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Adsorption of Glucose Oxidase to 3-D Scaffolds of Carbon Nanotubes: Analytical Applications
Abstract
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.
... Aiming to address this gap in knowledge and to gain a better understanding of the effects of the electric field on the driving forces and structural integrity of adsorbed proteins, this article describes results obtained with GOx. This protein was selected as a convenient probe for the adsorption experiments because it allows an easy route to evaluate potential structural modifications affecting its catalytic activity [16]. Additionally, GOx is commercially available and has a well-characterized structure and activity. ...
... Adsorption experiments were performed using a variable angle spectroscopic ellipsometer (WVASE, J.A. Woollam Co.; Lincoln, NE) following a procedure described elsewhere [16][17][18]. The basis of SE is the measurement of change 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. ...
... The collected data (amplitude ratio (W) and phase difference (D) as function of wavelength or time) were modeled using the WVASE software package (J.A. Woollam Co.; Lincoln, NE) and the mean square error (MSE, calculated by a built-in function in WVASE) was used to quantify the difference between the experimental and model-generated data. In agreement with previous reports, MSE < 15 was considered acceptable [16,17]. The ellipsometric measurements were interpreted using an optical model which describes the microstructure of the system under study in terms of the refractive index (n), extinction coefficient (k), and thickness (d). ...
... As a consequence, a significant number of proteins adsorbed to solid surfaces can be affected by surface-induced structural changes, leading to spreading (unfolding and refolding) which can be detrimental to the functionality of the adsorbed proteins. In addition, chemical modifications of the protein, use of hydrophilic substrates [5], and prudent selection of specific experimental conditions (that maximize protein immobilization rate) [6] can limit the spreading and help to preserve the activity of enzymes [7,8]. ...
... In order to determine the effect of the proposed films on the activity of immobilized GOx, the PS-b-P2VPcoated substrates (2 cm 2 geometric area) were first immersed in a solution containing 0.5 mg mL À1 of GOx (in 10 mM citrate buffer, pH = 4.2) for 30 min. These conditions were selected because the solution provides high buffer capacity around the isoelectric point of the protein, therefore maximizing the initial adsorption rate [5]. Then, the PS-b-P2VP/GOx substrates were gently rinsed with buffer (to remove any GOx loosely bound to the substrate), scanned using ellipsometry to determine the effective thickness, and placed in a quartz cuvette previously filled with a mixture of glucose, o-dianisidine, and HRP. ...
... As previously stated, the goal of this project was to mitigate post-immobilization conformational changes and maximize the catalytic activity of the resulting nanocomposite. Such information is critical for the development of catalytic surfaces and biosensors because the conditions selected for the immobilization can yield GOx films with a wide range of activity [5,[44][45][46][47]. Thus, the normalized catalytic activity of GOx immobilized to the selected films was evaluated spectrophotometrically. ...
... In the experiments herein described, the thickness of the OTCE was controlled by using different dilutions of the selected photoresist in PGMEA. As the previous models [30,31,[36][37][38] were not able to accurately describe the optical properties of the resulting OTCE (therefore precluding the calculation of the thickness by ellipsometry), the thickness of the substrates was initially investigated by AFM. Figure 1A shows a representative image of the film and the location of the groove used to calculate the thickness of the film. It is worth mentioning that at least five grooves were analyzed in each substrate, discarding those that did not reach the underlying Si/SiO 2 substrate. ...
... The particular importance of OTCE is that (besides being conductive, nanostructured, and uniform in terms of thickness) they enable the simultaneous use of optical and electrochemical techniques. Following previous papers from our group [30,36,38,57,58], it was considered important to investigate the adsorption of proteins at various electrode surfaces. This information could lead to the rational development of biosensors with improved sensitivity. ...
... Although the development of biosensors is outside the scope of the present manuscript, the possibility of applying the OTCE for such experiments is herein demonstrated. For these experiments, a previously described procedure was followed [30,36,38,57]. Briefly, the ellipsometry cell [59] was filled with buffer (10 mmol/L phosphate buffer, pH 4.7) and aligned. ...
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.
... Protein adsorption experiments were performed using a variable angle spectroscopic ellipsometer (WVASE; J.A. Woollam Co.; Lincoln, NE) following a procedure described elsewhere [16][17][18][19]. The spectroscopic ellipsometry (SE) fundamental is the measurement of change 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. ...
... The mean square error (MSE; calculated by a built-in function in WVASE) was used to measure the difference between the experimental and model-generated data. MSE values lower than 15 were considered acceptable and were in agreement with published reports [16,17]. The optical model used in this study to interpret the raw ellipsometric data was developed in the Garcia Lab and was introduced in earlier publications [10]. ...
The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon films (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential (OCP; control), +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and cyclic voltammetry (CV). Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (pre-coated with the collagen type I films) under standard cell culture conditions for 2 hours was affected by the extent of collagen pre-adsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential > +800 mV) however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion.
... To avoid electrostatic interactions between proteins and increase the adsorbed amount, the adsorption is usually performed at the IEP of the enzyme (4.2) [150]. It is also important to point out that under optimized conditions, most surfaces can be saturated in less than 60 min using a solution containing $0.1 mg mL À1 of GOx [148,151,152]. Aiming at increasing the adsorbed amount, but at the risk of losing enzymatic activity or increasing cost, a number of papers reported the immersion of the substrates for 8-24 h [149,153,154] and the use of solutions containing as much as 20 mg mL À1 [150] or 40 mg mL À1 [155] of the enzyme. ...
... Due to their exquisite electronic properties and adsorptive capacity, some of the most common nanomaterials used for the immobilization of GOx are carbon-based nanomaterials [156][157][158][159][160][161][162][163]. Using these substrates, GOx can be readily adsorbed by a combination of hydrophobic and (to a much lesser degree) electrostatic interactions forming a closely packed and irreversibly bound layer of protein with a thickness comparable to the size of the protein [148]. Despite the fact that several authors have reported direct electron transfer [164][165][166][167], Wooten et al. [168] concluded that GOx adsorbed on carbon nanotubes (CNT) yields a pair of surface-confined current peaks at À0.48 V that are not compatible with the direct electron transfer between the enzymatically active GOx and CNT. ...
... 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). ...
... Besides all the well-known advantages of CNT, our lab demonstrated that three-dimensional scaffolds of CNT can significantly increase the amount of enzymes adsorbed per unit area, preserve the catalytic activity of the adsorbed molecules, and provide significant improvements (300%) in the sensitivity of the resulting biosensors [29]. Furthermore, the conclusions of these studies allowed the rational selection of the experimental conditions required to develop a CNT-based biosensor for glutamate with a wide linear range (0.01-10 µM), low detection limit (10 nM, S/N≥3), fast response time (≤5 s), and good stability [30]. ...
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.
... While the ALG PODES beads showed a decrease of 40 ± 3 % in the response (after 10 h at room temperature), the color development decreased by 65 ± 2 % and by 67 ± 3 %, when beads composed of 5 % ALG or 1 % ALG were used. We hypothesize that these changes, which are somewhat in agreement with previous reports [71][72][73], could be attributed to slight conformational changes in the enzymes, induced by the ALG PODES (or ALG) matrix [74][75][76][77], that surrounds the proteins and (upon stabilization) creates a microenvironment that maintains their conformation and protects their active sites [78]. Considering that no color development was observed outside the beads, the release of the enzymes [79,80] or the redox dye to the solution is unlikely to be a significant contributor to the decrease within the investigated timescale. ...
... The mean square error (MSE, calculated by a built-in function in WVASE) was used to quantify the fitting accuracy and validate the optical model as well. In agreement with previous reports, [50,51] MSE < 15 was considered acceptable. ...
This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular‐wave signal with frequencies varying from 0.5 to 2 Hz. The basic experiment, which can be performed in less than 5 min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100 mV and superimposed to +800 mV (applied as a working potential) and varying the frequency in the 0.1–10000 Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1–1.0 Hz), results that are in line with the corresponding dielectric features of the protein.
... A f-MWCNT-PF nanocomposite electrode with xed geometrical dimensions (5 mm  2 mm) was used to immobilize GOx through EDC-NHS coupling reactions and enable the development of a bioelectrode (GOx-f-MWCNT-PF nanocomposite electrode). AFM is an important tool to study the surface features and the immobilization of biomolecules at the microscale.59,60 The nano-features of the f-MWCNT-PF and GOx-f-MWCNT-PF nanocomposites can be distinguished in the AFM images, which represent both a two-and three-dimensional reconstruction of the surface topography measured across a 1 mm 2 square pattern. ...
Soft, flexible and conductive interfaces, which can be used as electrode materials integrated with commercial electronic components and the human body for continuous monitoring of different analytes are in high demand in wearable electronics. In the present work, we explore the development of a functionalized multi-walled carbon nanotube‡‡Functionalized multi-walled carbon nanotube (f-MWCNT).
–parafilm§§Parafilm-M (PF).
nanocomposite (f-MWCNT–PF) for the fabrication of a flexible electrochemical platform for glucose detection. The f-MWCNT–PF nanocomposite was characterized by transmission electron microscopy, energy dispersive X-ray analysis, Fourier-transform infrared spectroscopy, Raman spectroscopy, and electrochemical techniques. A bioelectrode fabricated by immobilization of glucose oxidase at the f-MWCNT–PF surface was further characterized by atomic force microscopy and tested for glucose. The bioelectrode provides two linear regions of glucose detection: a linear range from 0.08 mM to 3 mM, with a correlation coefficient of 0.982 and a sensitivity of 35.322 μA mM⁻¹; and a linear range from 5 mM to 25 mM, with a correlation coefficient of 0.964 and a sensitivity of 9.346 μA mM⁻¹. The proposed method was successfully applied to measure glucose in blood serum samples, differentiating healthy and diabetic persons. Additionally, the lower detection region could be effectively applicable for glucose sensing in sweat or interstitial fluid samples. The flexible, water-repellant sensing platform can be used as a universal platform for analyte detection, demanding waterproof, conductive platforms for biosensing applications, as it is suitable for protein/enzyme immobilization.
... 1. Electrospinning the polymer solution followed by high-temperature pyrolysis of electrospun polymer fibers, 2. Directly electrospun the carbon-based nanomaterial such as graphene [80], carbon nanofibers (CNFs) [81], carbon nanotubes (CNTs) [82], and carbon foams [83] mixed into polymer precursor solutions. These carbon-based electrospun nanofibers possess high surface area [84], providing a larger amount of electron transfer sites [85], surface functionalization [86], enzyme loading [87], and providing a platform for additives [88]. In addition, carbon nanomaterials offer high thermal [88], mechanical [89], and chemical [90] stability which confer high stability for enzymatic and non-enzymatic sensors. ...
Recent developments in the biochemical and medicinal industries have been heavily focused on producing affordable glucose biosensors due to the continuous annual increase of diabetic patients worldwide. The development of a fast, accurate, and reliable glucose sensor will increase confidence in controlling diabetes mellitus and its associated health complications among the diabetic community. Electrospinning is a versatile method that can produce complex nanofibrous assemblies with attractive and functional characteristics from various polymers. Electrospun nanofibers demonstrated high efficiency in the immobilization of biological molecules, which can improve the sensing performance further. Integration of polymer electrospun nanofibers with metal nanoparticles, metal oxide or transition metal in producing nanobiocomposites is also a highly popular approach in the past few years. This report presents the current progress and research trends of the technique, focusing on various materials and fabrication strategies used to produce biosensing interfaces. This helps readers decide the suitable approach in designing highly sensitive, selective, fast, and inexpensive glucose sensors.
... The evidence of the entrapped species on the halloysites nanotubes was monitored using X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) to determine the degree of aluminol (AlOH) and the siloxane (SiOH) groups modification since these two functional groups will be key sites for bioactive compounds interaction. pH-dependent eluted samples were then tested on breast (MCF-7) cancer cell lines to investigate their inhibitory effects and the mechanism of inhibition were determined using cyclic voltammetry and flow cytometry analyses [38][39][40][41]. The results are reported here and show evidence of differential inhibitory effects of the bioactive compounds from the various pH conditions. ...
Traditional herbal medical practices continue to be part of the healthcare needs of the world especially residents of sub-Sahara Africa (sSA). However, the mechanism of action of the plant metabolites to elicit their potency continue to be a mystery due to the lack of standardized methods. The mechanism of plant bioactive compounds to cause cell death is gradually being linked to membrane polarization and depolarization behaviour. The current work seeks to probe the electrochemical response of model cells using bioactive compounds captured in bio-zeolites or membrane mimetics. The voltage and current fluctuations emanating from such studies will establish a correlation between cell death and membrane depolarization. It will be a useful biological interface sensing material with the potential to identify plant metabolites that can selectively detect and destroy diseased cells. Several model membranes have already been developed for biomedical applications and this new paradigm will elevate the usefulness of these model systems. The concept was investigated using extracts from Dioclea reflexa (DR) hook which belongs to the leguminous family. There are certain class of compounds in Dioclea reflexa (DR) that have clinical usefulness in both temperate and tropical regions, however the identity of the bioactive compounds responsible for inducing cell death continue to be a major challenge.
... Traditionally, diverse polymer-, carbon-, inorganic-, and hybrid-based ultralight porous three-dimensional (3D) materials have been used for enzyme immobilization [1][2][3][4]. Recently, modern porous 3D printing scaffolds with complex internal structures and channels have been the focus of much attention [5]. ...
For the first time, 3D chitin scaffolds from the marine demosponge Aplysina archeri were used for adsorption and immobilization of laccase from Trametes versicolor. The resulting chitin–enzyme biocatalytic systems were applied in the removal of tetracycline. Effective enzyme immobilization was confirmed by scanning electron microscopy. Immobilization yield and kinetic parameters were investigated in detail, in addition to the activity of the enzyme after immobilization. The designed systems were further used for the removal of tetracycline under various process conditions. Optimum process conditions, enabling total removal of tetracycline from solutions at concentrations up to 1 mg/L, were found to be pH 5, temperature between 25 and 35 °C, and 1 h process duration. Due to the protective effect of the chitinous scaffolds and stabilization of the enzyme by multipoint attachment, the storage stability and thermal stability of the immobilized biomolecules were significantly improved as compared to the free enzyme. The produced biocatalytic systems also exhibited good reusability, as after 10 repeated uses they removed over 90% of tetracycline from solution. Finally, the immobilized laccase was used in a packed bed reactor for continuous removal of tetracycline, and enabled the removal of over 80% of the antibiotic after 24 h of continuous use.
... Therefore, CNTs were added to the device not only to improve the electrochemical activity of the electrodes 41 but also to provide a convenient support to immobilize enzymes. [42][43][44] Rather than modifying the electrode's surface with the CNT, which would have negatively affected the adhesive properties of the surface, the CNTs were deposited on the paper substrate. The electrochemical response from 10 mM H 2 O 2 (also prepared in PBS) was measured in order to determine the optimum amount of CNTs required. ...
Electrochemical paper-based analytical devices represent an innovative and versatile platform for fluid handling and analysis. Nevertheless, the intrinsic structure of the paper can impose limitations to both the selection of the electrode material and the method selected to attach the electrodes to the device, potentially affecting the analytical performance of the device. To address these limitations, we herein propose carbon tape as a simple and low cost alternative to develop ePADs. The proposed material (in the form of tape or tabs) was first characterized using a combination of contact angle analysis, resistivity, Raman spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Upon this initial assessment, carbon tape was selected and modified with carbon nanotubes, to provide not only a better surface for proteins to adhere to, but also an enhanced electroactive surface. The analytical performance of the resulting device was assessed by integrating three enzymes that facilitate the oxidation of ethanol, glucose, and phenol, and by performing the detection of these analytes in beer samples. The resulting device, for which materials cost less than a dollar, represents a simple alternative material for ePADs, applied in this case to monitor three of the most important parameters during the production of beers.
... Such chemical modification can be utilized for fabricating more stable chemically modified electrode surfaces [20]. Further modifications of CNTs have been successfully achieved in improved sensitivity by tailoring the thickness of scaffolds [21], covalent immobilization of organophosphorus hydrolase (OPH) enzymes [22], or covalent modification of glucose oxidase (GOx) on carboxy-functionalized grapheme sheets [23] or graphemechitosan nanocomposite films [24]. Besides CNTs, sol gels/hydrogels have been extensively used for providing an excellent conducive base for enzyme immobilization in constructing the third-generation enzyme based biosensors. ...
Immense potentiality of biosensors in medical diagnostics has driven scientists in evolution of biosensor technologies and innovating newer tools in time. The cornerstone of the popularity of biosensors in sensing wide range of biomolecules in medical diagnostics is due to their simplicity in operation, higher sensitivity, ability to perform multiplex analysis, and capability to be integrated with different function by the same chip. There remains a huge challenge to meet the demands of performance and yield to its simplicity and affordability. Ultimate goal stands for providing point-of-care testing facility to the remote areas worldwide, particularly the developing countries. It entails continuous development in technology towards multiplexing ability, fabrication, and miniaturization of biosensor devices so that they can provide lab-on-chip-analysis systems to the community.
... After attachment to a solid surface, proteins often undergo conformational changes. 38,39 An adsorbed protein molecule with a perturbed state can facilitate adsorption of more protein molecules, leading to aggregation on the surface. Contact sliding of the bar abrades the formed protein layer (consisting of perturbed monomers and/or aggregates) off the surface, releasing the perturbed protein or protein aggregate into the bulk, which may be followed by further aggregation in solution, eventually leading to the formation of nanometer-and micron-sized aggregates. ...
This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non–contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.
... Additionally, random networks of drop cast CNT films often suffer from poor reproducibility compared to their well-defined GC or HOPG counterparts. 188 Nonetheless, CNT mesh electrodes can offer advantages such as increased loading of enzymes and transport of substrates/products. 189 Furthermore, the spontaneously adsorbed enzyme at CNT mesh electrodes retains their enzymatic activity. 190 Transition metals (such as those used for CNT growth) and N-doping significantly increase the network conductivity. ...
... As a compromise between signal magnitude and selectivity [45,46], the oxidation of hydrogen peroxide was performed at + 650 mV (vs. Ag|AgCl|KCl sat ) and followed by chronoamperometry. ...
... In agreement with previous reports, MSE < 15 were considered acceptable. 30,31 The ellipsometric measurements were interpreted using a previously developed optical model 13,14 allowing the description of the substrates in terms of the refractive index (n), extinction coefficient (k), and thickness (d). Consequently, five uniaxial layers with optical axes parallel to the surface substrate were considered in this optical model. ...
The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins, whereas myoglobin (Mb), α-
lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800 mV to the sorbent surface induced the formation of multiple layers of protein. These results suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.
... Scanning and transmission electron microscopy and atomic force microscopy may also be useful for evaluation of the coatings; however, these methods have an extremely low throughput and often require interfering treatments (such as drying, freezing, or deposition of a conductive layer) before an image can be made. 3,4,8 Recently developed suspended microchannel resonators (SMR) enable the measurement of the mass of single nanoparticles with precision in the range of femtogram to attogram. [9][10][11] In these systems, a suspension of particles is flushed through a microchannel inside the resonator. ...
We assessed the potential of a suspended microchannel resonator (SMR) to measure the adsorption of proteins to nanoparticles. Standard polystyrene beads suspended in buffer were weighed by a SMR system. Particle suspensions were mixed with solutions of bovine serum albumin (BSA) or monoclonal human antibody (IgG), incubated at room temperature for 3 h and weighed again with SMR. The difference in buoyant mass of the bare and protein-coated polystyrene beads was calculated into real mass of adsorbed proteins. The average surface area occupied per protein molecule was calculated, assuming a monolayer of adsorbed protein. In parallel, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and zeta potential measurements were performed. SMR revealed a statistically significant increase in the mass of beads because of adsorption of proteins (for BSA and IgG), whereas DLS and NTA did not show a difference between the size of bare and protein-coated beads. The change in the zeta potential of the beads was also measurable. The surface area occupied per protein molecule was in line with their known size. Presented results show that SMR can be used to measure the mass of adsorbed protein to nanoparticles with a high precision in the presence of free protein. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
... In agreement with previous reports, MSE < 15 was considered acceptable. 45,47 The ellipsometric measurements were interpreted using an optical model that considered the dielectric properties of Si (bulk, d ¼ 1 mm) and SiO 2 (d ¼ 2.1 AE 0.5 nm). A standard Cauchy function was added to describe the optical properties of the APTES layer (d ¼ 0.89 AE 0.06 nm). ...
This paper describes a silica nanoparticle-modified microfluidic paper-based analytical device (µPAD) with improved color intensity and uniformity for three different enzymatic reactions with clinical relevance (lactate, glucose, and glutamate). The µPADs were produced on Whatman grade 1 filter paper and using a CO2 laser engraver. Silica nanoparticles modified with 3-aminopropyltriethoxysilane (APTES) were then added to the paper devices to facilitate the adsorption of selected enzymes and prevent the washing away effect that creates color gradients in the colorimetric measurements. Here we show three different enzymatic assays for compounds. According to the results herein described, the addition of silica nanoparticles yielded to significant improvements in color intensity and uniformity. The resulting µPADs allowed for the detection of the three analytes in clinically-relevant concentration ranges with limits of detection (LOD) of 0.63 mM, 0.50 mM, and 0.25 mM for lactate, glucose, and glutamate , respectively. An example of an analytical application has been demonstrated for the semi-quantitative detection of all three analytes in artificial urine. The results demonstrate the potential of silica nanoparticles to avoid the washing away effect and improve the color uniformity and intensity in colorimetric bioassays performed on µPADs.
... 1-5, 21, 22 The DET-based electrochemical glucose sensing at GOx/CNT-based electrodes has also been debated with arguments on both sides of the issue depending on the specifics of experiments. [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] The present study focuses on the role of DET in glucose sensing at a GOx/CNT hybrid that was embedded in a bio-adhesive film of polysaccharide chitosan (CHIT) on the electrode surface. The CHIT provided a biocompatible, inert, and protective matrix for GOx and served as an effective dispersant for CNT. ...
The signal transduction and enzyme activity were investigated in biosensors based on the glucose oxidase (GOx) and carbon nanotubes (CNT) embedded in a bio-adhesive film of chitosan (CHIT). The voltammetric studies showed that, regardless of CHIT matrix, the GOx adsorbed on CNT yielding a pair of surface-confined current peaks at -0.48 V. The anodic peak did not increase in the presence of glucose in an O2-free solution indicating the lack of direct electron transfer (DET) between the enzymatically active GOx and CNT. The voltammetric peaks were due to the redox of enzyme cofactor flavin adenine dinucleotide (FAD), which was not the part of active enzyme. The presented data suggest that DET may not be happening for any type of GOx/CNT-based sensor. The biosensor was sensitive to glucose in air-equilibrated solutions indicating the O2-mediated enzymatic oxidation of glucose. The signal transduction relied on the net drop in a biosensor current that was caused by a decrease in a 4-e- O2 reduction current and an increase in a 2-e- H2O2 reduction current. The enzyme assays showed that CNT nearly doubled the retention of GOx in a biosensor while decreasing the average enzymatic activity of retained enzyme by a factor of 4-5. Such inhibition should be considered when using a protein-assisted solubilization of CNT in water for biomedical applications. The proposed analytical protocols can be also applied to study the effects of nanoparticles on proteins in assessing the health risks associated with the use of nanomaterials.
... Adsorption experiments were performed using a variable angle spectroscopic ellipsometer (WVASE, J.A. Woollam Co.; Lincoln, NE) following a procedure described elsewhere. 23,24 Spectroscopic ellipsometry (SE) has proven suitable to study adsorption of proteins in real time and provides useful information about the thickness, optical constants, and structure of the adsorbed film. More information regarding the principle of SE can be found elsewhere. ...
... However, the use of conjugation chemistries to link proteins to CNTs could lead to the aggregation of proteins by cross-linking and to loss of their activity, especially in the case of enzymes. This concern was addressed by the discovery that some proteins, including enzymes [58] and oligonucleotide probes [59], can be conjugated by simple physical adsorption on the inside of CNTs. Despite these developments, it is critical to understand the causes and consequence of the interactions between CNTs and protein conjugates, and efforts are being directed in that direction [60]. ...
Carbon-based nanomaterials and their derivatives have triggered extensive research and promise to be some of the key materials in nanotechnology. Unlike other existing materials, carbon nanomaterials have found many scientific and technological applications in diverse areas. The wide range of superior properties (mechanical, electrical, optical and thermal) that are inherent in carbon nanostructures, specifically nanotubes and graphene, and the simplicity of their structures have played an important role in the current rapid expansion of fundamental studies of these materials and their potential use in nanotechnology. Based on their novel electronic and optical properties and their controllable chemical functionality, carbon nanostructures are expected to be used in several applications, including nanoelectronics and energy research, as well as biomedical areas such as biosensors, cellular imaging, drug delivery, photothermal therapy and diagnostics. Realizing the immense potential of carbon nanomaterials for applications in biology and medicine, we shall review recent advances in this field and discuss the outlook for the future.
... The biosensor had a detection limit of 3.5 μM, with a linear range from 0.004 to 3.23 mM. In another effort, aimed at enhancing the amount of adsorbed enzyme, Nejadnik et al. [24] explored the use of CNT scaffolds for enzyme immobilization. It was hypothesized that the three-dimensional scaffolds can significantly increase the amount of enzyme adsorbed per unit area while preserving the catalytic activity of the adsorbed molecules . ...
The applications of biosensors range from environmental testing and biowarfare agent detection to clinical testing and cell analysis. In recent years, biosensors have become increasingly prevalent in clinical testing and point-of-care testing. This is driven in part by the desire to decrease the cost of health care, to shift some of the analytical tests from centralized facilities to “frontline” physicians and nurses, and to obtain more precise information more quickly about the health status of a patient. This article gives an overview of recent advances in the field of biosensors, focusing on biosensors based on enzymes, aptamers, antibodies, and phages. In addition, this article attempts to describe efforts to apply these biosensors to clinical testing and cell analysis.
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Biosensor for Point of care
Natural polymers offer not only low-cost, wide availability, biocompatibility, and biodegradability but also a rich chemical functionality towards custom modifications and new applications. Among those, sodium alginate (ALG) offers a number of competitive advantages, including the possibility to form beads by a simple process call spherification as well as their modification by a wide number of processes, including the possibility to mixing the alginate solution with deep eutectic solvents (DES). Thus, and considering the capacity of DES to stabilize enzymes, the main objective of this work was to design and fabricate a new PODES (using ALG as one of the components, ALGPODES) and to deploy this material as a new phase to develop colorimetric biosensors. The formed beads displayed a narrow size distribution (2.9 0.1 mm) and a permeable structure with large internal cavities (25-100 m). The applicability of these beads towards analytical applications (lab-on-a-bead) was demonstrated by integrating three sensing platforms and by their application in real food samples.
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.
In this work, optimization parameters were developed to capture plant metabolites from Dioclea Reflexa (DR) seed ex-tracts onto halloysites nanotubes (HNTs). A one-step pool of the crude extracts at neutral pH from the HNT lumen failed to elicit a reduction in breast cancer, Michigan Cancer Foundation-7 (MCF-7) cell viability. However, the pH-dependent elution of metabolites revealed that the acidic pH samples exhibited profound antiproliferative effects on the cancer cells compared to the basic pH metabolites using both trypan blue dye exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability test. pH~5.2 samples demonstrated by half-maximal inhibitory concentration (IC50) of 0.8 mg and a cyclic voltammetry oxidation peak potential and current of 234 mV and 0.45 µA, respectively. This indicates that the cancer cells death could be attributed to membrane polarization/depolarization effects of the sample. Fluorescence-activated cell sorting (FACS) studies confirmed that the plant metabolites affected breast cancer apoptotic signaling pathways of cell death. The studies proved that plant metabolites could be captured using simplified screening procedures for rapid drug discovery purposes. Such procedures, however, would require the integration of affordable analytical tools to test and isolate individual metabolites. Our approach could be an important strategy to create a library and database of bioactive plant metabolites based on pH values.
The effect of incorporating different types of carbon nanotubes into composite films of a redox polymer (FcMe2-C3-LPEI) and glucose oxidase (GOX) was investigated. The composite films were constructed by first forming a high-surface area network film of either single-walled carbon nanotubes (SWNTs) or multiwalled carbon nanotubes (MWNTs) on a glassy carbon electrode (GCE) by solution casting of a suspension of Triton-X-100 dispersed SWNTs. Next a glucose responsive redox hydrogel was formed on top of the nanotube-modified electrode by cross-linking FcMe2-C3-LPEI with glucose oxidase via ethylene glycol diglycidyl ether (EGDGE). Electrochemical and enzymatic measurements showed that composite films made with (7,6) SWNTs produced a higher response (3.3 mA/cm²) to glucose than films made with (6,5) SWNTs (1.8 mA/cm²) or MWNTs (1.2 mA/cm²) or films made without SWNTs (0.7 mA/cm²). We also show that the response of the composite films could be systematically varied by fabricating SWNT films with different weight ratios of (7,6) and (6,5) SWNTs. Optimization of the (7,6) SWNTs loading and the redox polymer-enzyme film produced a glucose response of 11.2 mA/cm². Combining the optimized glucose films with a platinum oxygen breathing cathode into a biofuel cell produced a maximum power density output of 343 μW/cm².
Tungsten-containing formate dehydrogenase (FoDH1) with a molecular mass of 170 kDa from Methylobacterium extorquens AM1 catalyzes the oxidation of formate (HCOO⁻) to carbon dioxide (CO2) with NAD⁺ as a natural electron acceptor in solution. FoDH1 does not produce any direct electron transfer (DET)-type bioelectrocatalytic wave at planar electrodes, but can adsorb on and communicate with mesoporous carbon electrodes. The curvature effect of mesoporous structures seems to increase the number of enzymes with orientations suitable for electrochemical communication. However, adsorption proceeds slowly on Ketjen Black-modified electrode and the catalytic current density remains low. Most probably, the size of the mesopores is too small to effectively trap FoDH1. The adsorbed FoDH1 catalyzes DET-type bioelectrocatalytic interconversion of the CO2/HCOO⁻ and NAD⁺/NADH redox couples. Most probably, one of the iron–sulfur clusters located near the enzyme surface communicates with mesoporous electrodes. When the communication proceeds effectively, FoDH1 behaves as a novel bidirectional catalyst for the substrates, since FoDH1 can realize fast uphill intramolecular electron transfer. The non-covalently bound flavin mononucleotide (FMN) cofactor in FoDH1 is dissociated from some FoDH1 molecules and adsorbs on the mesoporous electrode to give a symmetrical surface-confined redox wave. Although adsorbed FMN cannot participate in mediated electron transfer (MET)-type bioelectrocatalysis, dissociated FMN in solution works as a mediator for MET-type bioelectrocatalysis of the HCOO⁻ oxidation at planar electrodes.
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.
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.
In this work, a large-scale synthesis of a three-dimensional (3D) nitrogen-doped carbon nanotube (NCNT) film was achieved via electrospinning assisted by a chemical vapor deposition procedure. The resulting nanostructure with dense and uniform NCNTs was tightly bonded onto the electrospun carbon nanofiber matrix. Owing to the favorable 3D network structure and high nitrogen doping, the 3D NCNTs are a wonderful platform to immobilize hemin for biosensing. By directly dropping the flexible film onto the electrode surface without additional oxidant treatment, a H2O2 biosensor can be simply constructed. The novel biomimetic H2O2 biosensor has a low detection limit (0.03M S/N = 3) and a wide linear range (0.08-137.2M). The excellent synergic effect enables an enhanced electrochemical performance of the modified electrode with good biocompatibility and fast redox properties. In addition, the biosensor exhibited high reproducibility, good storage stability, and satisfactory anti-interference ability. The facile preparation method and attractive analytical performances make this robust electrode material promising for the development of effective electrochemical sensors.
Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 17 is July 11, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
An amperometric glucose biosensor based on immobilization of glucose oxidase on nitrogen-doped carbon nanotubes (N-CNTs) was successfully developed for the determination of silver ions. Upon exposure to glucose, a steady-state enzymatic turnover rate was detected through amperometric oxidation of the H2O2 byproduct, directly related to the concentration of glucose in solution. Inhibition of the steady-state enzymatic glucose oxidase reaction by heavy metals ions such as Ag(+), produced a quantitative decrease in the steady-state rate, subsequently creating an ultrasensitive metal ion biosensor through enzymatic inhibition. The Ag(+) biosensor displayed a sensitivity of 2.00 x 10(8) ± 0.06 M(-1), a limit of detection (σ = 3) of 0.19 ± 0.04 ppb, a linear range of 20 - 200 nM, and sample recovery at 101 ± 2%, all acquired at a low operating potential of 0.05 V (vs. Hg/Hg2SO4). Interestingly, the biosensor does not display a loss in sensitivity with continued use due to the % inhibition based detection scheme - loss of enzyme (from continued use) does not influence the % inhibition, only the overall current associated with the activity loss. The heavy metals Cu(2+) and Co(2+) were also detected using the enzyme biosensor, but found to be much less inhibitory, with sensitivities of 1.45 x 10(6) ± 0.05 M(-1) and 2.69 x 10(3) ± 0.07 M(-1), respectively. The mode of GOx inhibition was examined for both Ag(+) and Cu(2+) using Dixon and Cornish-Bowden plots, where a strong correlation was observed between the inhibition constants and the biosensor sensitivity.
The electrochemical behavior of hydrogen peroxide (H2O2) at carbon nanotubes (CNTs) and nitrogen-doped carbon nanotubes (N-CNTs) was investigated over a wide potential window. At CNTs, H2O2 will be oxidized or reduced at large overpotentials, with a large potential region between these two processes where electrochemical activity is negligible. At N-CNTs, the overpotential for both H2O2 oxidation and reduction is significantly reduced; however, the reduction current from H2O2, especially at low overpotentials, is attributed to increased oxygen reduction rather than the direct reduction of H2O2, due to a fast chemical disproportionation of H2O2 at the N-CNT surface. Additionally, N-CNTs do not display separation between observable oxidation and reduction currents from H2O2. Overall, the analytical sensitivity of N-CNTs to H2O2, either by oxidation or reduction, is considerably higher than CNTs, and obtained at significantly lower overpotentials. N-CNTs display an anodic sensitivity and limit of detection of 830 mA M(-1) cm(-2) and 0.5 μM at 0.05 V, and a cathodic sensitivity and limit of detection of 270 mA M(-1) cm(-2) and 10 μM at -0.25 V (V vs. Hg/Hg2SO4). N-CNTs are also a superior platform for the creation of bioelectrodes from the spontaneous adsorption of enzyme, compared to CNTs. Glucose oxidase (GOx) was allowed to adsorb onto N-CNTs, producing a bioelectrode with a sensitivity and limit of detection to glucose of 80 mA M(-1) cm(-2) and 7 μM after only 30 s of adsorption time from a 81.3 μM GOx solution.
The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800mV to the sorbent surface induced the formation of multiple layers of protein. These results also suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.
A versatile matrix was fabricated and utilized as a universal interface for the construction of enzyme-based biosensors. This matrix was formed on the gold electrode via combining self-assembled monolayer of 2,3-dimercaptosuccinic acid with gold nanoparticles. Gold nanoparticles were electrochemically deposited. Electrochemistry of three redox enzymes (catalase, glucose oxidase, and horseradish peroxidase) was investigated on such a matrix. The electrocatalytic monitoring of hydrogen peroxide and glucose was conducted on this matrix after being coated with those enzymes. On them the monitoring of hydrogen peroxide and glucose shows rapid response times, wide linear working ranges, low detection limits, and high enzymatic affinities. This matrix is thus a versatile and suitable platform to develop highly sensitive enzyme-based biosensors.
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 adsorption of flavin adenine dinucleotide (FAD) and glucose oxidase (GOx) onto carbon nanotube (CNT) and nitrogen-doped CNT (N-CNT) electrodes was investigated and found to obey Langmuir adsorption isotherm characteristics. The amount adsorbed and adsorption maximum are dependent on exposure time, the concentration of adsorbate, and the ionic strength of the solution. The formal potentials measured for FAD and GOx are identical, indicating that the observed electroactivity is from FAD, the redox reaction center of GOx. When glucose is added to GOx adsorbed onto CNT/N-CNT electrodes, direct electron transfer (DET) from enzyme active FAD is not observed. However, efficient mediated electron transfer (MET) occurs if an appropriate electron mediator is placed in solution, or the natural electron mediator oxygen is used, indicating that GOx is adsorbed and active on CNT/N-CNT electrodes. The observed surface confined redox reaction at both CNT and N-CNT electrodes is from FAD that either specifically adsorbs from solution, or adsorbs from the holo protein subsequently inactivating the enzyme. The splitting of cathodic and anodic peak potentials as a function of scan rate provides a way to measure the heterogeneous electron transfer rate constant (k(s)) using Laviron's method. However, the measured k(s) was found to be under ohmic control, not under the kinetic control of an electron transfer reaction, suggesting that k(s) for FAD on CNTs is faster than the measured value of 7.6 s(-1).
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.
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.
The aim of this review is to summarize the most relevant contributions in the development of electrochemical sensors based on carbon materials in the recent years. There have been increasing numbers of reports on the first application of carbon derived materials for the preparation of an electrochemical sensor. These include carbon nanotubes, diamond like carbon films and diamond film-based sensors demonstrating that the particular structure of these carbon material and their unique properties make them a very attractive material for the design of electrochemical biosensors and gas sensors.
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.
Peptidomimetic polymers consisting of poly-N-substituted glycine oligomers (polypeptoids) conjugated to biomimetic adhesive polypeptides were investigated as antifouling surface coatings. The polymers were immobilized onto TiO(2) surfaces via an anchoring peptide consisting of alternating residues of 3,4-dihydroxyphenylalanine (DOPA) and lysine. Three polypeptoid side-chain compositions were investigated for antifouling performance and stability toward enzymatic degradation. Ellipsometry and XPS analysis confirmed that purified polymers adsorbed strongly to TiO(2) surfaces, and the immobilized polymers were resistant to enzymatic degradation as demonstrated by mass spectrometry. All polypeptoid-modified surfaces exhibited significant reductions in adsorption of lysozyme, fibrinogen and serum proteins, and were resistant to 3T3 fibroblast cell attachment for up to seven days. Long-term in vitro cell attachment studies conducted for six weeks revealed the importance of polypeptoid side-chain composition, with a methoxyethyl side chain providing superior long-term fouling resistance compared to hydroxyethyl and hydroxypropyl side chains. Finally, attachment of both gram-positive and gram-negative bacteria for up to four days under continuous-flow conditions was significantly reduced on the polypeptoid-modified surfaces compared to unmodified TiO(2) surfaces. The results reveal the influence of polypeptoid side-chain chemistry on short-term and long-term protein, cell and bacterial fouling resistance.
This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the "inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions.
Thick matrices of fibrinogen with incorporation of a matrix metalloproteinase inhibitor were covalently bonded on functionalized silicon surfaces using an ethyl-3-dimethyl-aminopropyl-carbodiimide and N-hydroxy-succinimide affinity ligand coupling chemistry. The growth of the structure was followed in situ using dynamic ellipsometry and characterized at steady-state with spectroscopic ellipsometry. The growth was compared with earlier work on ex situ growth of fibrinogen layers studied by single wavelength ellipsometry. It is found that in situ growth and ex situ growth yield different structural properties of the formed protein matrix. Fibrinogen matrices with thicknesses up to 58 nm and surface mass densities of 1.6 microg/cm2 have been produced.
"Lab-on-a-chip" approaches based on the novel marriage between an electrokinetic microfluidic platform and nanotechnology is proposed for analytical domains. Conceptually, the analytical challenges are linked with the analytical promises offered from the integration of lab-on-a-chip and nanotechnologies. The analytical suitability of the electrokinetic microfluidic platform with multi-walled carbon nanotubes as detectors is proposed based on its dual format/use as a flow and separation system, independently. Two relevant applications of high significance, determination of total isoflavones and fast detection of antioxidant profiles were chosen to demonstrate their analytical potential. For both analytical uses, the target challenges, the strategy proposed, the expected role of microfluidics and carbon nanotubes and future prospects are discussed and demonstrated. A good analytical performance of the proposed microfluidic platform in terms of reliability, versatility and fast analytical solutions is demonstrated.
This paper describes a durable carbon nanotube (CNT) film for flexible devices and its mechanical properties. Films as thin as 10 nm thick have properties approaching those of existing electrodes based on indium tin oxide (ITO) but with significantly improved mechanical properties. In uniaxial tension, strains as high as 25% are required for permanent damage and at lower strains resistance changes are slight and consistent with elastic deformation of the individual CNTs. A simple model confirms that changes in electrical resistance are described by a Poisson's ratio of 0.22. These films are also durable to cyclic loading, and even at peak strains of 10% no significant damage occurs after 250 cycles. The scratch resistance is also high as measured by nanoscratch, and for a 50 μm tip a load of 140 mN is required to cause initial failure. This is more than 5 times higher than is required to cause cracking in ITO. The robustness of the transparent conductive coating leads to significant improvement in device performance. In touch screen devices fabricated using CNT no failure occurs after a million actuations while for devices based on ITO electrodes 400,000 cycles are needed to cause failure.These durable electrodes hold the key to developing robust, large-area, lightweight, optoelectronic devices such as lighting, displays, electronic-paper, and printable solar cells. Such devices could hold the key to producing inexpensive green energy, providing reliable solid-state lighting, and significantly reducing our dependence on paper.
A one-pot method has been developed to prepare magnetite nanoparticles decorated carbon nanotubes (CNTs) by thermal decomposition of iron chloride on CNTs templates in diethylene glycol. The morphological and structural characterizations indicate that magnetite nanoparticles are coated on the surfaces of the CNTs to form CNT-based nanocomposites. The density of magnetite nanoparticles on CNTs could be easily tuned by adjusting the weight ratio of iron chloride to CNTs. Magnetic measurements showed that the nanocomposites are superparamagnetic at room temperature and the magnetic properties of the samples can also be tuned by adjusting preparing conditions. The nanocomposites can be readily dispersed in water to form a stable solution and can be manipulated using an external magnetic field. As-synthesized nanocomposites may have potential applications in target–drug delivery, detection and separations, and in clinical diagnosis.
Over time, new materials have been used and incorporated in a wide variety of analytical processes. This century, technology has produced novel nanomaterials with unique properties whose use has increased in analytical sciences. Carbon nanostructures are among these new nanomaterials. This overview reports on the use of carbon nanomaterials, mainly fullerenes and carbon nanotubes, as sorbents in the analytical process. After a brief description of their main characteristics, we present their use in the development of selective membranes. Next, we describe their role as sorbent materials and stationary phases in chromatography and provide relevant examples. We also comment on the presence of carbon nanoparticles as components of electrophoretic buffers to improve both resolution and sensitivity of separations. Finally, we briefly describe other applications in which the sorption capabilities of carbon nanostructures play a role.
The uptake of glucose oxidase (GOx) onto a polycationic redox polymer (PAA- Os)-modified surface, by adsorption from dilute aqueous GOx solutions, was followed by the quartz crystal microbalance (QCM) and shows double exponential kinetics. The electrochemistry of the layer-by-layer-deposited redox-active polymer was followed by cyclic voltammetry in glucose-free solutions, and the enzyme, catalysis mediated by the redox polymer was studied in P-D-glucose-containing solutions. AFM studies of the different layers showed the existence of large two dimension enzyme aggregates on the osmium polymer for 1 muM GOx and less aggregation for 50 nM GOx solutions. When the short alkanethiol, 2,2'-diaminoethyldisulfide was preadsorbed onto gold, a monoexponential adsorption law was observed, and single GOx enzyme molecules could be seen on the surface where the enzyme was adsorbed from 50 nM GOx in water.
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.
We demonstrate the use of individual semiconducting single-wall carbon nanotubes as versatile biosensors. Controlled attachment of the redox enzyme glucose oxidase (GOx) to the nanotube sidewall is achieved through a linking molecule and is found to induce a clear change of the conductance. The enzyme-coated tube is found to act as a pH sensor with large and reversible changes in conductance upon changes in pH. Upon addition of glucose, the substrate of GOx, a steplike response can be monitored in real time, indicating that our sensor is also capable of measuring enzymatic activity at the level of a single nanotube. This first demonstration of nanotube-based biosensors provides a new tool for enzymatic studies and opens the way to biomolecular diagnostics.
Two distinct proteins, streptavidin and HupR, bind and form regular helical arrays on the surface of multiwalled carbon nanotubes under appropriate conditions (see picture). The decoration of the outer surface of these nanostructures with biological macromolecules was investigated by electron microscopy.
The interface between biological molecules and novel nanomaterials is important to developing new types of miniature devices for biological applications. Here, the streptavidin/biotin system is used to investigate the adsorption behavior of proteins on the sides of single-walled carbon nanotubes (SWNTs). Functionalization of SWNTs by coadsorption of a surfactant and poly(ethylene glycol) is found to be effective in resisting nonspecific adsorption of streptavidin. Specific binding of streptavidin onto SWNTs is achieved by co-functionalization of nanotubes with biotin and protein-resistant polymers.
Electrochemistry detection offers considerable promise for capillary-electrophoresis (CE) microchips, with features that include remarkable sensitivity, portability, independence of optical path length or sample turbidity, low cost and power requirements, and high compatibility with modern micromachining technologies. This article highlights key strategies in controlled-potential electrochemical detectors for CE microchip systems, along with recent advances and directions. Subjects covered include the design of the electrochemical detection system, its requirements and operational principles, common electrode materials, isolation from the separation voltage, derivatization reactions, typical applications, and future prospects. It is expected that electrochemical detection will become a powerful tool for CE microchip systems and will lead to the creation of truly portable (and possibly disposable) devices.
As general trends in current development of chemical analysis, including electroanalysis, one can indicate a search for methods fast and multianalyte, for miniaturized measuring devices, mechanization and automation of analytical processes. In all these trends a significant role is played by measurements in flow conditions. The major advantage of flow electroanalysis is a possibility of utilizing a kinetic discrimination in potentiometric measurements and enhancement of mass transport in voltammetric techniques. Flow injection techniques provide shortening of time of a single analytical determination due to reproducible use of transient signal from the detector without need of obtaining a steady-state equilibrium signal. Electrochemical detection in HPLC gives often improved selectivity and detection limit for electroactive solutes, whereas in capillary electrophoresis it allows a convenient design of portable, integrated chips for field application. This review presents state-of-the -art of flow electroanalysis based on 226 cited literature references
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.
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.
The electrochemical behavior of fullerene and fullerene derivatives are reviewed with special reference to their catalytic and sensor applications. Recent work on carbon nanotubes, used as catalyst supports in heterogeneous catalysis and sensor development is also presented. An overview of recent progress in the area of fullerene electrochemistry is included. Several cases of electrocatalytic dehalogenation of alkyl halides, assisted by the electrode charge transfer to fullerenes, are discussed. Research work on the electrocatalysis of biomolecules, such as hemin, cytochrome c, DNA, coenzymes, glucose, ascorbic acid, dopamine, etc. have also been considered. Based on the studies of the interaction of fullerenes, fullerene derivatives, and carbon nanotubes with other molecules and biomolecules in particular, the possibilities for the preparation of electrochemical sensors and their application in electroanalytical chemistry are highlighted.
The direct electrochemistry of catalase (Ct) was accomplished at a gold electrode modified with single-wall carbon nanotubes (SWNTs). A pair of well-defined redox peaks was obtained for Ct with the reduction peak potential at −0.414 V and a peak potential separation of 32 mV at pH 5.9. Both reflectance FT-IR spectra and the dependence of the reduction peak current on the scan rate revealed that Ct adsorbed onto the SWNT surfaces. The redox wave corresponds to the Fe(III)/Fe(II) redox center of the heme group of the Ct adsorbate. Compared to other types of carbonaceous electrode materials (e.g., graphite and carbon soot), the electron transfer rate of Ct redox reaction was greatly enhanced at the SWNT-modified electrode. The peak current was found to increase linearly with the Ct concentration in the range of 8×10−6–8×10−5 M used for the electrode preparation and the peak potential was shown to be pH dependent. The catalytic activity of Ct adsorbates at the SWNTs appears to be retained, as the addition of H2O2 produced a characteristic catalytic redox wave. This work demonstrates that direct electrochemistry of redox-active biomacromolecules such as metalloenzymes can be improved through the use of carbon nanotubes.
Carbon nanotubes (CNTs)-modified titania nanotube (NT) arrays are prepared by vapor-growing CNTs in the inner of titania NTs. Pt nanoparticles of ∼3 nm in diameter are uniformly decorated on the as synthesized titania-supported CNTs (TiO2/CNTs) electrode, showing remarkably improved catalytic activities for the oxidation of hydrogen peroxide. The consequent glucose biosensor fabricated by modifying TiO2/CNT/Pt electrode with glucose oxidase (GOx) presents a high sensitivity of 0.24 μA mM−1 cm−2 to glucose in the range of 0.006 mM to 1.5 mM with a response time of less than 3 s and a detection limit of 5.7 μM at 3 signal/noise ratio.
Discoveries of new materials have significant impact on development of new methods and instrumentation for chemical analysis. Based on 104 references, this article illustrates a growing number of applications of carbon nanotubes (CNTs) in analytical chemistry. The largest numbers of reported applications concern design of novel gas sensors, voltammetry, enzymatic biosensors, immunosensors and DNA probes. The sorptive properties of CNTs are also employed for analytical purposes in various ways. (c) 2005 Elsevier Ltd. All rights reserved.
The direct electrochemical and electrocatalytic properties of catalase (CAT) immobilized on the multi-wall carbon nanotubes (MWCNT) modified glassy carbon (MWCNT/GC) electrode was investigated in 0.1 M Tris–HCl buffer solution. It was found that CAT immobilized on the MWCNT/GC electrode could undergo a direct and quasi-reversible reaction with the formal potential (E0′) of −0.456 V. The electrochemical reaction involves the transfer of one electron and one proton. Furthermore, La3+ can affect the electrochemical behavior of the immobilized CAT depending on the concentration of La3+. The low concentration of La3+ can promote, but the high concentration of La3+ would inhibit the electrochemical reaction of the immobilized CAT. This is due to that the different concentrations of La3+ could increase or decrease the exposure extent of the heme group in CAT. CAT immobilized on the MWCNT/GC electrode could keep its biocatalytic activity for the H2O2 reduction. These results illustrated that MWCNT is a good carrier for immobilizing the biomicromolecules. This may be useful in the biosensors and biofuel cells.
As classical biosensing systems, amperometric glucose biosensors have been widely studied and developed since Clark and Lyons first proposed the concept of glucose-enzyme electrodes in 1962. Although glucose oxidase can selectively and specifically catalyze oxidation of glucose, amperometric detection can hardly distinguish the current generated by the enzymatic reaction from the currents generated by electroactive species in biological samples. Various methods have therefore been reported to improve the overall selectivity of enzyme-based biosensors. This review summarizes the methods developed for eliminating electrochemical interferences. We emphasize our efforts to improve the performance of biosensors.
Carbon nanotubes have begun to attract enormous interest in electrochemistry because of their small size and good electrochemical properties. The vast majority of studies thus far have used ensembles of carbon nanotubes to nanostructure macroscopic electrodes either with randomly dispersed nanotubes or with aligned carbon nanotubes. The resultant nanotube modified electrodes have most frequently been used for electro-analytical purposes such as the development of biosensors. This review introduces carbon nanotubes and approaches to nanostructuring electrodes with carbon nanotubes, discusses what is known of their electrochemical properties and briefly outlines some of the exciting applications to which they are being targeted for use.
Polarization dependence of the optical absorption properties of SWNTs is presented and investigated in detail for the energy range 0.5–6 eV. We found that the absorption peaks in the UV region at approximately 4.5 and 5.25 eV exhibit remarkable and different dependencies on the morphology of the SWNT film, or equivalently, on the incident light polarization relative to the SWNT axis. An analytical pathway to evaluate the physical degree of SWNT alignment for a vertically aligned SWNT film is developed with both transition dipoles parallel and perpendicular to the SWNT axis taken into account. This analytical procedure, coupled with polarized optical absorption measurements performed on the vertically aligned SWNT film grown on substrates, leads to the determination of the bare optical absorption cross-section of SWNTs for both parallel and perpendicular to SWNT axis. In the end, the proposed methodology for evaluating the SWNT film morphology is applied to investigate the transient change of the degree of alignment in the growth process of our vertically aligned SWNT films.
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.
To gain insights into the direct electron transfer (DET) mechanism of multi-walled carbon nanotubes (MWCNTs), we investigated the conformational changes that occur in proteins when they interact with MWCNTs. We used glucose oxidase (GOD) as an example. Using cyclic voltammetry measurements, the GOD that was immobilized on the MWCNT-modified carbon paper electrode exhibited apparent direct electrochemistry compared to that on the bare electrode without MWCNTs. The structural transformation of GOD upon adsorption on the MWCNTs was characterized spectrally. GOD was not denatured, and only small shifts of the wavenumber of the β-sheet structure were observed. There was a consistent tendency for the amount of α-helix to decrease and the β-sheet to increase. The α-helix content dropped from 21.2% to 19.6% as measured using Fourier transform infrared spectroscopy and from 27.1% to 25.9% as measured using circular dichroism. The reduction in the amount of α-helix led to a less shielded GOD active site and weakened the resistance of the electron transfer. These MWCNT-induced conformational changes could account for the DET between GOD and the MWCNT-modified electrode surface.
A novel biosensor, comprised of electrode of gold/multi-walled carbon nanotubes–glucose oxidase (Au/MWNTs–GOD), has been developed. The MWNTs were produced by microwave plasma enhanced chemical vapor deposition. The enzyme of GOD was immobilized using MWNTs. Performance and characteristics of the fabricated glucose biosensor were assessed with respect to response time, detection limit, pH value and storage stability. The results show that the fabricated biosensor is sensitive and stable in detecting glucose, indicating that MWNTs are a good candidate material for the immobilization of enzyme in glucose biosensor construction.
We report the measurement of the acidic sites in three different samples of commercially available full-length purified single-walled carbon nanotubes (SWNTs) – as obtained from CarboLex (CLI), Carbon Solutions (CSI) and Tubes@Rice (TAR) – by simple acid–base titration methods. Titration of the purified SWNTs with NaOH and NaHCO3 solutions was used to determine the total percentage of acidic sites and carboxylic acid groups, respectively. The total percentage of acidic sites in full length purified SWNTs from TAR, CLI and CSI are about 1–3%.
The preparation of a new type of finite carbon structure consisting of
needlelike tubes is reported. Produced using an arc-discharge
evaporation method similar to that used for fullerene sythesis, the
needles grow at the negative end of the electrode used for the arc
discharge. Electron microscopy reveals that each needle comprises
coaxial tubes of graphitic sheets ranging in number from two up to about
50. On each tube the carbon-atom hexagons are arranged in a helical
fashion about the needle axis. The helical pitch varies from needle to
needle and from tube to tube within a single needle. It appears that
this helical structure may aid the growth process. The formation of
these needles, ranging from a few to a few tens of nanometers in
diameter, suggests that engineering of carbon structures should be
possible on scales considerably greater than those relevant to the
fullerenes.
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.
A label-free bioelectronic detection of aptamer-thrombin interaction based on electrochemical impedance spectroscopy (EIS) technique is reported. Multiwalled carbon nanotubes (MWCNTs) were used as modifiers of screen-printed carbon electrotransducers (SPCEs), showing improved characteristics compared to the bare SPCEs. 5'amino linked aptamer sequence was immobilized onto the modified SPCEs and then the binding of thrombin to aptamer sequence was monitored by EIS transduction of the resistance to charge transfer (Rct) in the presence of 5 mM [Fe(CN)(6)](3-/4-), obtaining a detection limit of 105 pM. This study represents an alternative electrochemical biosensor for the detection of proteins with interest for future applications.
This work is aimed at studying the adsorption mechanism of short chain 20-mer pyrimidinic homo-ss-DNA (oligodeoxyribonucleotide, ODN: polyC(20) and polyT(20)) onto CNT by reflectometry. To analyze the experimental data, the effective-medium theory using the Bruggemann approximation represents a suitable optical model to account for the surface properties (roughness, thickness and optical constants) and the size of the adsorbate. Systematic information about the involved interactions is obtained by changing the physico-chemical properties of the system. Hydrophobic and electrostatic interactions are evaluated by comparing the adsorption on hydrophobic CNT and on hydrophilic silica and by modulating the ionic strength with and without Mg(2+). The ODN adsorption process on CNT is driven by hydrophobic interactions only when the electrostatic repulsion is suppressed. The adsorption mode results in ODN molecules in a side-on orientation with the bases (non-polar region) towards the surface. This unfavorable orientation is partially reverse by adding Mg(2+). On the other hand, the adsorption on silica is dominated by the strong repulsive electrostatic interaction that is screened at high ionic strength or mediated by Mg(2+). The cation-mediated process induces the interaction of the phosphate backbone (polar region) with the surface, leaving the bases free for hybridization. Although the general adsorption behavior of the pyrimidine bases is the same, polyC(20) presents higher affinity for the CNT surface due to its acid-base properties.
A novel electrochemical aptasensor for the detection of thrombin was developed on basis of the thrombin-binding aptamer (TBA) as a molecular recognition element and multi-walled carbon nanotubes (MWCNTs) as a carrier of the electrochemical capture probe. Amine-modified capture probe (12-mer) was covalently conjugated to the MWCNTs modified glassy carbon electrode (GCE). The target aptamer probe (21-mer) contains TBA (15-mer) labeled with ferrocene (Fc), which is designed to hybridize with capture probe and specifically recognize thrombin, is immobilized on the electrode surface by hybridization reaction. Introduction of the analyte thrombin triggered the dissociation of the aptamer probe labeled with Fc from the biosensors, led to a significant decrease in peak current intensity. Differential pulse voltammetry (DPV) was employed to detect the target analyte with different concentrations. The decreased peak current was in proportion to the concentration of thrombin in a range from 1.0x10(-12) to 5.0x10(-10)M with a detection limit of 5x10(-13)M. The present work demonstrates that using MWCNTs as a carrier for electrochemical capture probe is a promising way to amplify the electrochemical signal and to improve the sensitivity of the electrochemical aptasensor.
The electrochemical behavior of hydroquinone (HQ) was studied by cyclic voltammetry at a glassy carbon electrode (GCE) modified by a gel containing multi-walled carbon nanotubes (MWNTs) and room temperature ionic liquid (RTIL) of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF(6)). At the modified electrode, HQ showed a pair of quasi-reversible redox peaks. The cathodic peak current value (I(pc)) of HQ was 9.608 x 10(-4)A, which is 43 times larger than the one at the GCE, and 11 times larger than that of I(pc) at the MWNTs/GCE. Furthermore, the capabilities of electron transfer on these three electrodes were also investigated by electrochemical impedance spectroscopy (EIS), and the similar conclusion as cyclic voltammetry has drawn. Besides, we also characterized the surface morphology of the prepared composite film using the scanning electronic microscopy (SEM). The MWNTs were pulled away from the tangle in RTIL. The solvent effect of RTIL may be the reason of higher adsorption amount.
From diagnosis of life-threatening diseases to detection of biological agents in warfare or terrorist attacks, biosensors are becoming a critical part of modern life. Many recent biosensors have incorporated carbon nanotubes as sensing elements, while a growing body of work has begun to do the same with the emergent nanomaterial graphene, which is effectively an unrolled nanotube. With this widespread use of carbon nanomaterials in biosensors, it is timely to assess how this trend is contributing to the science and applications of biosensors. This Review explores these issues by presenting the latest advances in electrochemical, electrical, and optical biosensors that use carbon nanotubes and graphene, and critically compares the performance of the two carbon allotropes in this application. Ultimately, carbon nanomaterials, although still to meet key challenges in fabrication and handling, have a bright future as biosensors.
Carbon nanotubes (CNTs) have been incorporated in electrochemical sensors to decrease overpotential and improve sensitivity. In this review, we focus on recent literature that describes how CNT-based electrochemical sensors are being developed to detect neurotransmitters, proteins, small molecules such as glucose, and DNA. Different types of electrochemical methods are used in these sensors including direct electrochemical detection with amperometry or voltammetry, indirect detection of an oxidation product using enzyme sensors, and detection of conductivity changes using CNT-field effect transistors (FETs). Future challenges for the field include miniaturizing sensors, developing methods to use only a specific nanotube allotrope, and simplifying manufacturing.
This work reports on the performance of carbon nanotube modified screen-printed electrodes (SPE-MWCNT) for the selective determination of dopamine (DA) in the presence of ascorbic acid (AA) by adsorptive stripping voltammetry (AdSV). Several operating conditions and parameters were examined including the electrochemical pre-treatment and the previous AA interaction and DA accumulation in the presence AA at physiological conditions. Under the chosen conditions, DA peak current of differential pulse voltammograms increases linearly with DA concentration in the range of 5.0 x 10(-8) to 1.0 x 10(-6) mol L(-1) with a limit of detection of 1.5 x 10(-8) mol L(-1) in connection with 600s accumulation time. The sensitivity obtained for DA was independent from the presence or absence of AA; therefore, the proposed method can be readily applied to detect DA in real samples. The proposed methodology was successfully used for the quantification of DA in urine samples.
The relationship between the state of the surface of carbon nanotubes (CNTs) and their electrochemical activity was investigated using the enzyme cofactor dihydronicotinamide adenine dinucleotide (NADH) as a redox probe. The boiling of CNTs in water, while nondestructive, activated them toward the oxidation of NADH, as indicated by a shift in the anodic peak potential of NADH (E(NADH)) from 0.4 V to 0.0 V. The shift in E(NADH) was due to the redox mediation of NADH oxidation by traces of quinone species that were formed on the surface of treated CNTs. The harsher treatment that was comprised of microwaving CNTs in concentrated nitric acid had a similar effect on the E(NADH), and, additionally, it increased the anodic peak current of NADH. The latter correlated with the formation of defects on the surface of acid-microwaved CNTs, as indicated by their Raman spectra. The increase in current was discussed, considering the role of surface mediators on the buckled graphene sheets of acid-microwaved CNTs. The other carbon allotropes, including the edge-plane pyrolytic graphite, graphite powder, and glassy carbon, did not display a comparable activation toward the oxidation of NADH.
A nano-composite material consisting of amine functionalized multi-walled carbon nanotubes and a room temperature ionic-liquid (1-butyl-3-methylimidazolium tetrafluoroborate) was prepared and used to construct a novel catalase (Ct) based biosensor for the determination of hydrogen peroxide. The modified electrode exhibited a quasi-reversible cyclic voltammogram corresponding to the Fe(II)/Fe(III) redox couple in the heme prosthetic group of Ct with a formal potential of -460 mV in 0.1M phosphate buffer solution at pH 7.0. The nano-composite film showed an obvious promotion of the direct electron transfer between Ct and the underlying electrode. The apparent charge transfer rate constant and transfer coefficient for electron transfer between the electrode surface and enzyme were calculated as 2.23s(-1) and 0.45, respectively. The immobilized Ct exhibited a relatively high sensitivity (4.9 nA/nM) toward hydrogen peroxide. Under the optimized experimental conditions, hydrogen peroxide was detected in the concentration range from 8.6 to 140 nM with a detection limit of 3.7 nM at S/N=3. The modified electrode was stable for two weeks with no observable change in the cyclic voltammograms.
Recent years have provided numerous new examples of applying flow-through electrochemical detectors in chemical analysis. This review, based on about 250 original research papers cited from the current analytical literature, presents their application in flow analysis and capillary electrophoretic methods. Example applications are also given for arrays of electrochemical sensors in flow analysis and electrochemical detection in microfluidic systems. Potentiometric detection with ion-selective electrodes predominates in flow analysis carried out mostly in a flow-injection system, while amperometric and conductivity detections are most commonly employed in capillary electrophoresis.
The standard solution-depletion method is implemented with SDS-gel electrophoresis as a multiplexing, separation-and-quantification tool to measure competition between two proteins (i and j) for adsorption to the same hydrophobic adsorbent particles (either octyl sepharose or silanized glass) immersed in binary-protein solutions. Adsorption kinetics reveals an unanticipated slow protein-size-dependent competition that controls steady-state adsorption selectivity. Two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) are frequently observed depending on i, j solution concentrations. State 1 and State 2 are connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles with a downward inflection near 60 min of solution/adsorbent contact. Mass ratio of adsorbed i, j proteins (m(i)/m(j)) remains nearly constant between States 1 and 2, even though both m(i) and m(j) decrease in the transition between states. State 2 is shown to be stable for 24 h of continuous-adsorbent contact with stagnant solution whereas State 2 is eliminated by continuous mixing of adsorbent with solution. In sharp contrast to binary-competition results, adsorption to hydrophobic adsorbent particles from single-protein solutions (pure i or j) exhibits no detectable kinetics within the timeframe of experiment from either stagnant or continuously mixed solution, quickly achieving a single steady-state value in proportion to solution concentration. Comparison of binary competition between dissimilarly-sized protein pairs chosen to span a broad molecular-weight (MW) range demonstrates that selectivity between i and j scales with MW ratio that is proportional to protein-volume ratio (ubiquitin, Ub, MW=10.7 kDa; human serum albumin, HSA, MW=66.3 kDa; prothrombin, FII, 72 kDa; immunoglobulin G, IgG, MW=160 kDa; fibrinogen, Fib, MW=341 kDa). Results are interpreted in terms of a kinetic model of adsorption that has protein molecules rapidly diffusing into an inflating interphase that is spontaneously formed by bringing a protein solution into contact with a physical surface (State 1). State 2 follows by rearrangement of proteins within this interphase to achieve the maximum interphase concentration (dictated by energetics of interphase dehydration) within the thinnest (lowest volume) interphase possible by ejection of interphase water and initially-adsorbed proteins. Implications for understanding biocompatibility are discussed using a computational example relevant to the problem of blood-plasma coagulation.
Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of beta-D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide. Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications of glucose oxidase in biosensors have increased the demand in recent years. Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucose oxidase by fermentation is detailed, along with recombinant methods. Various purification techniques for higher recovery of glucose oxidase are described here. Issues of enzyme kinetics, stability studies and characterization are addressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.
The direct electrochemistry of catalase (CAT) at didodecyldimethylammonium bromide (DDAB) present on nafion dispersed multiwalled carbon nanotubes (MWCNTs-NF) modified glassy carbon electrode (GCE) has been reported. The presence of DDAB in MWCNTs-NF-CAT film enhances the surface coverage concentration of CAT (Fe(III/II)) to 48%. Similarly, in presence of DDAB, there is a 57% enhancement in electron transfer rate (ks) with 66% increase in CAT stability. (Fe(III/II)) redox couple exhibits linear dependence with the pH variation (-51 mV pH(-1)). The UV-vis absorption spectroscopy study reveals the entrapped CAT in DDAB film retains its native structure at MWCNTs-NF modified electrodes. Similarly, electrochemical impedance spectroscopy results confirm the co-existence of CAT and DDAB in the modified film. Further, scanning electron microscopy results reveal the structural morphological difference between various components in MWCNTs-NF-(DDAB/CAT) film. The cyclic voltammetry (CV) and amperometry (i-t curve) have been used for the measurement of electroanalytical properties of H2O2 by means of various film modified GCEs. The sensitivity values of MWCNTs-NF-(DDAB/CAT) film for H2O2 using CV (35.62 microA mM(-1)cm2) are higher than the values which are obtained for MWCNTs-NF-CAT film (2.74 mmicroA mM(-1)cm2). Similarly, the sensitivity values using i-t curve are 101.74 microA mM(-1)cm2 for MWCNTs-NF-(DDAB/CAT) and 74.69 microA mM(-1)cm2 for MWCNTs-NF-CAT film. Finally, the diffusion coefficient of H2O2 at MWCNTs-NF-(DDAB/CAT) film (3.4 x 10(-10) cm2 s(-1)) has been calculated using rotating disc electrode studies.
Carbon nanotubes (CNTs) possess preferential 'electrocatalytic' properties that affect the oxidation of enediol groups, establishing a relationship between electrocatalysis and chemical structure. Since this chemical structure occurs in analytes involved in high impact areas such as the pharmaceutical, cosmetic, and food safety industries, this preferential electrochemical behaviour was demonstrated using both standard and selected real-world samples. The oxygen-containing species present on the surface of CNTs and generated during acid treatment were responsible for an enhanced electron transfer reaction for these structures using a proton-assisted electron transfer mechanism, thus confirming their crucial role during the surface preparation process of electrocatalysis. The analytical benefits were that the inherent selectivity and sensitivity from these nanomaterials could be exploited for the direct detection of analytes in complex matrices, revealing their crucial role in the simplification of analytical processes.
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.