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Interaction of D-Amino Acid Oxidase with Carbon Nanotubes: Implications in the Design of Biosensors
Abstract
We have investigated the interaction of d-amino acid oxidase (DAAO) with single-walled carbon nanotubes (CNT) by spectroscopic ellipsometry. Dynamic adsorption experiments were performed at different experimental conditions. In addition, the activity of the enzyme adsorbed at different conditions was studied. Our results indicate that DAAO can be adsorbed to CNT at different pH values and concentrations by a combination of hydrophobic and electrostatic interactions. Considering that the highest enzymatic activity was obtained by adsorbing the protein at pH 5.7 and 0.1 mg x mL(-1), our results indicate that DAAO can adopt multiple orientations on the surface, which are ultimately responsible for significant differences in catalytic activity.
... These experiments are designed to give information on the initial adsorption rate, the biomolecule orientation, and the possible restructuring that the proteins may undergo in the short-term scale. Optical techniques such as ellipsometry [23] and reflectometry, [24,25] offer an optimal experimental setup to follow the adsorption process in this timescale. Both these techniques allow changing the experimental conditions (pH, ionic strength, adsorption time, etc.) that have a central effect on the protein adsorption process and ultimately affect the conformation and biological activity of the protein. ...
... Figure 3 shows the adsorption kinetics at different pH values (above, below, and at the protein isoelectric point) of albumin (BSA) biofunctionalized silica and carbon nanotube (CNT) substrates as measured by reflectometry [24] and D-amino acid oxidase (DAAO) biofunctionalized CNT determined by ellipsometry. [23] Electrostatic interactions dominate the adsorption process on the negative, hydrophilic silica substrate whereas on CNT, both electrostatic and hydrophobic interactions are present resulting in higher adsorbed amounts in steady-state conditions. Although both proteins interact with the sorbent substrates even under electrostatic repulsion, the initial adsorption rate and the saturation amount are significantly affected by the pH. ...
... [4,5] Ellipsometry also gives the thickness of the adsorbed protein layer that directly depends on the biomolecule orientation. [23,25] Scanning probe microscopy (as AFM and STM) performed in aqueous solution provides information on the orientation of the adsorbed proteins. [26,27] Thickness values determined in air usually are lower than those measured in aqueous solution because other variables, such as collapse of the adsorbed layer or reorientation at the air-solid interface, are involved. ...
This entry covers the different strategies used to biofunctionalize solid substrate with proteins and single-strand short DNA (ss-DNA), with special emphasis on the consequences of the adsorption process on the structure, orientation, and biological activity of the biomolecules. To this end, the discussion is divided in view of the two selected biomolecules and the basic biofunctionalization strategies, given by physical and chemical adsorption, as reported in the last decade. Among the great variety of proposed strategies to achieve chemical adsorption of proteins, the discussion is limited to the most used procedures resulting in either randomly (exposed protein side chains on derivatized substrates) or site-oriented (avidin-biotin, His-tag proteins-metal cations, and protein A(G)-antibodies) adsorbed proteins. On the other hand, much less research has been done with ss-DNA adsorbed (either physically or chemically) on sorbent substrates. Therefore, the discussion is focused on the effect of the sorbent substrate and solution conditions on ss-DNA orientation and conformation. Finally, the most common strategy involving thiol-gold chemistry is also particularly addressed to summarize ss-DNA chemical adsorption.
... Due to the simplicity and miniaturization capabilities of the instrumental setup as well as the potential to provide fast and sensitive detection in both in vitro and in vivo, amperometry1415161718 isone of the most advantageous ones. In particular, carbon nanotubes (CNT) offer a wide variety of additional advantages in the development of amperometric biosensors19202122 including high electrode area [23] and good electrical conductivity. Furthermore, recent studies have demonstrated that CNT can enhance the electrochemical reactivity of biomolecules and promote electron transfer reactions.2425262728 ...
... paper describes the development of an amperometric biosensor for L-glutamate based on the adsorption of L-glutamate oxidase (GlutOx) on a commercial screen-printed substrate containing the CNT. The experimental conditions to perform the adsorption of the enzyme on the CNT-based substrate were selected based on prior studies performed by our group. [22,293031 In addition to providing a robust platform that integrates the working, counter, and reference electrodes, the selected substrate could be easily integrated in a flowinjection analysis (FIA) system, which facilitated sample-handling operations and improved the overall throughput of the analytical method. The following sections pro ...
... The biosensor was rinsed with a buffer solution and stored in a pH 7.4 phosphate buffer solution at 4 °C, when not in use.Figure 6 shows that the biosensor retained 92% of its initial signal after 24 days. These results (obtained with a matrix containing CNT) are in good agreement with previous reports stating that although a fraction of the enzymes adsorbed to the CNT surface is denatured, the adsorption of enzymes to CNT is predominantly irreversible [2, 22, 23, 29, 30]. The biosensor's response to some of the most common potential interferences (ascorbic acid, L-cysteine, and acetaminophen) was also investigated.Figure 6 shows that at a concentration level of 100 μM (10 times higher than that of L-glutamate) the interference level of the three species was approximately 3 %. ...
The amperometric glutamate biosensor based on screen-printed electrodes containing carbon nanotubes (CNT), and its integration in a flow injection analysis system, is described herein. The sensor was fabricated by simply adsorbing enzyme glutamate oxidase (GlutOx) on a commercial substrate containing multi-wall CNT. The resulting device displayed excellent electroanalytical properties toward the determination of L-glutamate in a wide linear range (0.01-10 μM) with low detection limit (10 nM, S/N≥3), fast response time (≤5 s), and good operational and long-term stability. The CNT modified screen-printed electrodes have a potential to be of general interest for designing of electrochemical sensors and biosensors.
... Due to their small size and conductivity they can also be regarded as the smallest possible electrodes, with diameters as small as one nanometer [17]. When using CNT as substrates for biosensors it is crucial to understand the interaction between the recognition element (typically a protein) and the CNT because they can affect the protein conformation1819202122, cellular adhesion [23], and biocompatibility [18,24,25]. To understand the roots of these interactions, a detailed characterization of the CNT substrates must be performed. ...
... ed spectroscopy, and Raman spectroscopy are the most commonly reported characterization techniques [26]. The main problem associated with these techniques is their limitation to perform in-situ measurements of adsorption of biological molecules under physiological conditions. An alternative technique to perform in-situ measurements is ellipsometry. [21] Ellipsometry is an optical technique that measures changes in the reflectance and phase difference between the parallel (R P ) and perpendicular (R S ) components of a polarized light beam upon reflection from a surface. Using Equation 1, Equation 1 the intensity ratio of R P and R S can be related to the amplitude ratio (tan Ψ) and the ...
... To develop the model, the optical properties of two different substrates were investigated as a function of the wavelength and incidence angle using reflection and ellipsometric experiments. Finally, the model was used to gain insight into the interaction kinetics between a selected protein and carbon nanotubes [21]. ...
The paper describes a set of simple experiments performed to develop an optical model to describe Si/SiO(2) substrates coated with two transparent films of carbon nanotubes. The final goal is to use such optical model to investigate the interaction of proteins with carbon nanotubes. Experiments were performed to assess light reflection as a function of the wavelength or angle of incidence using two substrates (same material, different amounts) composed of oxidized carbon nanotubes. The experimental results indicate that the selected carbon nanotubes layers are anisotropic and significantly different from each other. Experiments performed by spectroscopic ellipsometry (as a function of the wavelength and incident angle) enabled the development of an Effective Medium Approximation model consisting in a two-fraction phase (arc-evaporated carbon and void space). Furthermore, the model enabled calculating the amount of protein adsorbed on the surface of the carbon nanotubes film.
... In order to obtain information related not only to the adsorption process but also the structure, thickness [21], optical constants, and microstructure of films [22], our lab is now focused on the use of variable angle spectroscopic ellipsometry (VASE) [23,24]. VASE enabled the possibility of extending the scope of the project to DNA [25] and other proteins such as albumin [26], D-amino acid oxidase [27], catalase [28], and glucose oxidase [29]. In general, our results demonstrated that the activity of enzymes adsorbed to CNT is not only exclusively proportional to the adsorbed amount of protein (which is the only variable optimized in most systems) but also to the initial adsorption rate (Figure 4). ...
... A) Effect of experimental conditions and B) initial adsorption rate on the enzymatic activity of Damino acid oxidase and catalase, respectively. Conditions as described in references [27] and [28]. A)Dynamic adsorption of BSA, fibrinogen, and collagen onto the nanostructured PDMS films. ...
Recent developments in materials, surface modifications, separation schemes, detection systems and associated instrumentation have allowed significant advances in the performance of lab-on-a-chip devices. These devices, also referred to as micro total analysis systems (µTAS), offer great versatility, high throughput, short analysis time, low cost and, more importantly, performance that is comparable to standard bench-top instrumentation. To date, µTAS have demonstrated advantages in a significant number of fields including biochemical, pharmaceutical, military and environmental. Perhaps most importantly, µTAS represent excellent platforms to introduce students to microfabrication and nanotechnology, bridging chemistry with other fields, such as engineering and biology, enabling the integration of various skills and curricular concepts. Considering the advantages of the technology and the potential impact to society, our research program aims to address the need for simpler, more affordable, faster and portable devices to measure biologically active compounds. Specifically, the program is focused on the development and characterization of a series of novel strategies towards the realization of integrated microanalytical devices. One key aspect of our research projects is that the developed analytical strategies must be compatible with each other; therefore, enabling their use in integrated devices. The program combines spectroscopy, surface chemistry, capillary electrophoresis, electrochemical detection and nanomaterials. This article discusses some of the most recent results obtained in two main areas of emphasis: capillary electrophoresis, microchip-capillary electrophoresis, electrochemical detection and interaction of proteins with nanomaterials.
... The effect of an external field on the catalytic activity There are a number of possible causes to explain the differences in catalytic activity described by Fig. 2, which not only refer to the amount of enzymes adsorbed on the surface, but also their conformation. 58 Considering the hydrophobic contributions to the adsorption process, and the enhancement provided by favorable electrostatic interaction free energies in Table 5 and Fig. 8, we do not expect to see major differences in trypsin coverage on the surface of the electrodes prepared under different applied potentials. This is further supported by comparing ∆G int with positive and negative electric fields in Fig. 8. ...
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.
... In this context, irrespective of the type of biosensor, reducing the size to micro-or nanoscale resulted in a better signal-to-noise ratio and, taken together with the smaller sample volume used, assay costs were lower. For instance, optical biosensors coupled to quantum dots (i.e., semiconductor crystals with peculiar quantum confinement effects), CNTs, and microfluidic-and graphene-based biosensors, as well as lab-on-a-chip devices, are widely reported in the literature for electrode fabrication [12,17,96], and have been also applied for detecting D-AAs. ...
D-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace.
... Despite catalyzing the same conversion of amino acids to keto acids and H 2 O 2 , D-amino acid oxidases and L-amino acid oxidases (L-AAO) are classified into different structural families, with D-AAO in the D-AAO structural family and L-AAO in the MAO family (7). Enzymes in both D-AAO and L-AAO have been developed and applied as biosensors for detection of food contamination in nutritional industries (9,10), as indicators for metabolic and neurological disorders (11), and as biocatalysts in pharmaceutical industries This article contains supporting information. * For correspondence: Pimchai Chaiyen, pimchai.chaiyen@vistec.ac.th. ...
L-Lysine oxidase/monooxygenase (L-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of L-amino acids, particularly L-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (H2O2), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of L-lysine and L-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from L-lysine), L-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from L-lysine and 4-aminobutanoic acid from L-ornithine). The reaction of reduced enzyme and oxygen yielding an imino acid and H2O2, with no detectable C4a-hydroperoxyflavin. Single turnover reactions in which L-LOX/MOG was first reduced by L-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H2O2 addition after enzyme denaturation. H2O2 addition to active L-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that H2O2 generated from L-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H2O2 resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on L-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation.
... Many quantitative methods have been reported to detect D-Ala [7,8], such as biosensor based on the linear relationship between the oxygen consumption of the oxidation reaction and the concentration of D-Ala was established [9]. According to selectively oxidation of D-Ala by D-α-Amino oxidase (DAAO) to produce H 2 O 2 [10,11], many detection methods have been reported, including colorimetric [12][13][14], electrochemiluminescence [15], electrochemical biosensors [9,16,17]. Some separation and determination methods based on the structural properties of D-Ala, such as chromatography [18][19][20], capillary electrophoresis [21] were also developed. ...
A sensitive fluorometric assay is described here for sensitive determination of D-alanine (D-Ala). The method is based on the use of poly[9,9-bis(6-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) and manganese dioxide (MnO2) nanosheets. The blue fluorescence of PFP, peaking at 422 nm, is absorbed by the MnO2 nanosheets due to an inner filter effect (IFE). In the presence of D-Ala and D-α-amino acid oxidase (D-AAO), the enzymatic oxidation leads to the production of H2O2, triggering the decomposition of MnO2 nanosheets and the recovery of the fluorescence of PFP. Under the optimum conditions, the nanosensor has a wide linear range from 1.0 nM to 1.0 mM with a detection limit of 0.35 nM. This is about 10–100 times lower than most previously reported methods. The recovery experiment was performed with spiked serum and gave accuracy rates from 97.2 to 109%. The standard deviation is from 2.3% to 7.2%.
Graphical abstractSchematic presentation of a sensitive “off-on” nanosensor for fluorimetric D-alanine detection.
... It has been proposed that evolved H 2 O 2 is eliminated during the production of 7ADCA [6]. DAAOs can also be used to catalyze the production of α-keto acids from essential D-amino acids [7,8], resolution of racemic mixtures of amino acids [9], and quantification of the D-amino acid content in biological fluids [10][11][12][13][14][15]. Cooperating with other enzymes, DAAOs promote the catalysis processes [16,17]. ...
d-amino acid oxidase (DAAO) and catalase (CAT) have been genetically modified by fusing them to an elastin-like polypeptide (ELP). ELP-DAAO and ELP-CAT have been separately immobilized on multi-walled carbon nanotubes (MWNTs). It has been found that the secondary structures of the enzymes have been preserved. ELP-DAAO catalyzed the oxidative deamination of d-alanine, and H2O2 was evolved continuously. When the MWNT-supported enzymes were used together, the generated hydrogen peroxide of ELP-DAAO could be decomposed in situ. The catalytic efficiency of the two immobilized enzymes was more than five times greater than that of free ELP-DAAO when the ratio of immobilized ELP-CAT to immobilized ELP-DAAO was larger than 1:1.
... DAAO was immobilized onto the surface of Au-PtNPs/ F-MWCNTs/GCE via Au-S and Au-N bonds and a combination of hydrophobic and electrostatic interactions. [40][41][42] The prepared DAAs biosensor had good performances in the enantioselective determination of DAAs at a low applied potential with high sensitivity, good selectivity, wide linear range, stability, and reproducibility. Other chemical reagents were analytical grade and used without further purification. ...
A new biosensor specified for D-amino acids (DAAs) based on the electrochemiluminescence (ECL) of luminol was proposed by coupling D-amino acid oxidase (DAAO) to in situ generate coreactant with functionalized Multi-walled carbon nanotubes (F-MWCNTs) and Au-Pt alloy nanoparticles as catalysts for the ECL reaction. The stepwise fabrication process of the biosensor was characterized via cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Furthermore, the Au–Pt nanostructures with different molar ratios between Au and Pt precursors were investigated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) and ECL technique, and the optimum Au/Pt ratio for ECL measurements was 1:1 with an optimized electrodeposition time at 400 s. The proposed biosensor showed excellent performance in the stereoselective recognition of D-alanine with a low detection limit of 1.67 × 10⁻⁹ M. Hence, this paper would provide a new platform on the specific detection of D-alanine with high sensitivity, good selectivity and acceptable stability.
... Non-covalent interactions do not require modifications while comprising many drawbacks, like weak attachment, random orientation, and conformational perturbation, reducing the biological activity and, hence, the molecular recognition capabilities of the adsorbed biomolecule. The non-covalent biofunctionalization is based on various adsorption interactions such as hydrophobic, electrostatic, hydrogen bonds, πstacking, and van der Waals either on pristine or modified nanoparticles and takes place fast and spontaneously [63][64][65]. Hydrophobic interactions appear as the result of the dehydration of apolar parts of the protein and the sorbent surfaces; they often lead to changes in the conformation of the adsorbed proteins. Upon adsorption, at one side of the protein molecule the surrounding aqueous solution is replaced by the nanoparticle. ...
Due to their negligible toxicity and buffering properties, layered double hydroxides
(LDHs) are commercially employed as antacids and they are increasingly studied as
vehicles in drug and gene delivery systems. The bioactive compounds (drugs,
biomolecules) are incorporated by intercalation or adsorption and released under
appropriate conditions by anion exchange, desorption and LDH layers dissolution. More
recently, their application as nanovehicles for cellular delivery has been developed based
on their synthesis in the nanometer scale. LDH nanoparticles are obtained by different
synthesis routes, but the more extended involves coprecipitation at either constant or
variable pH followed by hydrothermal aging in pure water. LDH based nanocarriers
present several advantages as drug and gene nanovehicles when compared to other
inorganic nanoparticles. First, LDH nanovehicles interact with the negatively charged cell
membrane due to the positive charge and the hydrophilic character of the nanoparticles
and their transfection is mainly produced by clathrin-mediated endocytosis. Secondly,
LDH nanovehicles are dissolved in mildly acid media into non-toxic species, releasing
their cargo and preventing the accumulation usually observed with polymers and other
inorganic vehicles. Finally, they are expected to avoid renal clearance and to allow long
circulation times due to their size. In this chapter, the strategies to obtain LDH
nanoparticles as well as the factors that determine the particle size distribution are
reviewed with emphasis on their application in drug and gene delivery. Hence, the
chapter highlights the physicochemical factors that affect the delivery process and the
proposed customization strategies to produce an optimal performance of LDH
nanoparticles as drug nanovehicles.
... d-Amino acid oxidases sever as the key enzymes involved in the two-step bioconversion of cephalosporin C to 7-aminocephalosporanic acid, the important intermediate for semisynthetic cephalosporin antibiotics (Pilone et al., 1995;Pollegioni et al., 2004). The reactions catalyzed by DAAOs can also be used to produce ␣-keto acids from essential damino acids (Upadhya et al., 2000;Fernández-Lafuente et al., 1998), to separate racemic mixtures of amino acids, and to detect and quantify d-amino acids content in foods and in biological fluids (Li and Zhang, 2000;Inaba et al., 2003;Domínguez et al., 2001;Mora et al., 2009;Pernot et al., 2008;Rosini et al., 2008). DAAO cooperating with other enzymes can promote the catalysis processes (Seo et al., 2012;Rachwalski et al., 2013). ...
... Using ellipsometry, Mora et al. [291] investigated the interaction of D-amino acid oxidase (DAAO) with SWCNT. In this case, dynamic adsorption experiments, performed as a function of the protein concentration, pH, and ionic strength, allowed them to conclude that DAAO can adopt multiple orientations on the surface and that the maximum catalytic activity can be obtained by controlling the adsorption conditions. ...
... Experiments were performed using a variable angle spectroscopic ellipsometer (WVASE, J.A. Woollam Co., Lincoln, NE) following a procedure described elsewhere. [55][56][57][58] Under these conditions, spectroscopic ellipsometry has proven suitable to study the kinetics of protein adsorption processes 59 and to calculate the optical constants, thickness, and microstructure of the adsorbed film. The sensitivity of the technique, critically evaluated elsewhere, 60 was also considered appropriate for the purpose of the present study. ...
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.
... The ellipsometer was also used to measure the ellipsometric angles (Ψ and Δ) as a function of the incident wavelength, according to a procedure described elsewhere. 3,4,37,38 In order to calculate the refractive index (n) and extinction coefficient (k) of the film, the collected data were modeled with WVASE (J. A. Woollam Company; Lincoln, NE), using an optical model composed of a layer of Si (bulk; d = 1 mm), a layer of SiO 2 (d = 2.104 nm), and the OTCE. Differences between the experimental and model-generated data were assessed by the mean square error (MSE), 39 a built-in function in WVASE. ...
... Spectroscopic ellipsometry was chosen because it has proven suitable to study adsorption of proteins, and provides useful information about the optical constants and structure of the adsorbed protein film [29,38394041. Dynamic adsorption experiments were performed in a commercial electrochemical cell (J.A. Woollam Co; Lincoln, NE) mounted directly on the vertical base of the ellipsometer [42]. In order to control the protein supply to the material substrate tested, the cell was modified by fixing a L-shaped stainless-steel tube (R = 0.254 mm) to the cell. ...
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.
... The authors stated that BSA molecules arriving at the CNT surface may adopt a preferred orientation with the positive and non-polar patches of the protein facing the hydrophobic sorbent surface, resulting in an attachment-controlled adsorption process. Later, Mora et al. investigated the relationship between the interaction phenomena (adsorption/desorption kinetics and amount) and the activity of adsorbed D-amino acid oxidase (DAAO) [173]. They found that the adsorption of DAAO to CNT is controlled by a combination of hydrophobic and electrostatic forces, and observed that the activity of the sensor was influenced not only by the adsorbed amount but also by the conformation adopted by the enzyme on the CNT surface. ...
The objective of this review is to provide a broad overview of the advantages and limitations of carbon-based nanomaterials with respect to analytical chemistry. Aiming to illustrate the impact of nanomaterials on the development of novel analytical applications, developments reported in the 2005-2010 period have been included and divided into sample preparation, separation, and detection. Within each section, fullerenes, carbon nanotubes, graphene, and composite materials will be addressed specifically. Although only briefly discussed, included is a section highlighting nanomaterials with interesting catalytic properties that can be used in the design of future devices for analytical chemistry.
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.
Low-cost, high-throughput, broadly useful photoresponsive enantiomeric excess (ee) sensing of amino acids remains challenging to date. Herein, based on the selective oxidation reaction of amino acid oxidase (AAO) to amino acid enantiomers (D/L-AA) and the oxidation reaction of substrate (H2O2) with aromatic boronic ester, we put forward a photoresponsive strategy for the determination of D/L-AA at a certain concentration. In this scheme, the substrate H2O2 produced by the enzyme-catalyzed reaction was determined by sensitive fluorescent and colorimetric response of ethyl-3-(3-(benzothiazol-2-yl)-5-methyl-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)-2-cyanoacrylate (HBT-PB) to reflect the enantiomeric content at a certain concentration. The photoresponsive probe HBT-PB was readily available and inexpensive with sensitive long-wavelength red fluorescence and colorimetric light response to H2O2, the detection limit (LOD) was estimated as 2.91 μM. The operation of the sensing method was simple and data collection and processing are straightforward. The practicability of the scheme was favorably confirmed by accurate and scientific analysis of methionine and Dopa samples. As a result, the scheme was not only suitable for high-throughput screening but also adaptable to low-cost and sensitive RGB colorimetric analysis platform (LOD of methionine and Dopa was calculated as 9.23 μM and 8.34 μM respectively) with modern plate readers, and possessed extremely high enantioselectivity and wide applicability which benefited from the specificity and efficiency of enzyme catalytic reaction.
Native D-amino acid oxidase (DAAO) that is expressed mostly as inclusion body and its toxicity for E. coli hamper efficient heterologous expression. In this study, the soluble expression of DAAO from Rhodosporidium toruloides (RtDAAO) was improved in E. coli through N-terminal modification, but the cell biomass was decreased. Then a trade-off between DAAO expression and biomass was achieved to obtain the highest volumetric activity of DAAO through regulated the number of N-terminus histidine residues. When variant ²H³G was fused with three N-terminus histidine residues, the volumetric activity was increased by 3.1 times and the biomass was not significant change compared with the wild type. Finally, the N-terminus disordered region of RtDAAO (HSQK) was replaced with HHHG and the variant enzyme activity reached 80.7 U/mL (with a 40 percent of inactive DAAO reduced) in a 7.5 L fermenter in 24 h.
Layered double hydroxides (LDHs) are clay-type materials with extraordinary properties, whose structure is composed of stacked inorganic metal oxide layers built by connected sheets. These layers hold usually charge deficiency which is balanced by the presence of anions (anionic clays) at the interlayer spacing. By taking advantage of this relevant property, many works across several fields have been dedicated to explore this by introducing anions inside the interlayer spacing of the clays. This includes exchange from simple anions to complex structures such as large polyoxoions or even metal complexes. The resulting tuned materials find wide use in several catalytic applications with the advantage that in some cases the heterogeneous process overcomes the homogeneous one.Important chemical reactions such as the Suzuki and Heck coupling reactions or other such as water splitting can be successfully catalyzed by such materials. Oxidation reactions including epoxidation of olefins and oxidation of alcohols to carbonyl compounds are also important chemical processes where layered double hydroxides can be efficient catalysts.From the economical point of view such catalysts feature high synthetic flexibility, tunable acid-base properties and ease of set-up and work-up, among others. These properties lead to the development of more efficient catalytic systems with improved yields and selectivity, which are valuable achievements in the present days by contributing towards the establishment of environmentally friendly technologies.
In this work, a bi-enzymatic biosensor allowed the total content of D-amino acids (DAAs) determination in highly relevant matrices involving bacteria. The strategy is based on the unique coimmobilization of D-amino acid oxidase (DAAO) and horseradish peroxidase (HRP) enzymes onto a multi wall carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) modified screen-printed electrode (SPCE). The greater amount of AuNPs deposited and hence the greater loading of both enzymes was observed when they were deposited after the activation of the carboxylated MWCNTs with EDC/Sulfo-NHS chemistry. These platforms provided a fast (300 s) and selective quantification of DAAs with excellent precision (RSD < 5%) and accuracy (Recoveries 100–104%) in bacterial samples. Collectively, the electrochemical bi-enzymatic biosensor become an universal, fast, sensitive and easy-to-use approach to determine total content of DAAs in complex matrices.
The present study describes a simple strategy to integrate electrochemical detection with an assembled microchip-capillary electrophoresis platform. The electrochemical cell was integrated with a microfluidic device consisting of five plastic squares interconnected with fused silica capillaries, forming a four-way injection cross between the separation channel and three side-arms (each of 15 mm in length) acting as buffer/sample reservoirs. The performance of the system was evaluated using electrodes made with either carbon ink, carbon nanotubes, or gold and under different experimental conditions of pH, capillary length, and injection time. Using this system it was possible to separate the neurotransmitters dopamine and cathecol and to quantify phenol from a real sample using a linear calibration curve with a calculated LOD of 0.7 µM. A similar concept was applied to determine glucose, by including a pre-reactor filled with beads modified with glucose oxidase (GOx). The latter system was used to determine glucose in a commercial sample, with a recovery of 95.2 %. Overall, the presented approach represents a simple, inexpensive, and versatile approach to integrate electrochemical detection with CE separations without requiring access to microfabrication facilities.
Due to their negligible toxicity and buffering properties, layered double hydroxides
(LDHs) are commercially employed as antacids and they are increasingly studied as
vehicles in drug and gene delivery systems. The bioactive compounds (drugs,
biomolecules) are incorporated by intercalation or adsorption and released under
appropriate conditions by anion exchange, desorption and LDH layers dissolution. More
recently, their application as nanovehicles for cellular delivery has been developed based
on their synthesis in the nanometer scale. LDH nanoparticles are obtained by different
synthesis routes, but the more extended involves coprecipitation at either constant or
variable pH followed by hydrothermal aging in pure water. LDH based nanocarriers
present several advantages as drug and gene nanovehicles when compared to other
inorganic nanoparticles. First, LDH nanovehicles interact with the negatively charged cell
membrane due to the positive charge and the hydrophilic character of the nanoparticles
and their transfection is mainly produced by clathrin-mediated endocytosis. Secondly,
LDH nanovehicles are dissolved in mildly acid media into non-toxic species, releasing
their cargo and preventing the accumulation usually observed with polymers and other
inorganic vehicles. Finally, they are expected to avoid renal clearance and to allow long
circulation times due to their size. In this chapter, the strategies to obtain LDH
nanoparticles as well as the factors that determine the particle size distribution are
reviewed with emphasis on their application in drug and gene delivery. Hence, the
chapter highlights the physicochemical factors that affect the delivery process and the
proposed customization strategies to produce an optimal performance of LDH
nanoparticles as drug nanovehicles
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.
The bioaffinity interaction between Histidine (His)-tag proteins and Ni(II) surface sites is exploited as a biofunctionalization strategy to achieve a better surface bioactivity than that provided by physical adsorption. This improved functioning is mainly ascribed to the presence of site-oriented proteins on the surface, induced by the interaction between the tag and the Ni(II) sites. In addition to the induced bioaffinity interaction, His-tag proteins are also spontaneously adsorbed (through hydrophobic and electrostatic interactions) on the substrate. These physically adsorbed proteins are randomly oriented and less bioactive, lowering the surface biorecognition capabilities. Therefore, the surface biofunctionalization based on His-Ni(II) interaction requires the optimization of the experimental conditions to promote the bioaffinity interaction while minimizing physical adsorption. This optimization can be achieved by properly selecting the adsorption conditions (solution pH and ionic strength, protein surface coverage, etc.) and the washing agents prior to the detection of the biorecognition event. This chapter is aimed at discussing experimental results related to the optimization of the bioaffinity interaction between a particular recombinant His-tag antigen and Ni(II) surface sites.
Density functional theory (DFT) was used to investigate the adsorption of lactic acid molecules on the surface of (4,4), (5,5), (6,6) and (7,7) single-walled carbon nanotubes (SWCNTs). A hybrid DFT method with the inclusion of dispersion correction was employed and the results compared to those obtained from the non-corrected DFT method. The energies and optimum distances for two different configurations were obtained after relaxation of the entire system. The calculations showed that the adsorption of lactic acid onto the outer wall of carbon nanotubes was thermodynamically favored. The adsorption of lactic acid outside the SWCNT with (4,4) chirality and via a vertical orientation to the tube axis above the center of a hexagon surface and through its hydroxyl group was the most stable state of physisorption with an adsorption energy of -13.39 kcal mol-1. Total density of states (TDOS) and projected density of states (PDOS) analysis in the vicinity of the Fermi level region suggested the electronic states to be contributed from SWCNTs rather than lactic acid. The DFT calculations also showed that non-covalent functionalization of SWCNTs with lactic acid could give rise to new impurity states in the DOS of pristine SWCNTs and suggested possible carrier doping of carbon nanotubes via selective adsorption of molecules. The global reactivity descriptors in the gas phase and solvent were calculated.
The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged d-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of d-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.
D-amino acid oxidase (DAAO) catalyzes oxidative deamination of D-amino acids to yield corresponding α-keto acids, producing hydrogen peroxide (H2O2). D-amino acid oxidase was genetically modified by fusion to an elastin-like polypeptide (ELP). For the enzyme immobilization, multi-walled carbon nanotubes (MWCNTs) were adopted as the model support. MWCNTs were functionalized with hematin. ELP-DAAO was immobilized on the functionalized CNTs by coupling to the hematin. The specific immobilization enabled ELP-DAAO in proximity to the hematin at a molecular distance. The molecular-distance proximity facilitated the immediate decomposition of H2O2 catalyzed by the hematin. The evolved oxygen was efficiently utilized to oxidize the reduced cofactor FDA of DAAO, and H2O2 was produced. The forming of H2O2- O2-H2O2 circle between the DAAO and hematin has been demonstrated to be the driving force to accelerate the deamination reaction. The enzyme kinetics has shown that the ELP-DAAO/hematin-CNTs conjugate exhibited a catalysis efficiency more than three times that of free ELP-DAAO, demonstrating its ability mimicking multi-enzyme catalysis. The methodology for highly specific immobilization of enzyme is not restricted to carbon nanotubes, and can be extended easily to other micro and nanomaterials as supports for specific immobilization of oxidases.
An electrochemical microfluidic strategy for the separation and enantiomeric detection of D-Methionine (D-Met) and D-Leucine (D-Leu) is presented. These D-amino acids (D-AAs) act as biomarkers involved in relevant diseases caused by Vibrio cholerae. On a single layout microfluidic chip (MC), highly compatible with extremely low biological sample consumption, the strategy allowed the controlled microfluidic D-AA separation and the specific reaction between D-amino acid oxidase (DAAO) and each D-AA biomarker avoiding the use of additives (i.e. cyclodextrins) for enantiomeric separation as well as any covalent immobilization of the enzyme into the wall channels or on the electrode surface such as in the biosensor-based approaches. Hybrid polymer/graphene-based electrodes were end-channel coupled to the microfluidic system to improve the analytical performance. D-Met and D-Leu were successfully detected becoming this proof-of-the-concept a promising principle for the development of point-of-care (POC) devices for in situ screening of V. cholerae related diseases.
A new biomaterial, based on silica organofunctionalized with p-phenylenediamine (p-PDA) and the enzyme peroxidase, was used in the development of an enzymatic solid-phase reactor. The analytical techniques used in the characterization showed that the organic ligand was incorporated into the silica matrix. Thus, the silica modified with p-PDA allowed the incorporation of peroxidase by the electrostatic interaction between the carboxylic groups present in the enzyme molecules and the amino groups attached to the silica. The enzymatic solid-phase reactor was used for chemical oxidation of phenols in 1, 4-benzoquinone that was then detected by chronoamperometry. The system allowed the analysis of hydroquinone with a detection limit of 83.6 nmol L ?1. Thus, the new material has potential in the determination of phenolic compounds river water samples.
Magnetic multiwalled carbon nanotubes (M-MWNTs) have been functionalized with mixed surfactants consisting of a sugar-based surfactant and an ionic surfactant. The synergistic effect of the two surfactants results in more surfactants adsorbed and the formation of a more disordered structure of assembled surfactants. These two aspects facilitate simultaneous hydrogen bonding and electrostatic interactions with proteins. It has been demonstrated that the M-MWNTs functionalized with mixed surfactants adsorbed more proteins than the M-MWNTs functionalized with single surfactants. The M-MWNTs functionalized with mixed surfactants have been tested for the adsorption of two proteins, bovine serum albumin (BSA) with a low isoelectric point and lysozyme with a high isoelectric point, and similar results have been obtained. Hence, carbon nanotubes functionalized with mixed surfactants have a wide range of potential applications for protein adsorption.
The geometrical structures, energetics and electronic properties of glycine molecules as well as dehydrogenated radical interaction with silicon carbide nanotubes (SiCNTs) are investigated based on density functional theory (DFT) for the first time. Different from the weak adsorption on CNTs, it is shown that glycine molecules tend to be chemisorbed onto SiCNTs. There are three patterns of the individual glycine molecule adsorbed on the (8, 0) SiCNT, including monodentate, cycloaddition and dissociative ones, with the latter two patterns (Eads ranges from -22.08 to -34.99 kcal mol(-1)) more stable than the monodentate one (Eads ranges from -8.16 to -21.14 kcal mol(-1)). In addition, we also investigated the adsorption of multiple glycine molecules on various zigzag (n, 0) (n = 7, 8, 9 and 10) SiCNTs. It is shown that totally n (n = 7, 8, 9 and 10) molecules can be chemisorbed on one circle of the wall of the SiCNT at most. And the Eads per glycine decreases gradually with the increasing tube diameter due to the curvature effects. For the adsorption of dehydrogenated glycine radicals, it is found that both the N-centered and C-centered ones can form stable complexes by attacking the (8, 0) SiCNT. Totally one monodentate and two bidentate adsorption configurations of the N-centered radical and three monodentate configurations of the C-centered one can be found. Note that the important half-metals can be obtained for the bidentate configurations from the N-centered radical due to the hybridization state of the radical and the tube in one spin channel crossing the Fermi level, while the p-type semiconductor can be produced for the monodentate configurations from the C-centered radical because the impurity state derived from the radical itself is closer to the edge of the valence band above the Fermi level, which may be applied in building electronic devices and metal-free catalysis. Finally, we found that the encapsulation of the glycine molecule is exothermic and thus energetically favourable in the SiCNTs with the diameter larger than the (9, 0) SiCNT. The present study is expected to create promising applications in nano-device building and biotechnology.
D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the D-forms of alanine (Ala) and serine (Ser). The permeability coefficient of D-Ala and D-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.
This work presents a systematic study on the relationship between the adsorption mechanism and the surface bio-activity of d-amino acid oxidase (pkDAAO). This rational approach is based on measuring the characteristic filling and relaxation times under different experimental conditions. With such a goal, real-time adsorption-desorption experiments at different degrees of surface coverage were performed tuning the electrostatic and hydrophobic interactions by changing the pH condition for the adsorption and the substrate properties (silica or gold). Surface bio-activity was measured in situ by amperometry using the bio-functional surface as the working electrode and ex situ by spectrophotometry. On both solid substrates, pkDAAO adsorption is a transport-controlled process, even under unfavorable electrostatic interactions (charged protein and substrate with the same sign) due to the high percentage of basic amino acids in the enzyme. On silica, the relaxation step is electrostatic in nature and occurs in the same time-scale as filling the surface when the substrate and the enzyme are oppositely charged at low surface coverage. Under unfavorable electrostatic conditions, the relaxation (if any) occurs at long time. Accordingly, the bio-activity of the native pkDAAO is preserved at any surface coverage. On gold, this step is driven by hydrophobic interactions (pH-independent) and the surface bio-activity is highly dependent on the degree of surface coverage. Under these conditions, the surface bio-activity is preserved only at high surfaces coverage. Our results clearly indicate that pkDAAO bio-functionalized surfaces cannot be coupled to amperometry because the analyte interferes the electrochemical signal. However, this simple bio-functionalized strategy can be joined to other detection methods.
An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0 mM, a high sensitivity of 32 μA mM−1 cm−2, and a response time of less than 5 s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose.
We have investigated the adsorption of hydrophobic amino acid valine/valine radicals with carbon nanotube (5,0) using density functional theory. Non-covalent interaction of valine with the nanotube exhibits differential binding strength with respect to the functional group of valine present close to the nanotube surface and it has enhanced the conductivity of the tube. Covalent interaction of valine radicals with the CNT introduced local sp3 hybridization at the adsorption site. In some of the cases nitrogen atom of the valine radical has broken the C–C bond and introduces a seven member ring in the adsorption site to retain the sp2 bonding network.
Different strategies used to biofunctionalize CNTs with proteins, from direct physical adsorption on pristine CNTs to chemical treatments to achieve covalent interaction, are described. The discussion is focused on the consequences of the adsorption process on the structure and properties of both proteins and CNTs. On this base, recent developments in CNTs-proteins based biosensors (electrochemical and optical) and drug delivery systems are reviewed.
According to the aim to compose combinatorial material by adsorption of carbon nanotubes onto the structured CeO2 surface the interaction of the armchair (5,5) and zigzag (8,0) nanotubes with the (001) and (111) surfaces of CeO2 islands have been investigated by theoretical methods. The thermodynamics of the adsorption were studied at the low surface coverage region. The interaction energy between the nanotube and the different CeO2 surfaces shows significant increase when the size of the interface reaches 7–8 unit cells of CeO2 and it remains unchanged in the larger interface region. However, the entropy term of the adsorption is significantly high when the distances of CeO2 islands are equal to 27nm (adsorption of armchair (5,5) nanotube) or 32nm (adsorption of zigzag (8,0) nanotube). This property supports adsorption of nanotubes onto CeO2 surfaces which possesses a very specific surface morphology. A long-wave vibration of nanotubes was identified as background of this unexpected phenomenon. This observation could be applicable in the development of such procedures where the nanotube adsorption parallel to the surface is aimed to perform.
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.
Carboxylated single-walled carbon nanotubes (c-SWNTs) were synthesized prosperously in order to improve dispersion of raw carbon nanotubes. Then, bovine serum albumin (BSA) was used as the template protein to study the biocompatibility of c-SWNTs by UV-Vis, fluorescence and circular dichroism (CD) spectroscopic methods at the molecular level. Results from fluorescence spectrum showed obvious decreases in fluorescence intensity of BSA induced by c-SWNTs, indicating the occurrence of interaction between BSA and c-SWNTs. Static quenching effect of c-SWNTs was verified by linear Stern-Volmer plots and K(SV) values. Thermodynamic parameters at different temperatures demonstrated that the interaction between c-SWNTs and BSA was mainly favored by hydrophobic force. In addition, Na(+) interfered with the quenching effect of c-SWNTs, which revealed that electrostatic force played a role in binding roles of BSA to c-SWNTs simultaneously. The results of UV and synchronous fluorescence spectrum validated that hydrophobicity of amino acid residues expressly increased with the addition of c-SWNTs. The content of α-helix structure in BSA decreased by 14.06% with c-SWNTs viewed from CD spectrum. Effect of SWNTs on the conformation of BSA could be controlled by the surface chemistry of SWNTs.
This manuscript describes results related to the characterization of electrodes modified with a composite of acetylcholinesterase covalently bound to carbon nanotubes (CNT). The characterization was performed by computational methods and complemented by cyclic voltammetry, infrared spectroscopy, and electrochemical impedance spectroscopy. In-silico simulations enabled the identification of the binding site and the calculation of the interaction energy. Besides complementing the computational studies, experimental results obtained by cyclic voltammetry showed that the addition of CNT to the surface of electrodes yielded significant increases in effective area and greatly facilitated the electron transfer reactions. These results are also in agreement with impedance spectroscopy data, which indicated a high apparent rate constant, even after the immobilization of the enzyme. These results lend new information about the physical and chemical properties of biointerfaces at the molecular level, specifically about the mechanism and consequences of the interaction of a model enzyme with CNT.
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.
In this chapter, the basic concepts behind ellipsometry and spectroscopic ellipsometry are discussed along with some instrument
details. Ellipsometry is an optical technique that measures changes in the reflectance and phase difference between the parallel
(R
P) and perpendicular (R
S) components of a polarized light beam upon reflection from a surface. Aside from providing a simple, sensitive, and nondestructive
way to analyze thin films, ellipsometry allows dynamic studies of film growth (thickness and optical constants) with a time
resolution that is relevant to biomedical research. The present chapter intends to introduce ellipsometry as an emerging but
highly promising technique, that is useful to elucidate the interactions of proteins with solid surfaces. In this regard,
particular emphasis is placed on experimental details related to the development of biomedically relevant conjugated surfaces.
Results from our group related to adsorption of proteins to nanostructured materials, as well as results published by other
research groups, are discussed to illustrate the advantages and limitations of the technique.
We have systematically investigated the noncovalent and covalent adsorption of alanine and alanine radicals, respectively, onto a (5, 0) single-walled carbon nanotube using first-principles calculation. It was found that XH···π (X = N, O, C) interactions play a crucial role in the non-ovalent adsorption and that the functional group close to the carbon nanotube exhibits a significant influence on the binding strength. Noncovalent functionalization of the carbon nanotube with alanine enhances the conductivity of the metallic (5, 0) nanotube. In the covalent adsorption of each alanine radical onto a carbon nanotube, the binding energy depends on the adsorption site on CNT and the electronegative atom that binds with the CNT. The strongest complex is formed when the alanine radical interacts with a (5, 0) carbon nanotube through the amine group. In some cases, the covalent interaction of the alanine radical introduces a half-filled band at the Fermi level due to the local sp
3 hybridization, which modifies the conductivity of the tube.
D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acid D-isomers with absolute stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Recently, the extraordinary functional plasticity of DAAO has become evident; in turn, boosting research on this flavoprotein. Protein engineering has allowed for a redesign of DAAO substrate specificity, oxygen affinity, cofactor binding, stability, and oligomeric state. We review recent developments in utilizing DAAO, including as a biocatalyst for resolving racemic amino acid mixtures, as a tool for biosensing, and as a new mechanism of herbicide resistance. Perspectives for future biotechnological applications of this oxidative biocatalyst are also outlined.
D: -Amino acid oxidase (DAAO) in mammal kidney regulates the renal reactive oxygen species (ROS) levels directly and plays a leading role in the development of ROS-mediated renal pathologic damages based on its crucial role in the oxidative deamination of D: -amino acids and the consequent generation of H(2)O(2). Quantitative measurement of DAAO activity in the process of renal ischemia, which could help to understand the molecular mechanisms of this gripping acute renal disease, was conducted through the determination of chiral substrate by capillary electrophoresis (CE) in our study. In this study, a chiral ligand exchange CE method was explored with Zn(II)-L: -alaninamide complex as the chiral selector to investigate DAAO activity by determining the decreased concentration of the chiral substrate of DAAO-mediated enzymatic reaction. Then, the change of DAAO activity following 60-min acute renal ischemia in rats was observed with the proposed method. The study showed that the operation of renal ischemia resulted in a 45.49 ± 8.30% (n = 8) decrease in the DAAO-induced consumption of substrate, indicating a sharp decrease in renal DAAO activity following this acute renal injury. This phenomenon, with the possible reason of metabolic acidosis, could pave a new way for the study of oxidative stress in the development of renal ischemia injury.
The adsorption conditions used to immobilize catalase onto thin films of carbon nanotubes were investigated to elucidate the conditions that produced films with maximum amounts of active catalase. The adsorption kinetics were monitored by spectroscopic ellipsometry, and the immobilized catalase films were then assayed for catalytic activity. The development of a volumetric optical model used to interpret the ellipsometric data is discussed. According to the results herein discussed, not only the adsorbed amount but also the initial adsorption rates determine the final catalytic activity of the adsorbed layer. The results described in this paper have direct implications on the rational design and analytical performance of enzymatic biosensors.
By using femtosecond laser pulses at a wavelength range from 720 to 780 nm, we have observed absorptive and refractive nonlinearities in a film of multiwalled carbon nanotubes grown mainly along the direction perpendicular to the surface of quartz substrate. The z-scans show that both absorptive and refractive nonlinearities are of negative and cubic nature in the laser irradiance range from a few to 300 GW∕cm2. The magnitude of the third-order nonlinear susceptibility, χ(3), is of an order of 10−11 esu. The degenerate pump–probe measurement reveals a relaxation time of ∼2 ps.
Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.
To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50 degrees C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.
Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.
D-Amino acids administered to animals are absorbed by the intestine and transported through the blood-stream to solid tissues where they are oxidized in vivo by D-amino acid oxidase and D-aspartate oxidase to produce the same compounds they do in vitro; i.e. NH3, H2O2, and the keto acid corresponding to the amino acid ingested. In the liver and kidneys of the animals, an inverse relationship exists between the occurrence of D-amino acids and these oxidative enzymes. For example, younger animals have lower amounts of these oxidases and consequently higher concentrations of free D-amino acids compared to adult animals. If the ingested D-amino acids are not metabolized by these enzymes, they will accumulate in the tissues and may provoke serious damage, e.g. suppression of the synthesis of other essential enzymes and inhibition of the growth rate of the animals. A specific enzyme induction for these D-amino acid oxidases exists in young rats following ingestion of free D-amino acids by the mother. Specifically, when a mother rat ingests D-Ala or D-Asp during pregnancy and suckling, an increase in D-amino acid oxidase or D-aspartate oxidase is observed in the liver and kidneys of the baby rats. These results suggest that the in vivo biological role of these oxidases in animals is to act as detoxifying agents to metabolize D-amino acids which may have accumulated during aging.
D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction. The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.
D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.
Covering the huge developments in sensor technology and electronic sensing devices that have occurred in the last 10 years, this book uses an open learning format to encourage reader understanding of the subject. An invaluable distance learning book. Applications orientated providing invaluable aid for anyone wishing to use chemical and biosensors. Key features and subjects covered include the following: Sensors based on both electrochemical and photometric transducers; Mass-sensitive sensors; Thermal-sensitive sensors; Performance factors for sensors; Examples of applications; Detailed case studies of five selected sensors; 30 discussion questions with worked examples and 80 self-assessment questions; 140 explanatory diagrams; An extensive bibliography.
The adsorption of bovine serum albumin (BSA) onto relatively hydrophobic TiO2 surfaces was studied by ellipsometry as a function of pH and BSA concentration. Titanium oxide layers were electrochemically grown on Ti disc electrodes. When fast attachment of BSA onto TiO2 takes place, the adsorption can be considered as occurring in two different steps. The first step is fast and is the result of the direct adsorption of the protein molecules that attach to the surface without changing their conformation. The second process is slow and lasts for several hours. In this process, the adsorbed amount remains constant, whereas the thickness of the layer increases and its refractive index decreases with time. The changes in this second step are due mainly to rearrangements in the adsorbed layer produced by variations in the conformation and structure of the adsorbed molecules. The main conformational changes take place in the direction normal to the surface because lateral molecule–molecule interactions impede significant lateral expansion. Adsorption from BSA solutions of low concentration does not appear to lead to significant reconformation of the protein layer. Comparison with adsorption on powdered TiO2 indicates that the adsorbed amount and the effective area occupied by an adsorbed BSA molecule can remain about constant even when strong surface reconformation takes place.
Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.
We present Brownian dynamics simulations of the displacement of a protein monolayer by competitive adsorption. The protein film is modelled as a network of spherical bonded particles adsorbed at a fluid interface. Spherical displacer particles, which have a stronger affinity for the interface than the protein film particles, are introduced into the system through the sub-phase. At early stages, these particles diffuse to the interface and are adsorbed in the gaps in the network. Soon thereafter, however, further adsorption initiates displacement of the film particles, ultimately leading to the complete removal of the protein layer from the surface. We study the evolution of the number and size of the displacer islands formed at the interface. The introduction of a direct long-range repulsion between film and displacer particles is shown to lead to a phase-separation-type behaviour at intermediate time scales. Further comparisons with the displacement of a non-bonded monolayer are also presented.
Carbon nanotubes (CNTs) were functionalized and employed in an electrochemical cell to serve as a biosensor to specifically detect either lactate or pH in an electrolyte solution of artificial sweat. They were functionalized with the carboxyl group (COOH) to detect pH and the enzyme lactate oxidase (LOX) to detect lactate. All CNT samples were characterized to compare the electrodes before and after functionalization. Fourier transform infrared spectroscopy (FTIR) was used to verify the attachment of both COOH and LOX to the respective carbon nanotubes samples. Scanning electron microscopy (SEM) was used to examine the structure of the CNT–lactate electrode. Square wave voltammetry proved to be the best template to use to sense these target analytes. The functionalized CNT–COOH electrode displayed a linear response to pH 1–10, with a negative voltage shift corresponding to an increase in pH. Two types of lactate sensors were fabricated, both of which exhibited an increase in current corresponding to an increase in lactate concentration. The functionalized CNT–LOX on a glassy carbon electrode displayed an amperometric response in the range of 1–4 mM lactate. The CNT–LOX on a silicon/indium tin oxide (Si/ITO) substrate displayed an amperometric response in the range of 0.01–0.05 M lactate.
Bioapplications of carbon nanotubes have been predicted and explored ever since the discovery of these one-dimensional carbon allotropes. Indeed, carbon nanotubes have many interesting and unique properties potentially useful in a variety of biological and biomedical systems and devices. Significant progress has been made in the effort to overcome some of the fundamental and technical barriers toward bioapplications, especially on issues concerning the aqueous solubility and biocompatibility of carbon nanotubes and on the design and fabrication of prototype biosensors. In this article we take a comprehensive look at the advances in this fast-moving and exciting research field. We review the current status of available methodologies for the aqueous dispersion and solubilization of carbon nanotubes, discuss the results on modifications of carbon nanotubes with various biological and bioactive species, and highlight some of the recent achievements in the fabrication and evaluation of carbon nanotube-based bioanalytical devices.
Total internal reflection fluorescence (TIRF) and ellipsometry have been used to study competitive protein adsorption to a hydrophobic model surface prepared by radio frequency plasma deposition of hexamethyl disiloxane on silicon. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) at concentrations corresponding to 1/100 of those in blood plasma were investigated. It is shown that by employing the combination of ellipsometry and TIRF, information on both the total adsorbed amount and the composition of the adsorbed protein layer can be obtained. It was found that adsorbed HSA is not displaced by IgG and/or Fgn to any large extent. IgG and HSA dominate the adsorption from the ternary protein mixture, although fibrinogen is also present in the adsorbed layer to a smaller extent.
We report on a complete characterization of the optical dispersion properties of conducting thin films of single-wall carbon nanotubes (SWCNTs). The films studied exhibit sheet resistances between 50 and 1000 Ω∕sq and optical transparencies between 65% and 95% on glass and quartz substrates. These films have the potential to replace transparent conducting oxides in applications such as photovoltaics and flat-panel displays; however, their optical properties are not sufficiently well understood. The SWCNT films are shown to be hole conductors, potentially enabling their use as hole-selective contacts and allowing alternative device designs. The fundamental optical, morphological, and electrical characteristics of the films are presented here, and a phenomenological optical model that accurately describes the optical behavior of the films is introduced. Particular attention is paid to ellipsometry measurements and thorough evaluation of the reflection and absorption spectra of the films.
Neither the adsorbed amount per unit actual surface area nor the structural stability of hen egg lysozyme is influenced by increasing the nanometer-scale roughness (5 < Rave < 60 nm) of a series of model substrates. Seven poly(ether sulfone) (PES) ultrafiltration membranes of increasing mean pore size with the same surface chemistry were chosen as model rough surface substrates. Topographical images, using atomic force microscopy, combined with attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR) and sessile captive bubble contact angle measurements were used to characterize the surface properties of the substrates. ATR/FTIR spectroscopy together with a newly developed optimization algorithm for predicting the content of secondary structure motifs is used to correlate the secondary structure and amount of adsorbed lysozyme with the substrate surface roughness. From the adsorption measurements, the net adsorbed amount (total minus nonspecific adsorbed amount) of lysozyme corresponded to approximately one monolayer of coverage for all the substrates independent of the roughness. Although lysozyme was structurally disturbed through adsorption to PES substrates, no significant changes in its secondary structure were observed with the increasing roughness.
Purified carbon nanotubes are new carbon allotropes, sharing similarities with graphite, that have recently been proposed for their potential use with biological systems as probes for in vitro research and for diagnostic and clinical purposes. However the biocompatibility of carbon nanotubes with cells represents an important problem that, so far, remains largely uninvestigated. The objective of this in vitro study is to explore the cytocompatibility properties of purified carbon nanofibres with cardiomyocytes. Cardiac muscle cells from a rat heart cell line H9c2 (2-1) have been used. Highly purified single-walled nanotubes (SWNTs) were suspended at the concentration of 0.2 mg ml−1 by ultrasound in complete Dulbecco's modified Eagle's medium, and administered to cells to evaluate cell proliferation and shape changes by light microscopy, cell viability by trypan blue exclusion, and apoptosis, determined flow cytometrically by annexin/PI staining. Microscopic observation evidenced that carbon nanotubes bind to the cell membrane, causing a slight modification in cell shape and in cell count only after three days of treatment. Cell viability was not affected by carbon nanotubes in the first three days of culture, while after this time, cell death was slightly higher in nanotube-treated cells (p = ns). Accordingly, nanotube treatment induced little and non-significant change in the apoptotic cell number at day 1 and 3. The effect of nanotubes bound to cells was tested by reseeding treated cardiomyocytes. Cells from a trypsinized nanotube-treated sample showed a limited ability to proliferate, and a definite difference in shape, with a high degree of cell death: compared to reseeded untreated ones, in SWNT-treated samples the annexin-positive/PI-negative cells increased from 2.9% to 9.3% in SWNT (p<0.05, where p<0.05 defines a statistically significant difference with a probability above 95%), and the annexin-positive/PI-positive cells increased from 5.2% to 18.7% (p<0.05). However, overtime cells from a trypsinized nanotube-treated sample continued to grow, and partially recovered the original shape. In conclusion our results demonstrate that highly purified carbon nanotubes possess no evident short-term toxicity and can be considered biocompatible with cardiomyocytes in culture, while the long-term negative effects, that are evidenced after reseeding, are probably due to physical rather than chemical interactions.
Porous silicon layers with a one-dimensional lateral gradient in pore size are prepared by electrochemical etching and characterized by spectroscopic ellipsometry in the visible to near-infrared region. The ellipsometer is equipped with a micro-spot option giving a lateral resolution of approximately 100 μm. By matching multiple-layer-model calculations to the laterally-resolved variable angle of incidence spectroscopic ellipsometry data, the thickness variation along the gradient as well as the in-depth porosity profile is mapped. Upon exposure to a protein solution, protein adsorption occurs on top of the porous silicon layer. At the high-porosity region of the gradient also penetration of protein molecules into the porous layer takes place. Ellipsometry data are recorded after protein exposure and variations of protein adsorption along the porous silicon gradient is modeled as well as the in-depth profile of protein penetration.
Three different approaches are proposed to increase the resistance of enzymes against hydrogen peroxide. (a) Multipoint covalent immobilization. Through this technique, enzyme rigidity would be greatly increased and hence, any conformational change on the enzyme structure involved before or after oxidation with hydrogen peroxide becomes greatly prevented. (b) Oriented immobilization on supports having large internal surfaces. The immobilization of enzymes, through different areas of their surface on solid supports with internal morphology composed by large surfaces, promotes a certain masking of the enzyme areas that are very close to the support surface. In this way, the accessibility of hydrogen peroxide to such protein areas becomes greatly restricted. (c) Additional chemical modification of immobilized enzyme derivatives with polymers. By adding thick barriers surrounding the whole enzyme molecule, the effective concentration of hydrogen peroxide in the proximity of the most sensitive residues may be strongly reduced. Multipoint covalently immobilized d-amino acid oxidase (DAAO) from Rhodotorula gracilis on glyoxyl-agarose is 11-fold more stable than native enzyme against the deleterious effect of hydrogen peroxide. On the other hand, DAAO from Trigonopsis variabilis was not stabilized by rigidification but it could be highly stabilized by an adequate combination of the best orientation on the support plus an additional modification with poly-aldehyde polymers.
The desorption of proteins from liquid interfaces depends on the conditions under which they have been adsorbed. At low concentrations, the adsorption process takes a comparatively long time and the molecules arriving at the interface have enough space and time to adsorb and unfold. In contrast, adsorption from higher concentrated solutions is faster and adsorbing molecules strongly compete from the beginning of the process. The rate of desorption is studied as a function of the adsorption layer coverage in order to understand to what extend protein adsorption is reversible. The experimental findings cannot give a clear answer on the reversibility, however, the theoretical analysis shows that desorption rates for proteins are many orders of magnitude lower than those for usual surfactants.
The application of ellipsometry of the study of the adsorption behavior of proteins and synthetic macromolecules at the air-water interface has been investigated. It is shown that for macromolecules the amount adsorbed per unit area, Γ, as determined by ellipsometry, only has a well-defined physical meaning if the refractive-index increment remains constant up to high concentrations present in the adsorbed layer. It has been found experimentally that this conditioned is fulfilled for proteins. The ellipsometric Γ values of some protein agree satisfactorily with those obtained by two independent techniques has been used to investigate the adsorption from solution of κ-casein, bovine serum albumin, and polyvinyl alcohol. For bovine serum albumin, Γ reaches a plateau value of 2.9 mg/m2 for concentrations ≥ 0.05 wt%. The thickness of the adsorbed molecules. For κ-casein, Γ steadily increases with increasing centration and multilayers are formed. The technique provides interesting information on conformational changes in adsorbed macromolecules, on the rate of the process, and on the conditions under which these occur.
A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.
An amperometric third-generation hydrogen peroxide biosensor was designed by immobilizing hemoglobin (Hb) on a glassy carbon electrode modified with multiwall carbon nanotubes (MWNT). The direct electron transfer of the Hb immobilized on the MWNT-modified electrode was observed. The formal potential of the immobilized Hb was −0.241 V versus Ag/AgCl (3 M NaCl) and the heterogeneous electron transfer rate constant was 0.58 s−1 in a 0.20 M acetate buffer solution (pH 5.4). The immobilized Hb exhibited excellent electrocatalytic activity to reduce hydrogen peroxide in the absence and presence of oxygen, which facilitated designing an amperometric third-generation biosensor for hydrogen peroxide. In the presence of oxygen, the response to hydrogen peroxide of the designed biosensor at a potential of −0.35 V was linear in the concentration range from 6.0 × 10−6 to 6.0 × 10−3 M, and the detection limit was 1.2 × 10−6 M. The relative standard deviation was 2.4% for nine successive assays at 1.0 × 10−5 M hydrogen peroxide. The designed biosensor was applied to the determination of hydrogen peroxide in pharmaceutical injections with high sensitivity and rapid response.
Carbon nanotubes (CNT) are well-ordered, high aspect ratio allotropes of carbon. The two main variants, single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT) both possess a high tensile strength, are ultra-light weight, and have excellent chemical and thermal stability. They also possess semi- and metallic-conductive properties. This startling array of features has led to many proposed applications in the biomedical field, including biosensors, drug and vaccine delivery and the preparation of unique biomaterials such as reinforced and/or conductive polymer nanocomposites. Despite an explosion of research into potential devices and applications, it is only recently that information on toxicity and biocompatibility has become available. This review presents a summary of the performance of existing carbon biomaterials and gives an outline of the emerging field of nanotoxicology, before reviewing the available and often conflicting investigations into the cytotoxicity and biocompatibility of CNT. Finally, future areas of investigation and possible solutions to current problems are proposed.
Total internal reflection ellipsometry (TIRE) in spectroscopic mode in the wavelength range 400–1200 nm is employed in situ at a solid/liquid interface for investigation of protein adsorption on thin semitransparent gold films. In this configuration, the surface plasmon resonance phenomenon gives a large enhancement of the thin film sensitivity. Adsorption of a monolayer of the protein ferritin is monitored kinetically in situ and results in a change in the ellipsometric parameter Δ of more than 90° compared to 3° in similar ellipsometric measurements on gold substrates. This large sensitivity demonstrates a potential for sensor applications. The ferritin layer optical function is modeled with a Cauchy dispersion model resulting in a layer thickness of 9.2 nm, in good agreement with the dimension of the ferritin molecules. A transition layer between the protein film and the gold layer is necessary to introduce in the model to account for interactions between the protein layer and the gold film. The large sensitivity of TIRE for thin layers opens up a pathway to analyze in detail the structure of thin protein layers provided that a further development of the experimental setup and the model for the protein layer is carried out.
Highly fluorescent enantiomeric derivatives of d/l-serine from naphthalene-2,3-dicarboxaldehyde were resolved by hydroxypropyl-γ-cyclodextrin modified capillary electrophoresis using a saccharide as an enhancing chiral selector. Four saccharides, i.e. d-(+)-glucose, d-(−)-fructose, β-lactose, and sucrose were tested. Similar enhancing effects were observed. Coupled with laser induced fluorescence detection, this separation was applied to the determination of d-serine in neural samples including rat brain tissues, Aplysia ganglia, microdialysates from rat brain, and Aplysia individual neurons. High levels of d-serine were found in certain rat brain sections including hippocampus and striatum. d-Serine was also found to occur in Aplysia ganglia, but interestingly, it was not detected in single neurons isolated from Aplysia ganglia.
A selective dual enzyme electrode system for the monitoring of sugars in flow injection was developed. The working electrodes were based on cellobiose dehydrogenase (CDH) and oligosaccharide dehydrogenase (ODH) ‘wired’ with an osmium-based redox polymer on solid graphite electrodes. In each case the respective enzyme and poly(1-vinylimidazole) (PVI) in which every tenth mer is complexed with osmium (4,4′-dimethylbpy)2Cl, (denoted PVI10dmeOs) were crosslinked with poly(ethylene glycol) diglycidyl ether (PEGDGE). The two enzyme electrodes had distinct but different selectivities and sensitivities towards a number of investigated sugars. The CDH-modified electrodes responded, with relative activities, for cellobiose (100%), lactose (62.5%), maltose (1.2%), glucose (1%) and for higher cellodextrines (Glu3–6) while the ODH electrodes responded to all investigated sugars except d-fructose and the higher cellodextrins with relative activities for d-glucose (100%), cellobiose (60.4%), lactose (47.3%), maltose (40.6%), l-arabinose (26.1%), maltotriose (24.0%), d-galactose (21.9%), d-xylose (13.1%) and d-mannose (8.9%), 1 mM concentrations each. Linear calibration curves for cellobiose were obtained between 25 μM and 3 mM for the CDH- and ODH-modified electrodes with a sensitivity of 7.9 and 1.4 μA mM−1 cm−2, respectively. The dual electrode system was also used as an end column detector for detection of various sugars after their separation in a size-exclusion Chromatographic system.
Understanding protein adsorption at electrode surface is important for biosensor development, in particular for piezoelectric quartz crystal (PQC) microbalance (QCM) sensor. Two proteins, bovine serum albumin (BSA) and fibrinogen were monitored in situ for their electrode-surface processes using both QCM and electrochemical impedance method. Conductive polymer films, which are based on de-doping polyaniline (PAN), ions-doped PAN (PAN + ClO4−) or sodium dodecyl sulfate (SDS) doped PAN (PAN + SDS) were prepared by electrochemical methods. The effect of the polymer film thickness and doping agents of PAN on protein adsorption were investigated. The maximum adsorptions of proteins onto PAN, PAN + ClO4− and PAN + SDS films were measured, with results showing 0.71, 0.65 and 0.99 μg/cm2 for BSA and 2.73, 1.85 and 4.07 μg/cm2 for fibrinogen, respectively. The results suggest an end-on type adsorption of BSA to produce a mono-layered structure onto all three PAN films. As for fibrinogen, only the ClO4−-doped PAN film was adsorbed in an end-on type mono-layered structure. For the adsorption of fibrinogen on the other two PAN films, the adsorbed protein molecules formed multi-layered structure. The adsorption of proteins onto the PAN films was found to follow the Langmuir isotherm. Results from adsorption kinetics studies showed two consecutive steps in the adsorption of BSA and fibrinogen on PAN films. BSA and fibrinogen molecules were firstly adsorbed onto the films before undergone rearrangement. The adsorption equilibrium constants were 1.64 × 106, 9.51 × 105 and 2.42 × 106 for BSA adsorption and 2.74 × 108, 2. 43 × 108 and 2.52 × 108 for fibrinogen adsorption onto ClO4−-doped PAN, de-doping PAN and SDS-doped PAN films, respectively.
The study of the adsorption of albumin on a platinum rotating disk electrode was performed using impedance measurements. The kinetics of the adsorption was investigated by recording, during 15 min after protein addition, the variation with time of the electrochemical double layer capacitance, Cd. The resulting curve was computed and a model of two consecutive reactions occuring at the interface was proposed to describe the adsorption reaction. The corresponding theoretical equation used to fit the data showed five parameters: three capacitance (of the bare surface, of the surface covered with the reversibly adsorbed proteins, and of the surface covered with the irreversibly adsorbed proteins) and two time constants. The influence of the shear rate on these parameters was studied within the range 0–1728st- and the two time constants, corrected for the mass transfer contribution, showed a shear dependence. Different possibilities were considered for the decrease in capacitance that goes with the adsorbed protein denaturation reaction.
This review paper outlines the important issues with regards to the development of biosensors for the monitoring of biological warfare agents (BWAs) starting with the basic components of biosensors and the features of BWAs, which are compatible with detection using biological recognition molecules. The advantages and limitations of biosensors are discussed followed by the current state of the art in biosensors for detecting BWAs. Finally the developments required to enable biosensors to become more effective at providing early warning of possible biological attack and advances towards these developments are covered.
Carbon-based thin films, such as amorphous carbon (a-C) and amorphous hydrogenated diamond-like carbon (a-C:H or DLC) are considered as excellent candidates for use as bicompatible coatings on biomedical implants. This arises not only due to their excellent properties, but also due to their chemical composition containing only carbon and hydrogen, which are biological compatibles. The aim of this work is the comparative study of the haemocompatibility of the carbon-based thin films developed by magnetron sputtering under various deposition conditions. Haemocompatibility is one of the most important properties, together with the tissue compatibility and corrosion and wear resistance, that determine the biocompatibility of the artificial implants.
This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology.
In surface biology there are numerous studies carried out using single wavelength ellipsometry, especially in the area of macromolecular adsorption on solid surfaces. The results obtained contribute significantly to the understanding of the basic mechanisms of adsorption and surface dynamics of organic molecules, especially of proteins. An example of an area of great importance is biomaterials, where ellipsometry is used as a tool in the process of acquiring knowledge about the biological acceptance of new as well as currently used implant materials. In the area of affinity biosensors, ellipsometry has been suggested as a potential readout principle. Ellipsometry is also a tool in emerging technologies, such as surface molecular engineering with the aim to construct molecular superstructures with predesigned biological functions and to interface biology with electronics. However, in most cases when ellipsometry is applied in biology, it has been used for surface mass determination. The potential in using spectroscopic data for resolving microstructural and dynamic information has not been exploited fully. From the above perspective, this report reviews the use of spectroscopic ellipsometry for studies in surface biology and highlights the advantages it offers. Two main themes are developed. The first is spectroscopy on monolayers of macromolecules with emphasis on determination of their dielectric functions and microstructure. A specific example discussed is ferritin adsorption on gold. The results, including dynamics of both the surface mass and layer microstructure, indicate an adsorption model based on a two-state adsorption mechanism. The second theme is ellipsometrically based biosensor systems. The discussion covers aspects of what imaging ellipsometry can provide in this context and is exemplified by results from affinity biosensor and gas sensor systems.
The albumin adsorption on non-hydrogenated amorphous carbon films with different diamond-like character (i.e. sp3 content) is addressed. The films were produced by ion beam assisted deposition and by filtered cathodic vacuum arc deposition to obtain a wide range of sp3 contents. A combination of the spectroscopic ellipsometry, Raman spectroscopy and elastic recoil detection analysis was used for characterization of the films. It is shown that an increase of the deposition temperature leads to a decrease of the film band gap, density and a shift of the Raman G-band position. The wettability of the film is not influenced by its sp3 content. Albumin adsorption on the surface depends more on its wetting behavior than on the sp3 content. In addition, Ar ion treatment of the layers can be used to reduce the amount of adsorbed proteins.
As functional materials, chitin and chitosan offer a unique set of characteristics: biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacterial properties, heavy metal ions chelation, gel forming properties and hydrophilicity, and remarkable affinity to proteins. Owing to these characteristics, chitin- and chitosan-based materials, as yet underutilized, are predicted to be widely exploited in the near future especially in environmentally benign applications in systems working in biological environments, among others as enzyme immobilization supports. This paper is a review of the literature on enzymes immobilized on chitin- and chitosan-based materials, covering the last decade. One hundred fifty-eight papers on 63 immobilized enzymes for multiplicity of applications ranging from wine, sugar and fish industry, through organic compounds removal from wastewaters to sophisticated biosensors for both in situ measurements of environmental pollutants and metabolite control in artificial organs, are reviewed.
Seven years have passed since one of us (W.N.) published the last comprehensive review on the mechanism of globular protein adsorption to solid/water interfaces. Since that time, annual contributions to the field have steadily increased and substantial progress has been made in a number of important areas. This review takes a fresh look at the driving force for protein adsorption by combining recent advances with key results from the past. The analysis indicates that four effects, namely structural rearrangements in the protein molecule, dehydration of (parts of) the sorbent surface, redistribution of charged groups in the interfacial layer, and protein surface polarity usually make the primary contributions to the overall adsorption behavior.
Haemocompatibility is one of the most important properties, together with the tissue compatibility, corrosion and wear resistance that determine the biocompatibility of the artificial implants. Carbon-based thin films, such as amorphous carbon and amorphous hydrogenated diamond-like carbon (a-C:H or DLC) are considered as excellent candidates in order to be used as biocompatible coatings on biomedical implants. The aim of this work is to develop a methodology in order to study the protein adsorption phenomenon on thin films and to explore the optical properties of two basic blood plasma proteins, human serum albumin (HSA) and fibrinogen (Fib) and their adsorption mechanisms on amorphous hydrogenated carbon (a-C:H) thin films. Two techniques advantageous for the study of biological samples are used: Vis–UV spectroscopic ellipsometry (SE) and atomic force microscopy (AFM).a-C:H Films are grown with rf reactive magnetron sputtering. Static and real-time SE measurements are made. In the energy range of Vis–UV, proteins are almost transparent, while they present an absorption peak at higher energies. Different protein adsorption behaviour is observed on amorphous hydrogenated carbon films deposited under different conditions. This is probably due to their different surface structure, composition and topography of the surface and its interaction with the protein molecule. Adsorption phenomenon is studied through AFM technique as well. AFM results are in accordance with those derived by SE. The combination of the two techniques provides us a more accurate description of protein adsorption mechanisms.
The aim of this study was the fabrication and characterization of biomembranes by the phase inversion (PI) method followed by their subsequent casting onto screen-printed electrodes (SPE) for biomedical applications. The combination of multiwalled carbon nanotubes (MWCNT) as a transducer with polysulfone (PSf) polymer enables easy incorporation of biological moieties (hormones or antibodies), providing a 3D composite with high electrochemical response to corresponding analytes. Antibody/MWCNT/PSf biosensors were characterized by confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM), and electrochemical methods. For biomedical purposes, human chorionic gonadotropin (hCG) hormone was tested by competitive immunoassay. The detection limit was determined to be 14.6 mIU/mL with a linear range up to 600 mIU/mL. We concluded that the easy and fast incorporation of biomolecules by the PI method, as well as their stability and distribution throughout the 3D polysulfone composite, are testament to the utility for the versatile fabrication of biosensors for clinical diagnosis.
Optical anisotropy in single-walled carbon nanotube thin film networks is reported. We obtain the real and imaginary parts of the in-(parallel) and out-of-plane (perpendicular) complex dielectric functions of the single-walled carbon nanotube (SWNT) thin films by combining transmission measurements at several incidence angles with spectroscopic ellipsometry data on different substrates. In sparse networks, the two components of the real part of the complex dielectric constant (epsilon1 parallel and epsilon1 perpendicular) were found to differ by 1.5 at 2.25 eV photon energy. The resulting angular dependence (from 0 to 70 degrees incidence angles) of transmittance is reflected in the conversion efficiency of organic solar cells utilizing SWNT thin films as the hole conducting electrodes. Our results indicate that, in addition to the transparency and sheet resistance, factors such as the optical anisotropy must be considered for optical devices incorporating SWNT networks.
D-Amino acid oxidase [D-amino acid: O 2 oxidoreductase (deaminating), EC 1.4.3.3] holoenzyme migrated as a single band in zone electrophoresis on an agarose plate at several pH values from 5.5 to 9.5. The isoelectric point was found at pH 6.24.
In disc electrophoresis in polyacrylamide gel, the holoenzyme migrated also as a single band at pH 8.9, but separated into two bands at pH 8.2, both of which showed the enzymatic activity without the addition of the coenzyme, FAD.
In the experiments of isoelectric focusing, two bands were detected in both the holoenzyme and the apoenzyme.
Ultracentrifugal analysis indicated the occurrence of monomer and dimer of the holoenzyme. It was also indicated that the monomer-dimer equilibrium shifted towards monomer formation by the lowering of pH, temperature, or both of the solution and towards dimer formation as the holoenzyme was mixed with benzoate, a substrate-substitute.
A method based on microchip capillary electrophoresis with amperometric detection was developed for the rapid separation and direct detection of oxidizable aromatic amino acids (without prior derivatization). The working electrode was a thick-film carbon strip electrode positioned opposite the outlet of the separation channel. Factors influencing the separation and detection processes were examined and optimized. The five aromatic amino acids, tyrosine, 5-hydroxytryptophan, tryptophan, p-aminobenzoic acid, and m-aminobenzoic acid, can be well separated within 5 min using a separation voltage of 2000 V and a 25 mM phosphate buffer (pH 7.0) run buffer containing 50 mM sodium dodecylsulfate. Most favorable amperometric detection was obtained at +0.95 V. Linear calibration plots are observed for micromolar concentrations of the oxidizable amino acids. The new protocol offers good stability and for reproducibility, with relative S.D. of less than 5% for both migration times and peak currents (n=8). It should be useful for the analysis of aromatic amino acids, as desired for life sciences.
Angle and spectra resolved surface plasmon resonance (SPR) images of gold and silver thin films with protein deposits is demonstrated using a regular computer screen as light source and a web camera as detector. The screen provides multiple-angle illumination, p-polarized light and controlled spectral radiances to excite surface plasmons in a Kretchmann configuration. A model of the SPR reflectances incorporating the particularities of the source and detector explain the observed signals and the generation of distinctive SPR landscapes is demonstrated. The sensitivity and resolution of the method, determined in air and solution, are 0.145 nm pixel(-1), 0.523 nm, 5.13x10(-3) RIU degree(-1) and 6.014x10(-4) RIU, respectively, encouraging results at this proof of concept stage and considering the ubiquity of the instrumentation.
A method for the quantitative determination of serum D-amino acids in the range 0.5-20 nmol is described. In the method alpha-keto acids, derived from D-amino acids by D-amino acid oxidase, are measured as hydrazones. The method is unresponsive to the presence of a large excess of L-amino acids. It allows a fast assay in a small amount of specimen (0.1 ml), with good reproducibility and accuracy.
1. Minute but appreciable amounts of d-amino acids were detected in normal human plasma.
2. The content was significantly higher in an elderly population (age 76 ± 6 years, mean ± sd, n = 41) than in a younger group (age 42 ± 4 years, n = 26), i.e. 6.9 ± 4.8 nmol/ml (mean ± sd, range 0–18.8 nmol/ml) and 2.5 ± 1.8 nmol/ml (range 0–6.3 nmol/ml) for the elderly and the younger groups, respectively.
3. Elevation of plasma d-amino acid level was observed in a group of patients with renal disease (3.6–52.6 nmol/ml), in proportion to the serum level of creatinine (n = 50, r = 0.726, P < 0.001), β2-microglobulin (n = 34, r = 0.551, P < 0.005), and to glomerular filtration rate (n = 39, r = 0.556, P < 0.001).
The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form
with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 Å resolution
with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was
found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was
located in the typical βαβ dinucleotide binding motif, with the α-helix dipole stabilizing the pyrophosphate negative charge.
The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is
within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene
ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or α-helix dipole near
N(1) of flavin. Hydrogen bonds were observed at C(2)=O, N(3)-H, C(4)=O, and N(5) of the flavin ring.
The interaction of proteins with surfaces is important in separation and purification procedures as well as in metabolism and its regulation. The degree of binding to a given surface in principle depends on the precise amino acid composition of the protein, although very little is presently known about the relationship between amino acid sequence and binding. Here we report accurate measurements of the kinetics of adsorption of two closely homologous serum albumins (human and bovine) to a hydrated metal oxide surface, using an accurate integrated optics technique. Marked differences between the two proteins are observed. The results are analyzed using a model involving two bound forms, reversible and irreversible. The two forms are identified as two orientations of the protein with respect to the surface which make differing numbers of hydrogen bonds to the surface. These numbers were estimated on the basis of the measured desorption rate constants. The interfacial binding energy was calculated from the quotient of the adsorption and desorption rate constants and compared with the value calculated from surface energy available data. Remarkably, substitution of phosphate buffer for HEPES buffer causes dramatic changes in the adsorption, abolishing the irreversible mode completely for human serum albumin.
A proteolytic enzyme, alpha-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solutions on solid surfaces with different hydrophobicities and morphologies. With both enzymes the affinity of adsorption is larger for the more hydrophobic surface. Water-soluble, flexible oligomers grafted on the sorbent surface cause a decrease in enzyme adsorption. CD spectroscopy and differential scanning calorimetry (DSC) indicate severe structural perturbations in the enzymes resulting from adsorption. The CD spectra reflect an average of the structure of the whole protein population. The DSC data allow additional conclusions to be drawn on the heterogeneity in the conformational states of the adsorbed enzymes. The degree of structural perturbation, that is the fraction of the adsorbed molecules of which the structure is perturbed, is lower at a surface that (1) is less hydrophobic, (2) contains water-soluble flexible oligomers and (3) is more covered by the protein. The specific activities of the enzymes are decreased on adsorption, more or less following the extent of structural perturbation. Unlike in solution, in the adsorbed state the heat-induced inactivation process is not identical with the heat-induced unfolding process. Furthermore, when the enzymes are adsorbed their specific activities are much less sensitive to temperature variation.
The real and imaginary parts of the complex refractive index, ñ(ω)=n(ω)+ik(ω), of arc-evaporated carbon films have been obtained over the range of photon energies ħω from 0.5 to 62.0 eV. Values of k(ω) obtained from transmission measurements in this energy range were combined with values of k(ω) from the literature in the infrared and soft-x-ray regions. A Kramers-Kronig analysis then yielded the values of n(ω). The density of the arc-evaporated carbon films was found to be 1.90±0.05 g cm-3 by the ‘‘sink-float’’ method, and their thicknesses were determined optically. A sum-rule calculation yielded the effective numbers of valence and core electrons to be 4.2 and 1.8, respectively. The experimental values determined for ñ(ω) have been used to estimate values of the inelastic mean free path Λ(E) for electrons of energy E from 200 to 3000 eV in amorphous carbon. Good agreement is found between Λ(E) and experimentally determined values of electron attenuation length L(E) from the literature.