Development of bimetal-grown multi-scale carbon micro-nanofibers as an immobilizing matrix for enzymes in biosensor applications

This study describes the development of a novel bimetal (Fe and Cu)-grown hierarchical web of carbon micro-nanofiber-based electrode for biosensor applications, in particular to detect glucose in liquids. Carbon nanofibers (CNFs) are grown on activated carbon microfibers (ACFs) by chemical vapor deposition (CVD) using Cu and Fe as the metal catalysts. The transition metal-fiber composite is used as the working electrode of a biosensor applied to detect glucose in liquids. In such a bi-nanometal-grown multi-scale web of ACF/CNF, Cu nanoparticles adhere to the ACF-surface, whereas Fe nanoparticles used to catalyze the growth of nanofibers attach to the CNF tips. By ultrasonication, Fe nanoparticles are dislodged from the tips of the CNFs. Glucose oxidase (GOx) is subsequently immobilized on the tips by adsorption. The dispersion of Cu nanoparticles at the substrate surface results in increased conductivity, facilitating electron transfer from the glucose solution to the ACF surface during the enzymatic reaction with glucose. The prepared Cu-ACF/CNF/GOx electrode is characterized for various surface and physicochemical properties by different analytical techniques, including scanning electron microscopy (SEM), electron dispersive X-ray analysis (EDX), Fourier-transform infrared spectroscopy (FTIR), BET surface area analysis, and transmission electron microscopy (TEM). The electrochemical tests show that the prepared electrode has fast response current, electrochemical stability, and high electron transfer rate, corroborated by CV and calibration curves. The prepared transition metal-based carbon electrode in this study is cost-effective, simple to develop, and has a stable immobilization matrix for enzymes.     

A bioconjugated polyglycerol dendrimer with glucose sensing properties

In this work, the biological and electrochemical properties of glucose biosensor based on polyglycerol dendrimer (PGLD) is presented. Streptokinase (SK), glucose oxidase (GOx) and phosphorylcholine (PC) were immobilized onto PGLD to obtain a blood compatible bioconjugate with glucose sensing properties. The bioconjugated PGLD was entrapped in polyaniline nanotubes (PANINT's) through template electrochemical polymerization of aniline. PANINT's were used as electron mediator due to their high ability to promote electron-transfer reactions involving GOx. Platelet adhesion, fibrinolytic activity and protein adsorption were studied by in vitro experiments to examine the interaction of blood with PGLD biosensor. The PGLD biosensor exhibits a strong and stable amperometric response to glucose. The enzyme affinity for the substrate (K (M) (app) ) indicates that the enzyme activity was not significantly altered after the bioconjugation of GOx with PGLD dendrimer. The bioelectrochemical properties suggest that the bioconjugated PGLD developed in this work appears to be a good candidate for providing interfaces for implantable biosensors, especially oxidoreductase-based sensors.     

Layer-by-layer self-assembly of glucose oxidase and Os(Bpy)2CIPyCH2NH-poly(allylamine) bioelectrode

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 beta-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 microM 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.     

Removal of organic compounds by alginate gel beads with entrapped activated carbon

The adsorption of alginate gel (AG) beads and AG with activated carbon entrapped (AG–AC) beads prepared using different types of metal ions were investigated by measuring the removal of several organic compounds with different charges and size. AG–AC beads prepared in a CaCl₂ solution adsorbed strongly positively charged compounds as well as electrically neutral and low molecular weight compounds such as p-chlorophenol. However, a high molecular weight humic acid was not adsorbed by AG–AC. The AG–AC selectively adsorbed p-chlorophenol from a humic acid solution. The adsorption capacity obtained from the adsorption isotherm of AC entrapped in AG was compared with that of AC. The AG–AC beads prepared in a solution of FeCl₃ were able to specifically adsorb negatively charged gallic acid. Thus, entrapping AC into AG resulted in the selective adsorption.     

Preparation of amino-functionalized mesostructured cellular foams and application as hosts for large biomolecules

Large mesopores cellular foam (LMCFs) materials were synthesized using microemulsion templating in acidic solutions. The amine functional groups were attached to channels of LMCFs materials via post-synthesis grafting. The structural and chemical properties of these prepared materials were characterized by TEM, XRD, FTIR and nitrogen adsorption. These resulting materials had disordered mesopores with well-defined large mesopore. The bovine serum albumin (BSA) and glucose oxidase (GOx) were used for adsorption experiment. The biomolecule was immobilized by covalently couple to the interior surface of amino-functionalized mesostructured cellular foams (AF-MCFs). The results showed that AF-MCFs had high-capacity bioimmobilization ability.     

Enzyme-immobilized Langmuir-Blodgett film for a biosensor

A lipid-protein monolayer for a biosensor was prepared utilizing a Langmuir- Blodgett technique. The enzyme glucose oxidase was used as the protein. Three types of lipid were chosen to change the surface charge of the polar group. The enzyme was immobilized on the lipid monolayer by adsorption from the subphase solution onto the lipid monolayer on the air/water interface. It was found that the lipid-enzyme interaction was dominated by electrostatic forces, and the characteristics of the film can be controlled by expansion and recompression of the adsorbed monolayer. Finally, a glucose sensor was fabricated by depositing the film onto a hydrogen peroxide electrode.     

水热法制备炭球—活性炭复合材料

以商品活性炭和葡萄糖为原料, 采用水热合成方法, 在活性炭表面和孔内合成纳米炭球, 制得富含含氧官能团的炭球—活性炭复合材料. 通过低温液氮(N₂/77K)吸附测定了炭球—活性炭复合材料的比表面积和孔容、孔径分布. 以SEM观测材料表面形貌. 采用FTIR、XPS分析复合材料的表面官能团结构. 以水相中无机Cr(VI)的去除测试材料的吸附性能. 结果表明：葡萄糖水热处理后在活性炭表面生成炭球, 活性炭孔隙结构降低, 炭球尺寸和分布受葡萄糖溶液浓度影响较大, 活性炭表面生成以—OH为主的含氧官能团. 炭球—活性炭复合材料对Cr(VI)的单位质量和单位面积吸附容量最高分别为原料活性炭的近4倍和95倍.     

Glucose biosensor based on the microcantilever

Diagnosis and management of diabetes require quantitative and selective detection of blood glucose levels. We report a technique for micromechanical detection of biologically relevant glucose concentrations by immobilization of glucose oxidase (GOx) onto a microcantilever surface. Microfabricated cantilevers have traditionally found utility in atomic force microscope imaging. During the past decade, however, microcantilevers have been increasingly used as transducers in chemical-sensing systems. This paper describes the combination of this technology with enzyme specificity to construct a highly selective glucose biosensor. The enzyme-functionalized microcantilever undergoes bending due to a change in surface stress induced by the reaction between glucose in solution and the GOx immobilized on the cantilever surface. Experiments were carried out under flow conditions. The common interferences for glucose detection in other detection schemes have been tested and have shown to have no effect on the measurement of blood glucose level by this technique.     

Removal of hexavalent chromium from aqueous solution using activated carbon prepared from walnut shell biomass through alkali impregnation processes

Walnut (Juglans regia) is a commonly used nutrient industrial crop but the shell of the walnut has no economic value. Hence to revamp the waste walnut shell biomass to useful product, activated carbon (AC) was prepared from J. regia shells by impregnating with NaOH. Different ACs were prepared by varying the impregnation ratio of char:NaOH as 1:1 (AC1), 1:3 (AC2), and 1:5 (AC3). The effect of impregnation ratios on the adsorptive properties of ACs for the adsorption of hexavalent chromium [Cr(VI)] was studied. The ACs were characterized by SEM, surface functionality, and zero point charge. Langmuir, Freundlich, Temkin, and Dubinin–Radushkevitch isotherm were used to interpret the batch equilibrium data. The adsorption of Cr(VI) onto ACs followed Langmuir isotherm model. Kinetic data followed pseudo second-order rate equation. Intraparticle diffusion model and Boyd plot were used to study the mechanism of the adsorption reaction. The adsorption was both by film diffusion and intraparticle diffusion. The rate-controlling step was predicted as external mass transfer. Thermodynamic parameters were also estimated. Overall, AC with higher impregnation ratio (AC3) possessed better adsorption properties compared to AC2 and AC1.     

Effect of surface acidic oxides of activated carbon on adsorption of ammonia

The influence of surface acidity of activated carbon (AC) was experimentally studied on adsorption of ammonia (NH(3)). Coconut shell-based AC was modified by various acids at different concentrations. There were five different acids employed to modified AC, which included nitric acid, sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid. Acidic functional groups on the surface of ACs were determined by a Fourier transform infrared spectrograph (FTIR) and by the Boehm titration method. Specific surface area and pore volume of the ACs were measured by a nitrogen adsorption apparatus. Adsorption amounts of NH(3) onto the ACs were measured by a dynamic adsorption system at room temperature according to the principle of the ASTM standard test method. The concentration of NH(3) in the effluent stream was monitored by a gas-detecting tube technique. Experimental results showed that adsorption amounts of NH(3) on the modified ACs were all enhanced. The ammonia adsorption amounts on various activated carbons modified by different acids are in the following order: nitric acid>sulfuric acid>acetic acid approximately phosphoric acid>hydrochloric acid. It is worth to note that the breakthrough capacity of NH(3) is linearly proportional to the amount of acidic functional groups of the ACs.     

Activated carbon with excellent chromium(VI) adsorption performance prepared by acid-base surface modification

In the present work, activated carbon (AC) with excellent Cr(VI) adsorption performance especially at low concentrations was prepared by an acid-base surface modification method. Raw activated carbon (AC(0)) was first oxidized in boiling HNO(3) (AC(1)), then treated with a mixture of NaOH and NaCl (AC(2)). Batch equilibrium and continuous column adsorption were conducted to evaluate the adsorption performance. Boehm titration, elemental analysis, and N(2)/77K adsorption isotherm methods were used to characterize the surface properties and pore structure of modified ACs. The results revealed that the modified AC exhibited excellent Cr(VI) adsorption performance in terms of adsorption capacity and adsorption rate: AC(2)>AC(1)>AC(0). Modification caused S(BET) to decrease and the total number of surface oxygen acidic groups to increase. HNO(3) oxidization produced positive acid groups, and subsequently NaOH treatment replaced H(+) of surface acid groups by Na(+), and the acidity of AC decreased. The main cause of higher Cr(VI) adsorption capacity and rate for AC(2) was the presence of more oxygen surface acidic groups and suitable surface acidity. HNO(3)-NaOH modification shows potential for the preparation of high quality AC for the effective removal of low concentrations of Cr(VI).     

Synthesis of ordered large-pore mesoporous carbon for Cr(VI) adsorption

Highly ordered mesoporous carbon with large accessible pores (OMC-P) was prepared by using laboratory-made poly(ethylene oxide)-b-polystyrene diblock copolymer as template via the evaporation-induced self-assembly method. The OMC-P was first used as adsorbent for removal of Cr(VI) ion from aqueous solution. Adsorption behavior was studied as a function of time, concentration of adsorbate, temperature, and pH. The kinetics of adsorption of Cr(VI) ion onto OMC-P is well fit to the pseudo-second order model. The Cr(VI) ion adsorption is favored at lower temperatures and at initial acid pH values in the equilibrium. The Freundlich and the Langmuir isotherm fit the equilibrium data satisfactorily. The influence of porosity on equilibrium adsorption capacity was investigated on three types of carbon materials, namely, OMC-P, ordered mesoporous carbon templated from amphiphilic triblock copolymer F127 (OMC-F) and commercial activated carbon (AC). The prepared OMC-P exhibits much higher adsorption performance than the other two carbons.     

A novel catalyst for synthesis of styrene: carbon nanofibers immobilized on activated carbon

Carbon NanoFibers (CNFs) with hierarchically structure have been immobilized onto Activated Carbon (AC) by impregnation with an aqueous solution of Fe(CH3COO)2, reduction and subsequent chemical vapor decomposition of ethylene. The morphology of the CNFs can be modulated by adjusting the pH of the Fe(CH3COO)2 solution used for impregnating the AC. A stable yield of 35% in the oxidative dehydrogenation of ethylbenzene to styrene was obtained at a temperature of 673 K, around 200 K lower than the current industrial process. The immobilized CNFs on AC catalysts combine the catalytic properties of the carbon nanofibers and the suprastructure of the AC host. The final material is an easy to handle active catalyst, with an open structure of immobilized CNFs avoiding the pressure drop problem, which is typically observed for fine powder forms of CNFs. The immobilized CNFs on AC are attractive for gas-phase fixed-bed industrial applications.     

Perchlorate adsorption and desorption on activated carbon and anion exchange resin

The mechanisms of perchlorate adsorption on activated carbon (AC) and anion exchange resin (SR-7 resin) were investigated using Raman, FTIR, and zeta potential analyses. Batch adsorption and desorption results demonstrated that the adsorption of perchlorate by AC and SR-7 resin was reversible. The reversibility of perchlorate adsorption by the resin was also proved by column regeneration test. Solution pH significantly affected perchlorate adsorption and the zeta potential of AC, while it did not influence perchlorate adsorption and the zeta potential of resin. Zeta potential measurements showed that perchlorate was adsorbed on the negatively charged AC surface. Raman spectra indicated the adsorption resulted in an obvious position shift of the perchlorate peak, suggesting that perchlorate was associated with functional groups on AC at neutral pH through interactions stronger than electrostatic interaction. The adsorbed perchlorate on the resin exhibited a Raman peak at similar position as the aqueous perchlorate, indicating that perchlorate was adsorbed on the resin through electrostatic attraction between the anion and positively charged surface sites.     

Improvement of homogeneity of analytical biodevices by gene manipulation

Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with K(m) and K(cat) similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that the homogeneity of the analytical biodevices could be considerably improved by using the proposed method.     

DNA as a support for glucose oxidase immobilization at Prussian blue-modified glassy carbon electrode in biosensor preparation

An amperometric glucose biosensor has been developed using DNA as a matrix of Glucose oxidase (GOx) at Prussian-blue (PB)-modified glassy carbon (GC) electrode. GC electrode was chemically modified by the PB. GOx was immobilized together with DNA at the working area of the PB-modified electrode by placing a drop of the mixture of DNA and GOx. The response of the biosensor for glucose was evaluated amperometrically. Upon immobilization of glucose oxidase with DNA, the biosensor showed rapid response toward the glucose. On the other hand, no significant response was obtained in the absence of DNA. Experimental conditions influencing the biosensor performance were optimized and assessed. This biosensor offered an excellent electrochemical response for glucose concentration in micro mol level with high sensitivity and selectivity and short response time. The levels of the relative standard deviation (RSDs), (<4%) for the entire analyses reflected a highly reproducible sensor performance. Through the use of optimized conditions, a linear relationship between current and glucose concentration was obtained up to 4 x 10(-4) M. In addition, this biosensor showed high reproducibility and stability.     

Enhanced colorimetric detection on porous microarrays using in situ substrate production

A new technique is reported for the enhanced colorimetric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the microarray porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H(2)O(2) kit used for peroxidase label detection. The obtained target limit of detection is then 50 times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary/noncomplementary signal ratio.     

Glucose oxidase-modified carbon-felt-reactor coupled with peroxidase-modified carbon-felt-detector for amperometric flow determination of glucose

Glucose oxidase (GOx) and horseradish peroxidase (HRP) were covalently immobilized on a porous carbon-felt (CF) by using cyanuric chloride (CC) as a linking reagent. The resulting GOx-modified-CF (GOx-ccCF) was used as column-type enzyme reactor and placed on upstream of the HRP-ccCF-based H₂O₂ flow-detector to fabricate amperometric flow-biosensor for glucose. Sensor setting conditions and the operational conditions were optimized, and the analytical performance characteristics of the resulting flow-biosensor were evaluated. The chemical modification of the GOx via CC was found to be effective to obtain larger catalytic activity as compared with the physical adsorption. Under the optimized conditions (i.e., volume ratio of the GOx-ccCF-reactor to the HRP-ccCF-detector is 1.0; applied potential is − 0.12 V vs. Ag/AgCl; carrier pH is 6.5; and carrier flow rate is 4.3 ml/min), highly selective and quite reproducible peak current responses toward glucose were obtained: the RSD for 30 consecutive injections of 3 mM glucose was 1.04%, and no serious interferences were observed for fructose, ethanol, uric acid, urea and tartaric acid for the amperometric measurements of glucose. The magnitude of the cathodic peak currents for glucose was linear up to 5 mM (sensitivity, 6.38 ± 0.32 μA/μM) with the limit detection of 9.4 μM (S/N = 3, noise level, 20 nA). The present GOx-ccCF-reactor and HRP-ccCF-detector-coupled flow-glucose biosensor was utilized for the determination of glucose in beverages and liquors, and the analytical results by the sensor were in fairly good agreement with those by the conventional spectrophotometry.     

Hierarchical carbon nanostructure design: ultra-long carbon nanofibers decorated with carbon nanotubes

Hierarchical carbon nanostructures based on ultra-long carbon nanofibers (CNF) decorated with carbon nanotubes (CNT) have been prepared using plasma processes. The nickel/carbon composite nanofibers, used as a support for the growth of CNT, were deposited on nanopatterned silicon substrate by a hybrid plasma process, combining magnetron sputtering and plasma-enhanced chemical vapor deposition (PECVD). Transmission electron microscopy revealed the presence of spherical nanoparticles randomly dispersed within the carbon nanofibers. The nickel nanoparticles have been used as a catalyst to initiate the growth of CNT by PECVD at 600°C. After the growth of CNT onto the ultra-long CNF, SEM imaging revealed the formation of hierarchical carbon nanostructures which consist of CNF sheathed with CNTs. Furthermore, we demonstrate that reducing the growth temperature of CNT to less than 500°C leads to the formation of carbon nanowalls on the CNF instead of CNT. This simple fabrication method allows an easy preparation of hierarchical carbon nanostructures over a large surface area, as well as a simple manipulation of such material in order to integrate it into nanodevices.     

Modification of indium tin oxide electrodes with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis

Indium tin oxide electrodes were modified with DNA, and the guanines in the immobilized nucleic acid were used as a substrate for electrocatalytic oxidation by Ru(bpy)3(3+) (bpy = 2,2'-bipyridine). Nucleic acids were deposited onto 12.6-mm2 electrodes from 9:1 DMF/water mixtures buffered with sodium acetate. The DNA appeared to denature in the presence of DMF, leading to adsorption of single-stranded DNA. The nucleic acid was not removed by vigorous washing or heating the electrodes in water, although incubation in phosphate buffer overnight liberated the adsorbed biomolecule. Acquisition of cyclic voltammograms or chronoamperomograms of Ru(bpy)3(2+) at the modified electrodes produced catalytic signals indicative of oxidation of the immobilized guanine by Ru(III). The electrocatalytic current was a linear function of the extent of modification with a slope of 0.5 microA/pmol of adsorbed guanine; integration of the current-time traces gave 2.2+/-0.4 electrons/guanine molecule. Use of long DNA strands therefore gave steep responses in terms of the quantity of adsorbed DNA strand. For example, electrodes modified with a 1497-bp PCR product from the HER-2 gene produced detectable catalytic currents when as little as 550 amol of strand was adsorbed, giving a sensitivity of 44 amol/mm2.