Increasing amperometric biosensor sensitivity by length fractionated single-walled carbon nanotubes

In this work the sensitivity-increasing effect of single-walled carbon nanotubes (SWCNTs) in amperometric biosensors, depending on their average length distribution, was studied. For this purpose the SWCNTs were oxidatively shortened and subsequently length separated by size exclusion chromatography. Transmission electron micrographs of different fractions of SWCNTs were collected. Diaphorase "wired" to an osmium redox polymer was blended with the shortened SWCNTs of different lengths. Depending on the average length of the SWCNTs the sensitivity of the amperometric biosensor model system towards oxidation of 1,4-dihydronicotinamide adenine dinucleotide (NADH) was increased by a factor of five. The best performance was achieved with SWCNTs of medium length. The linear range for NADH detection was between 5muM and 7mM, the maximum sensitivity was 47nAmuM(-1)cm(-2), and the detection limit was 1muM. The biosensor exhibited excellent electrocatalytic properties. Even at relatively high NADH concentrations the oxidative current was limited by the diffusion rate of NADH.     

Use and improvement of microbial redox enzymes for environmental purposes

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed.     

Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.     

Electrochemical growth of gold nanoparticles on horizontally aligned carbon nanotubes: a new platform for ultrasensitive DNA sensing

A new platform based on electrochemical growth of Au nanoparticles on horizontally aligned single walled carbon nanotube (SWCNT) array was developed for ultrasensitive DNA detection. The as-prepared DNA-functionalized SWCNT-Au platform, in which every gold-coated SWCNT acts as an isolated micro electrode, could detect lower than 10 zmol complimentary 10-base DNA, which corresponded to having 6 DNA molecules in a 1 mL sample solution. For a 1-base mismatched DNA, the experimental detection limit was 100 amol. A linear relationship between the change of charge transfer resistance and target DNA concentration was achieved at low concentration range. Over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship with respect to the logarithm of the target DNA concentration. The sensor also showed great stability and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining Au nanoparticles with the on-site fabricated SWCNT array represents a promising platform for achieving ultrasensitive biosensor.     

Reactions of copper macrocycles with antioxidants and HOCl: potential for biological redox sensing

A series of simple copper N₂S₂ macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.     

Glucose/O2 biofuel cell based on enzymes, redox mediators, and multiple-walled carbon nanotubes deposited by AC-electrophoresis then stabilized by electropolymerized polypyrrole

In this study, we developed an automated strategy to manufacture an enzyme BFC powered by glucose/O(2). The bioanode consists of GOx enzyme and PQQ redox mediator adsorbed over night on MWCNTs then deposited by means of AC-electrophoresis at 30 Hz and 160 V(p-p) and, finally stabilized by electropolymerized polypyrrole. The biocathode is constructed from LAc enzyme and ABTS redox mediator adsorbed over night on MWCNTs, then electrophoretically deposited under AC-electric field at 30 Hz and 160 V(p-p) and, finally stabilized by electrodeposited polypyrrole. The BFC was studied under air in phosphate buffer solution pH 7.4 containing 10 mM glucose and in human serum with 5 mM glucose addition at the physiological temperature of 37°C. Under these conditions, the maximum power density reaches 1.1 μW · mm(-2) at a cell voltage of 0.167 V in buffer solution and 0.69 μW · mm(-2) at cell voltage of 0.151 V in human serum. Such automated BFCs have a great potential to be optimized, miniaturized to micro and nanoscale devices suitable for in vivo studies.     

Use of enzymes for improvement of feed

Investigations of the addition of enzymes to traditional feed-mixtures, for improvement of feed utilization and of growth of domestic animals, have been performed. A survey of the literature on the effects of enzyme addition is shown in tabular form.Experiments with rats fed on barley diets, with and without enzyme addition, showed no significant difference between the diets, but a tendency towards feed improvements by the addition of some of the enzymes, e.g., amylase, cellulase and protease. Some of the experiments with chickens fed on a barley-based diet, with the addition of either cellulase and/or pectinase or protease, showed increases in growth of up to 7% (P < 0.05), and improvements in feed utilization of 6% (P < 0.01). In one experiment on chickens fed on a barley of better quality than that in the above-mentioned experiments, no improvements were found by adding an enzyme mixture, consisting of cellulase, pectinase and protease, to the feed.The results thus suggest that a better feed-utilization by use of enzyme addition is obtainable only if the feed mixture is composed of less-digestible feed ingredients. The amount of added enzymes strongly influences the price of the resultant feed, and in the experiments showing feed improvement, the break-even price of the enzymes is less than 26 Dkr/kg enzyme. The use of enzymes for making slowly digested and less-expensive products (waste products) applicable as feed components will be examined in future work     

Electrochemical biosensors for biocontaminant detection consisting of carbon nanotubes,platinum nanoparticles,dendrimers, and enzymes

The presented approach provides the advanced development of effective, rapid, and versatile electrochemical sensors for a small amount of analytes on potential, cheap, and disposable printed chips. The electrocatalytic activity of this biosensor revealed the feasible detection of hydrogen peroxide at low potential (∼0.09 V) and the detection of a biocontaminant inhibitor (organophosphorus pesticide) in a wide range of concentrations. This efficiency comes from the chemical immobilization of catalysts (Pt nanoparticles) and electron transfer-enlarging materials (carbon nanotubes) on an electrode. Especially, dendrimers raise the stable conjugation of enzymes (acetylcholinesterase/choline oxidase/peroxidase) as well as nanoparticles and carbon nanotubes on an electrode.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study

In this work, novel conductive composite nanofiber mesh possessing reactive groups was electrospun from solutions containing poly(acrylonitrile-co-acrylic acid) (PANCAA) and multi-walled carbon nanotubes (MWCNTs) for redoxase immobilization, assuming that the incorporated MWCNTs could behave as electrons transferor during enzyme catalysis. The covalent immobilization of catalase from bovine liver on the neat PANCAA nanofiber mesh or the composite one was processed in the presence of EDC/NHS. Results indicated that both the amount and activity retention of bound catalase on the composite nanofiber mesh were higher than those on the neat PANCAA nanofiber mesh, and the activity increased up to 42%. Kinetic parameters, K(m) and V(max), for the catalases immobilized on the composite nanofiber mesh were lower and higher than those on the neat one, respectively. This enhanced activity might be ascribed to either promoted electron transfer through charge-transfer complexes and the pi system of carbon nanotubes or rendered biocompatibility by modified MWCNTs. Furthermore, the immobilized catalases revealed much more stability after MWCNTs were incorporated into the polymer nanofiber mesh. However, there was no significant difference in optimum pH value and temperature, thermal stability and operational stability between these two immobilized preparations, while the two ones appeared more advantageous than the free in these properties. The effect of MWCNTs incorporation on another redox enzyme, peroxidase, was also studied and it was found that the activity increased by 68% in comparison of composite one with neat preparation.     

An amperometric biosensor based on a composite of single-walled carbon nanotubes, plasma-polymerized thin film, and an enzyme

We report on an amperometric biosensor that is based on a nanocomposite of carbon nanotubes (CNT), a nano-thin plasma-polymerized film (PPF), and glucose oxidase (GOx) as an enzyme model. A mixture of the GOx and a CNT film is sandwiched with 10-nm-thick acetonitrile PPFs. Under PPF layer was deposited onto a sputtered gold electrode. To facilitate the electrochemical communication between the CNT layer and GOx, CNT was treated with nitrogen or oxygen plasma. The resulting device showed that the oxidizing current response due to enzymatic reaction was 4-16-fold larger than that with only CNT or PPF, showing that the PPF and/or plasma process is an enzyme-friendly platform for designing electrochemical communication from the reaction center of GOx to the electrode via CNTs. The optimized glucose biosensor showed high sensitivity (sensitivity of 42 microA mM(-1)cm(-2), correlation coefficient of 0.992, linear response range of 0.025-2.2 mM, and a detection limit of 6 microM at signal/noise ratio of 3, +0.8 V versus Ag/AgCl), high selectivity (almost no interference by 0.5 mM ascorbic acid) for glucose quantification, and rapid response (<4 s to reach 95% of maximum response). Additionally, the devices showed a small and stable background current (0.35+/-0.013 microA) compared with the glucose response (ca. 10 microA at 10mM glucose) and suitable reproducibility from sample-to-sample (<3%, n=4).     

Applications of carbon nanotubes for cancer research

Carbon nanotubes have many unique properties such as high surface area, hollow cavities, and excellent mechanical and electrical properties. Interfacing carbon nanotubes with biological systems could lead to significant applications in various disease diagnoses. Significant progress in interfacing carbon nanotubes with biological materials has been made in key areas such as aqueous solubility, chemical and biological functionalization for biocompatibility and specificity, and electronic sensing of proteins. In addition, the bioconjugated nanotubes combined with the sensitive nanotube-based electronic devices would enable sensitive biosensors toward medical diagnostics. Furthermore, recent findings of improved cell membrane permeability for carbon nanotubes would also expand medical applications to therapeutics using carbon nanotubes as carriers in gene delivery systems. This article reviews the current trends in biological functionalization of carbon nanotubes and their potential applications for breast cancer diagnostics. The article also reports the applications of confocal microscopy for use in understanding the interactions of biological materials such as antibodies on carbon nanotubes that are specific to surface receptors in breast cancer cells. Furthermore, a nanotube-field-effect transistor is demonstrated for electronic sensing of antibodies that are specific to surface receptors in cancer cells.     

Computational sensitivity investigation of hydrogel injection characteristics for myocardial support

Biomaterial injection is a potential new therapy for augmenting ventricular mechanics after myocardial infarction (MI). Recent in vivo studies have demonstrated that hydrogel injections can mitigate the adverse remodeling due to MI. More importantly, the material properties of these injections influence the efficacy of the therapy. The goal of the current study is to explore the interrelated effects of injection stiffness and injection volume on diastolic ventricular wall stress and thickness. To achieve this, finite element models were constructed with different hydrogel injection volumes (150 µL and 300 µL), where the modulus was assessed over a range of 0.1 kPa to 100 kPa (based on experimental measurements). The results indicate that a larger injection volume and higher stiffness reduce diastolic myofiber stress the most, by maintaining the wall thickness during loading. Interestingly, the efficacy begins to taper after the hydrogel injection stiffness reaches a value of 50 kPa. This computational approach could be used in the future to evaluate the optimal properties of the hydrogel.     

Disposable electrodes modified with multi-wall carbon nanotubes for biosensor applications

This paper deals with the development of a disposable electrochemical sensor for the detection of hydrogen peroxide, using screen-printed carbon-based electrodes (SPCEs) modified with multi-wall carbon nanotubes (MWCNs) dispersed in a polyethylenimine (PEI) mixture. The modified sensors showed an excellent electrocatalytic activity towards the analyte, respect to the high overvoltage characterising unmodified screen-printed sensors. The composition of the PEI/MWCNT dispersion was optimised in order to improve the sensitivity and reproducibility. The optimised sensor showed good reproducibility (10% RSD calculated on three experiments repeated on the same electrode), whereas a reproducibility of 15% as RSD was calculated on electrodes from different preparations. Preliminary experiments carried out using glucose oxidase (GOD) as biorecognition element gave rise to promising results indicating that these new devices may represent interesting components for biosensor construction.     

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV-Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H(2)O(2) in the linear range from 5×10(-6) to 1×10(-3) mol L(-1) with a detection limit of 2.1×10(-6) mol L(-1) (based on the S/N=3).     

Probing astroglia with carbon nanotubes: modulation of form and function

Carbon nanotubes (CNTs) have shown much promise in neurobiology and biomedicine. Their structural stability and ease of chemical modification make them compatible for biological applications. In this review, we discuss the effects that chemically functionalized CNTs, applied as colloidal solutes or used as strata, have on the morpho-functional properties of astrocytes, the most abundant cells present in the brain, with an insight into the potential use of CNTs in neural prostheses.     

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k_cat and K_m, selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H₂O₂ production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.     

Modification of proportion sensitivity testing method for ethionamide

Standardized methodology for drug susceptibility testing of second line drugs is vital for treatment of multi/extensively drug resistant tuberculosis. Discrepancy between laboratory methods and clinical interpretation is well established for bacteriostatic drugs such as ethionamide. Optimization of the standard proportion sensitivity testing (PST) method for ethionamide was under taken in 235 Mycobacterium tuberculosis isolates from new and previously treated pulmonary tuberculosis patients. An additional higher concentration of 57 μg/ml was evaluated against at the standard 40 μg/ml concentration in PST method. Performance parameters and agreement between the two drug concentrations was higher indicating the efficiency of PST method at its present format at 40 μg/ml and additional higher concentration of 57 μg/ml as an alternative when required.     

Activity of key redox enzymes in rabbits with different typologic features

It has been found that the activity of LDG, PDG, IDG, SDG, NADN-DG, NADFN-DG, CO, G-6-FDG and 6-FGDG in the cerebral cortex, liver and kidneys of rabbits with a strong type of nervous activity, as a rule, is higher than in the animals with a weak type of the nervous system. Between the animals with different types of the nervous system distinctions are also revealed in the character of regulation of a number of metabolic cycles. It is suggested that typological features of the nervous system are determined by the character of metabolic processes.