
Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to
explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively
discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and
computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined.
This communication describes a simple way to improve the sensitivity of spectroscopic ellipsometry, when applied to monitor the adsorption of proteins to solid surfaces. The method described herein is based on the reaction of a commercially available dye (Coomassie brilliant blue G) with the adsorbed proteins and the subsequent analysis by spectroscopic ellipsometry. In order to demonstrate the potential advantages of this method, the adsorption of bovine serum albumin to an antifouling coating was also investigated. According to our results, the modification with the dye significantly affects the optical properties of the adsorbed protein layer, which can be represented using a simple optical model (Lorentz). In general, the proposed modification increases the sensitivity of the detection by 2.5 ± 0.4-fold and enables the analysis of thin layers of adsorbed protein not obtainable by conventional methods. These results particularly reveal the importance of the proposed modification for the evaluation of low adsorbing substrates and antifouling coatings.
Interactions between hydrophobic surfaces at nanometer separation distances in aqueous solutions are important in a number of biological and industrial processes. Force spectroscopy studies, most notably with the atomic force microscope and surface-force apparatus, have found the existence of a long range hydrophobic attractive force between hydrophobic surfaces in aqueous conditions that cannot be explained by classical colloidal science theories. Numerous mechanisms have been proposed for the hydrophobic force, but in many cases the force is an artifact due to the accumulation of submicroscopic bubbles at the liquid-hydrophobic solid interface, the so called nanobubbles. The coalescence of nanobubbles as hydrophobic surfaces approach forms a gaseous capillary bridge, and thus a capillary force. The existence of nanobubbles has been highly debated over the last 15 years. To date, experimental evidence is sound but a theoretical understanding is still lacking. It is the purpose of this review to bring together the many experimental results on nanobubbles and the resulting capillary force in order to clarify these phenomena. A review of pertinent nanobubble stability and formation theories is also presented.
There is a striking disparity between the heart-shaped structure of human serum albumin (HSA) observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH. These two contrasting views could be reconciled if the protein were flexible enough to change its conformation in solution from that found in the crystal. To investigate this possibility we recorded the rotational motions in real time of an erythrosin-bovine serum albumin complex (Er-BSA) over an extended time range, using phosphorescence depolarization techniques. These measurements are consistent with the absence of independent motions of large protein segments in solution, in the time range from nanoseconds to fractions of milliseconds, and give a single rotational correlation time (BSA, 1cP, 20C)=402ns. In addition, we report a detailed analysis of the protein hydrodynamics based on two bead-modeling methods. In the first, BSA was modeled as a triangular prismatic shell with optimized dimensions of 84848431.5Å, whereas in the second, the atomic-level structure of HSA obtained from crystallographic data was used to build a much more refined rough-shell model. In both cases, the predicted and experimental rotational diffusion rate and other hydrodynamic parameters were in good agreement. Therefore, the overall conformation in neutral solution of BSA, as of HSA, should be rigid, in the sense indicated above, and very similar to the heart-shaped structure observed in HSA crystals. This work was financed by projects PB96852 and PB961106 from the DGICYT and grant 01578/CV/98 from the Fundación Séneca (Región de Murcia). Peer reviewed
Cytochrome c (Cyt) is a small soluble heme protein with a hexacoordinated heme and functions as an electron shuttle in the mitochondria and in early events of apoptosis when released to the cytoplasm. Using molecular dynamics simulations, we show here that biologically relevant electric fields induce an increased mobility and structural distortion of key protein segments that leads to the detachment of the sixth axial ligand Met80 from the heme iron. This electric-field-induced conformational transition is energetically and entropically driven and leads to a pentacoordinated high spin heme that is characterized by a drastically lowered reduction potential as well as by an increased peroxidase activity. The simulations provide a detailed atomistic picture of the structural effects of the electric field on the structure of Cyt, which allows a sound interpretation of recent experimental results. The observed conformational change may modulate the electron transfer reactions of Cyt in the mitochondria and, furthermore, may constitute a switch from the redox function in the respiratory chain to the peroxidase function in the early events of apoptosis.
Typescript. Thesis (Ph. D.)--Wayne State University, 2001. Includes bibliographical references (leaves 144-146).
In contrast to previous reports claiming bovine serum albumin (BSA) denaturation at mercury surfaces, recently it has been shown that BSA and other proteins do not denature as a result of adsorption to the mercury electrodes at alkaline and neutral pH values. In this pH range, constant current chronopotentiometry (CPS) with mercury or solid amalgam electrodes can be used to distinguish between native, denatured and damaged BSA. Here we show that at acid pH values (around pH 4.5) native and denatured BSA yield almost the same CPS responses suggesting denaturation of native BSA at the electrode surface. Under these conditions BSA is, however, not denatured at the electrode at accumulation potentials (E(A) values) close to the potential of zero charge, but at E(A) values more negative than -0.8 V, after destabilization of the surface-attached BSA by electroreduction of some disulfide groups at about -0.48 V and by electric field effects at more negative potentials.
The interaction of beta-amyloid (Abeta) peptides with externally applied electric fields (EF) of varying strengths was investigated by means of molecular dynamics (MD) simulations. The results suggest that the EF favors the switch of Abeta-peptides from helical to beta-sheet conformation, and a mechanism is tentatively proposed. Switching off the field does not restore the original conformation.
Functionalization of carbon nanotubes (CNTs) with proteins is often a key step in their biological applications, particularly in biosensing. One popular method has used the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to covalently conjugate proteins onto carboxylated CNTs. In this article, we critically assess the evidence presented in these conjugation studies in the literature. As CNTs have a natural affinity for diverse proteins through hydrophobic and electrostatic interactions, it is therefore important to differentiate protein covalent attachment from adsorption in the immobilization mechanism. Unfortunately, many studies of conjugating proteins onto CNTs using EDC lacked essential controls to eliminate the possibility of protein adsorption. In studies where the attachment was claimed to be covalent, discrepancies existed and the observed immobilization appeared to be due to adsorption. So far, bond analysis has been lacking to ascertain the nature of the attachment using EDC. We recommend that this approach of covalent immobilization of proteins on CNTs be re-evaluated and treated with caution.
A method for determination of the orientation of adsorbed structure-stable proteins using Total Internal Reflection Fluorescence is outlined. The theory has been elaborated for orientation studies on adsorbed free base cytochrome c, of which the prophyrin can be used as an intrinsic fluorescent label. The ratio of fluorescence intensities at two polarization modes of the incident light (the transverse magnetic and the transverse electric polarization mode, respectively) gives a relation between the orientation angles of the porphyrin relative to the interface. As an illustration of the theory, experimental results on the adsorption of cytochrome c at an optically transparent SnO2 film electrode are presented. It is concluded that the orientation of the molecules can only be affected by the interfacial potential during the process of adsorption, but, once adsorbed, the orientation cannot be changed anymore by variation of the potential.
The adsorption of fibrinogen on to platinum and carbon and of albumin on to carbon was investigated for various changes of the surface by recording the variations of the double-layer capacitance of the electrochemical interface during adsorption, as a function of time. The rate of the second step of the adsorption decreased with increasing negative charge on to platinum but was charge-independent on to carbon. In contrast, on both surfaces, the rate of the first step and the area of the electrode surface in close contact with adsorbed proteins were both found to increase with increasing negative charge of the surface, although at pH 7.4 albumin and fibrinogen are negatively charged. The hypothesis of ion coadsorption inside the proteic layer has been proposed to account for this result.
Highly purified preparations of defatted bovine serum albumin monomer and dimer were prepared by gel filtration on Sephadex G-100. Sedimentation equilibrium experiments demonstrated that the monomer fraction was homogeneous with respect to mass and had an anhydrous molecular weight of 66,700 ± 400. Sedimentation velocity experiments showed that the concentration dependence of the sedimentation coefficient is dependent upon ionic strength but the value at infinite dilution is not. From this and other studies it was concluded that conformation of the isoionic monomer is not dependent upon ionic strength. After considering the methods of combining various kinds of data on hydrodynamic properties of rigid macromolecules to calculate shape parameters, a new set of functions, the γ functions, was defined. They are especially useful in calculating axial ratios of macromolecules. This is discussed and illustrated with data from the above experiments and from rotary diffusion constants reported earlier. Comparison of our calculated axial ratios for bovine serum albumin with values taken from or based upon earlier literature leads to a choice of 3.0-3.5 as the best available value. It is concluded that hydrated bovine serum albumin monomer can be represented adequately as a prolate ellipsoid of revolution with major axis 2a = 140 Å and minor axes 2b = 40 Å. Within experimental limitations, this is not inconsistent with Bloomfield's linear aggregate of three spheres. Data on the dimer are consistent with a side-to-side aggregation of such a monomer, with about 50 % overlap.
The secondary structure and the thermostability of bovine serum albumin (BSA), before adsorption and after homomolecular displacement from silica and polystyrene particles, are studied by circular dichroism spectroscopy and differential scanning calorimetry. The structural perturbations induced by the hydrophilic silica surface are reversible, i.e. BSA completely regains the native structure and stability after being exchanged. On the other hand, the adsorption on, and subsequent desorption from, polystyrene particles causes irreversible changes in the stability and (secondary) structure of BSA. The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA. The alpha-helix content is reduced while the beta-turn fraction is increased in the exchanged molecules. Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces. The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.
The reversibility of the adsorption-desorption cycle was established by comparing the thermostability (determined by differential scanning calorimetry) and secondary structure (obtained by circular dichroism spectroscopy) of BSA before adsorption, adsorbed on, and exchanged from silica particles. Circular dichroism was also measured as a function of temperature at a given wavelength. Adsorbed BSA presents a higher thermostability and a lower alpha-helix content than the native protein while it regains its conformation when released from the surface back into the solution; the homomolecular exchange is reversible.The changes in ellipticity (at a given wavelength) as a function of the temperature show that the thermal denaturation of native, adsorbed, and exchanged BSA proceeds in two steps. For the dissolved protein, the first step up to 50 degrees C involves a slight change in the structure while in the 50-90 degrees C temperature range the actual unfolding takes place. For the adsorbed BSA, the first step proceeds up to 60 degrees C and includes some intermolecular association between the adsorbed protein molecules, which may be responsible for the increased thermostability. The unfolding occurs in the 60-90 degrees C range; it is less cooperative and involves a lower enthalpy change than the native protein. Adsorbed BSA presents the same secondary structure as that observed for dissolved BSA that has passed a heating-cooling cycle. Copyright 2001 Academic Press.
Spectroscopic methods provide a powerful tool for studying the properties of proteins at interfaces. The protein accumulated in one adsorbed layer is frequently less than the minimum mass of protein required by a detection method. In such a case (as is the case in circular dichroism spectroscopy) the sorbent material is usually supplied as dispersion. However, light scattering by the dispersed particles often interferes with the measurement of the circular dichroism of the protein. Therefore, there is a strong need for an experimental setup that enables these measurements to be made using flat surfaces. An example of such a setup is the multiplate quartz cell presented here. The potential of this multiplate quartz cell is shown by some preliminary circular dichroism measurements of IgG adsorbed on different types of surfaces. Copyright 2000 Academic Press.
Imaging of hydrophobic surfaces in water with tapping mode atomic force microscopy reveals them to be covered with soft domains, apparently nanobubbles, that are close packed and irregular in cross section, have a radius of curvature of the order of 100 nm, and a height above the substrate of 20-30 nm. Complementary force measurements show features seen in previous measurements of the long-range hydrophobic attraction, including a jump into a soft contact and a prejump repulsion. The distance of the jump is correlated with the height of the images. The morphology of the nanobubbles and the time scale for their formation suggest the origin of their stability.
This study presents the use of complementary colorimetric and amperometric techniques to measure the quantity of protein or enzyme immobilised onto a carbon paste electrode modified with a layer of electrodeposited polyaniline. By applying a solution of bovine serum albumin at 0.75 mg/ml, efficient blocking of the electrode from electroactive species in the bulk solution could be achieved. When the horseradish peroxidase was immobilised on the electrode, optimal amperometric responses from hydrogen peroxide reduction were achieved at approximately the same concentration. The mass of enzyme immobilised at this solution concentration was determined by a colorimetric enzyme assay to be equivalent to the formation of a protein monolayer. Under these conditions, amperometric responses from the immobilised layer are maximised and non-specific bulk solution interactions are minimised. At higher immobilised protein concentrations, diminished amperometric responses may be due to inhibited diffusion of hydrogen peroxide to enzyme which is in electronic communication with the electrode surface, or impeded electron transfer.