Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor

The formation of covalently linked composites of multi–walled carbon nanotubes (MWCNT) and glucose oxidase (GOD) with high-function density for use as a biosensing interface is described. The reaction intermediates and the final product were characterized by using FT–IR spectroscopy, and the MWCNT-coated GOD nanocomposites were examined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Interestingly, it was found that the GOD–MWCNT composites are highly water soluble. Electrochemical characterization of the GOD–MWCNT composites that were modified on a glassy carbon electrode shows that the covalently linked GOD retains its bioactivity and can specifically catalyze the oxidation of glucose. The oxidation current shows a linear dependence on the glucose concentration in the solution in the range of 0.5–40 mM with a detection limit of 30 μM and a detection sensitivity of 11.3 μA/mMcm². The present method may provide a way to synthesize MWCNT related composites with other biomolecules and for the construction of enzymatic reaction-based biofuel cells and biosensors. Supported by grants from the National Natural Science Foundation of China (NSFC, No. 20125515; 90206037; 20375016) and the Natural Science Foundation of Jiangsu Province (Grant No. BK 2004210)     

铕敏化荧光法测定人体血清和尿液中的芦氟沙星

:研究了测定人体血清和尿样中芦氟沙星 ( RFX)的铕敏化荧光法。在HAc- Na Ac缓冲溶液中 ,RFX与 Eu3 +、EDTA形成三元配合物 ,产生了 Eu2 +的特征荧光 ,据此建立了直接、快速测定人体血清和尿液中的芦氟沙星含量的方法。测定在血清和尿液中 RFX浓度的线性范围分别为 2 .5× 1 0 -8～ 1 .0× 1 0 -6mol· L-1和 1 .0× 1 0 -7～ 5.0× 1 0 -6mol· L-1,检出限为 6.6× 1 0 -9mol· L-1和 1 .5× 1 0 -8mol· L-1。     

Spectrofluorimetric determination of levofloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine levofloxacin is proposed and applied to determine the substance in tablets and spiked human urine and serum. The fluorimetric method allow the determination of 20–3000 ng ml⁻¹ of levofloxacin in aqueous solution containing acetic acid–sodium acetate buffer (pH 4) with λ_exc=292 and λ_em=494 nm, respectively. Micelle enhanced fluorescence improves the sensibility and allows levofloxacin direct measurement in spiked Human serum (5 μg ml⁻¹) and urine (420 μg ml⁻¹), in 8 mM sodium dodecyl sulphate solutions at pH 5.     

Fluorescence and terbium-sensitised luminescence determination of garenoxacin in human urine and serum

Fluorescence and terbium-sensitised luminescence properties of new quinolone garenoxacin have been studied. The fluorimetric method allows the determination of 0.060-0.600 μg ml⁻¹ of garenoxacin in aqueous solution containing HCl/KCl buffer (pH 1.5) with λ_exc=282 nm and λ_em=421 nm. Micellar-enhanced fluorescence was also studied, leading to a higher than 400% increase in analytical signal in presence of 12 mM sodium dodecyl sulphate (SDS), allowing the determination of 0.020-0.750 μg ml⁻¹ of garenoxacin. The terbium-sensitised luminescence method allows the determination of 0.100-1.500 μg ml⁻¹ of garenoxacin in 12 mM SDS solution containing 0.08 M acetic acid/sodium acetate buffer (pH 4.1) and 7.5 mM Na₂SO₃ (chemical deoxygenation agent), with λ_exc=281 nm and λ_em=546 nm. Relative standard deviation (R.S.D.) values for the three methods were in the range 1.0-2.0%. The proposed procedures have been applied to the determination of garenoxacin in spiked human urine and serum.     

Construction of glucose biosensor based on sorption of glucose oxidase onto multilayers of polyelectrolyte/nanoparticles

A new approach to constructing an enzyme-containing film on the surface of a gold electrode for use as a biosensor is described. A basic multilayer film (BMF) of (PDDA/GNPs) _n /PDDA was first constructed on the gold electrode by electrostatic layer-by-layer self-assembly of poly(diallyldimethylammonium chloride) (PDDA) and gold nanoparticles (GNPs). Glucose oxidase (GOx) was then sorbed into this BMF by dipping the BMF-modified electrode into a GOx solution. The assembly of the BMF was monitored and tested via UV-vis spectroscopy and cyclic voltammetry (CV). The ferrocenemethanol-mediated cyclic voltammograms obtained from the gold electrode modified with the (PDDA/GNPs) _n /PDDA/GOx indicated that the assembled GOx remained electrocatalytically active for the oxidation of glucose. Analysis of the voltammetric signals showed that the surface coverage of active enzyme was a linear function of the number of PDDA/GNPs bilayers. This result confirmed the penetration of GOx into the BMF and suggests that the BMF-based enzyme film forms in a uniform manner. Electrochemical impedance measurements revealed that the biosensor had a lower electron transfer resistance (R _et) than that of a sensor prepared by layer-by-layer assembly of PDDA and GOx, due to the presence of gold nanoparticles. The sensitivity of the biosensor for the determination of glucose, which could be controlled by adjusting the number of PDDA/GNPs bilayers, was investigated.     

Enzyme co-immobilization for the sequential determination of lactic acid and glucose in serum

An enzymatic method for the sequential determination of lactic acid and glucose is proposed. Sample matrix effects are overcome by using an internally coupled valve system. The problem arising from the dissimilar concentrations of the two analytes commonly occurring in serum is solved by applying the scale-expansion technique with a diode-array spectrophotometer. The determination ranges are 10–400 and 2–100 μg ml^?1 for lactic acid and glucose, respectively (r.s.d. 1.63 and 2.30%; n=11). Mixtures of these compounds in ratios up to 1:10 can be readily resolved, which allows their determination in serum with good results.     

Optimization of a glucose biosensor setup based on a Ni/Al HT matrix

An amperometric glucose biosensor was developed using an anionic clay matrix of hydrotalcitic nature (Ni/Al-NO₃ HT) as enzyme support, which was electrochemically synthesized at −0.90 V versus SCE, using a rotating disk Pt electrode to assure homogeneity of the electrodeposition suspension. The biorecognition element was glucose oxidase (GOx) immobilized on HT during the electrosynthesis, which was followed by cross-linking with glutaraldehyde vapours to avoid the enzyme release.The performances of the biosensor, in terms of sensitivity to glucose calculated from the slope of the calibration curve, are dependent on parameters related to the electrodeposition.An experimental design was applied to detect the optimal conditions of electrosynthesis in order to optimize the glucose biosensor performance. The factors taken into account were enzyme concentration and Ni/Al molar ratio. A full factorial design was performed to study linear interactions between factors and their quadratic effects and the optimal setup was evaluated by the isoresponse curves. The significant factors were enzyme concentration (linear and quadratic terms) and the interaction between enzyme concentration and Ni/Al molar ratio. Under the optimized electrodeposition conditions, the reproducibility of the biosensor fabrication was very good, being the RSD of the sensitivity about 5%.     

A fully integrated graphene-polymer field-effect transistor biosensing device for on-site detection of glucose in human urine

Convenient and rapid self-measurement of the glucose level in the body is of great significance for diabetics to know their health conditions in time. In view of this, a polymer functionalized graphene field-effect transistor (P-GFET) portable biosensing device is demonstrated for glucose monitoring. The polymer is synthesized by acrylamide/3-acrylamidophenylboronic acid (AAPBA)/N, N-dimethylaminopropyl acrylamide. In the presence of glucose, the P-GFET shows Dirac point shifts and current changes as a result of the covalent bond between glucose and AAPBA in the synthesized polymer on graphene. The sensitivity of this P-GFET sensor can increase while the density of AAPBA in polymer increases. The used sensor could regain the detection capability after hydrochloric acid treatment due to the reversible reaction between polymer and glucose. In addition, the chemisorption interaction between polymer and glucose, which is stronger than physisorption interaction with other objects in urine, has been supported by the density functional theory study. The P-GFET shows high sensitivity of 822 μA1cm^?21mM^?1 with a limit of detection of 1.9 μM during human urine glucose monitoring. The sensor holds a detection range of 0.04–10 mM and good reusability over 20 times. With the customized portable real-time measurement capability in urine, our P-GFET sensor can offer advantages over current glucose detection methods.     

An optical glucose biosensor based on entrapped-glucose oxidase in silicate xerogel hybridised with hydroxyethyl carboxymethyl cellulose

An optical glucose biosensor was fabricated by entrapping glucose oxidase (GOx) within the xerogel that was derived from tetraethylorthosilicate and hybridised with hydroxyethyl carboxymethyl cellulose polymer. The entrapped-GOx was mainly characterised with its long-lasting apparent biocatalytic activity as compared to that being entrapped in only sol-gel matrix. The biocatalytic activity of the entrapped-enzyme has extended its shelf lifetime up to 3 years. This long-term stability was closely correlated with the reduction in the shrinkage process of the hybrid gel being used. In conjunction with an optical oxygen transducer, the entrapped-GOx was assembled as an optical glucose biosensor comprised a sample flow system with which the dissolved oxygen in the sample could be precisely controlled and varied. The analytical working range was tuneable within 9.0 μM-100 mM range depending on the dissolved oxygen concentration in the test solution. The time taken to reach a 95% steady signal was 6-9 min at flow rate of 1.0 mL min⁻¹. The glucose biosensor has been satisfactorily applied to the determination of glucose contents of urine samples.     

Characterization of different diamond-like carbon electrodes for biosensor design

Diamond-like carbon (DLC) films are gaining big interest in electrochemistry research area. DLC electrodes made with different ratio of sp³/sp² carbon hybridization or doped with different percentages of nickel were characterized electrochemically by cyclic voltammetry and by amperometric measurements towards hydrogen peroxide. SiCAr1 and SiCNi5% were chosen as sensitive transducers for the elaboration of amperometric glucose biosensors. Immobilization of glucose oxidase was carried out by cross-linking with glutaraldehyde. Measurements were made at a fixed potential + 1.0 V in 40 mM phosphate buffer pH 7.4. SiCAr1 seems to be more sensitive for glucose, 0.6875 μA/mM, than SiCNi5%, 0.3654 μA/mM. Detections limits were 20 μM and 30 μM, respectively. Apparent Michaelis-Menten constants were found around 3 mM. Forty-eight percent and 79% of the original response for 0.5 mM glucose remained after 10 days for both biosensors, respectively.     

Automatic flow system for the sequential determination of copper in serum and urine by flame atomic absorption spectrometry

In this work, a fully automated flow system exploiting the advantages of the association of multi-pumping, multicommutation, binary sampling and merging zones, to accomplish the sequential determination of copper in serum and urine by flame atomic absorption spectrometry, is described. The developed flow system allowed multiple tasks, such as serum samples preparation (samples and standard solutions viscosity adjustment), serum copper (SCu) measurement, urine copper (UCu) pre-concentration and its subsequent elution and measurement, to be carried out sequentially. The implemented flow manifold presented a modular configuration consisting on two quasi-independent modules, each one accountable for a specific sample manipulation and whose combined operation under computer control enabled the determination of copper in a wide concentrations range.Once optimised and with a sample consumption of about 0.250 mL of serum and 7 mL of urine, the developed flow system allowed linear calibration plots up to 5 mg L⁻¹ with a detection limit of 0.035 mg L⁻¹ for SCu and linear calibration plots up to 300 μg L⁻¹ with a detection limit of 0.67 μg L⁻¹ for UCu. The sampling rate varied according to the module employed and was about 360 determinations h⁻¹ (SCu module), 12 determinations h⁻¹ (UCu module) or 24 determinations h⁻¹ (12 urine and 12 serum samples; UCu and SCu modules simultaneously). Repeatability studies (R.S.D.%, n = 10) showed good precision for UCu at concentrations of 25 μg L⁻¹ (2.54%), 50 μg L⁻¹ (0.90%) and 100 μg L⁻¹ (1.62%) as well as for SCu at concentrations of 0.25 mg L⁻¹ (8.11%), 1 mg L⁻¹ (3.11%) and 5 mg L⁻¹ (0.90%). A comparative evaluation showed a good agreement between the results obtained in the analysis of UCu and SCu (n = 18) by both the developed methodology and the reference procedures. Accuracy was further evaluated by means of the analysis of reference samples (Seronorm™ Trace Elements Urine and Seronorm™ Trace Elements Serum) and the obtained results complied with the certified values.     

Simple, rapid and sensitive spectrofluorimetric determination of diflunisal in serum and urine based on its ternary complex with terbium and EDTA

A very simple, rapid and highly sensitive fluorimetric method for the determination of diflunisal in serum and urine is described. The method is based on the formation of a ternary complex between diflunisal, Tb³⁺ and EDTA in alkaline aqueous solutions. This complex exhibits very intense terbium ion luminescence with a main emission maximum at 546 nm when excited at 284 nm. Optimum conditions for the complex formation have been investigated. The detection limit for diflunisal is 2.4 μg 1⁻¹, while the range of application is 0.01–6.00 mg 1⁻¹. The method has been successfully applied for the determination of diflunisal in untreated human serum and urine samples. Analytical recoveries from serum and urine samples spiked with diflunisal were in the ranges of 96.8–101.2% and 98.0–102.0%, respectively.     

Disposable luminol copolymer-based biosensor for uric acid in urine

A new electrochemiluminescent (ECL) disposable biosensor for uric acid was manufactured by immobilization in a double-layer design of luminol as a copolymer with 3,3′,5,5′-tetramethylbenzidine (TMB) and the enzyme uricase in chitosan on gold screen-printed cells. The good mechanical and improved electroluminescent characteristics of the new copolymer poly(luminol–TMB) make it possible to determine uric acid by measuring the growing ECL emission with the analyte concentration. The combination of enzymatic selectivity with ECL sensitivity results in a disposable analytical device with a linear range for uric acid from 1.5 × 10⁻⁶ to 1.0 × 10⁻⁴ M, a limit of detection of 4.4 × 10⁻⁷ M and a precision of 13.1% (1.0 × 10⁻⁵ M, n = 10) as relative standard deviation. Satisfactory results were obtained for uric acid determination in 24 h-urine samples compared to a reference procedure. This uric acid biosensor can be used as a low-cost alternative to conventional methods.     

硫堇-牛血清白蛋白氧化还原复合膜修饰葡萄糖生物传感器的研究

通过交联法和自组装法制备了一种双酶型葡萄糖生物传感器.首先以牛血清白蛋白-戊二醛为交联剂以实现对辣根过氧化物酶(HRP)的固载,再利用凝集素-糖蛋白的识别作用将葡萄糖氧化酶(GOD)分子组装到电极表面,制得双酶型的葡萄糖生物传感器.采用原子力显微镜(AFM)考察了复合膜的性质,同时采用循环伏安法和计时电流法考察了该传感...     

Optical fiber biosensor coupled to chromatographic separation for screening of dopamine, norepinephrine and epinephrine in human urine and plasma

An optical fiber biosensor has been developed for the determination of catecholamines (dopamine, norepinephrine and epinephrine) based on the recognition capacity of the enzyme laccase. In this study, a glass tube constituted by a fused silica fiber coated with a film of polystyrene/divinylbenzene resin (PS/DVB) was used for catecholamines separation. Firstly, the analyzer was tested for calibration and its analytical performance for catecholamines detection was compared with a classical analytical method, namely high performance liquid chromatography-electrochemical detector (HPLC-ED). The developed analytical device shows a high potential for catecholamines quantification with a detection limit of 2.1, 2.6 and 3.4 pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. The analytical sensitivity, inferred from the slope of the calibration curves established for a range of concentrations between 5 and 125 pg mL⁻¹, was found to be 0.344, 0.252 and 0.140 dB/pg mL⁻¹ for dopamine, norepinephrine and epinephrine, respectively. Furthermore, catecholamines speciation with the PS/DVB fiber was completely achieved in 3 min. The analytical performance of the reported sensor was also evaluated and found adequate for catecholamines determination in human urine and plasma samples.     

Investigation of organic phase biosensor for measuring glucose in flow injection analysis system

The aim of our present work was to develop a flow-through measuring apparatus for the determination of glucose content as model system in organic media and to compare the properties of the biosensor in organic and in aqueous solutions. Glucose oxidase (GOx) enzyme was immobilized on a natural protein membrane in a thin-layer enzyme cell, made of Teflon. The enzyme cell was connected into a flow injection analyzer (FIA) system with an amperometric detector. After optimizing the system the optimal flow rate was found at 0.8 ml min⁻¹. In this case 50-60 samples were measured per hour. Adding ferrocene monocarboxylic acid (FMCA) to acetonitrile and to 2-propanol the optimal concentration was 5 mg l⁻¹, while in the case of tetrabutylammonium-p-toluenesulfonate (TBATS) the optima were 2.7 and 8.0 mg l⁻¹, respectively. With 6% buffer in acetonitrile containing FMCA more than 100 samples could be measured with the enzyme cell without any loss of activity. Measuring the hydrogen peroxide content produced in 2-propanol, the optimal concentration of buffer solution was at about 20%. The linear measuring range was 0-0.5 mM glucose in acetonitrile and 0-1.0 mM in 2-propanol.Glucose concentration of oily food samples was measured and compared with results obtained by the reference UV-photometric method. The correlation between the results measured by the two methods was very good with correlation coefficient (r) as high as 0.976.     

Direct electrochemistry of glucose oxidase in a colloid Au-dihexadecylphosphate composite film and its application to develop a glucose biosensor

Colloid Au (Au(nano)) with a diameter of about 10 nm was prepared and used in combination with dihexadecylphosphate (DHP) to immobilize glucose oxidase (GOD) onto the surface of a graphite electrode (GE). The direct electrochemistry of GOD confined in the composite film was investigated. The immobilized GOD displayed a pair of redox peaks with a formal potential of -0.475 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 150 mV s(-1). The GOD in the composite film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction peak current of dissolved oxygen decreased, which could be developed for glucose determination. A calibration linear range of glucose was 0.5-9.3 mM with a detection limit of 0.1 mM and a sensitivity of 1.14 microA mM(-1). The glucose biosensor showed good reproducibility and stability. The general interferences that coexisted in human serum sample such as ascorbic acid and uric acid did not affect glucose determination.     

超薄硅溶胶-凝胶修饰膜的制备与表征

报道了一种制备硅溶胶－凝胶膜（sol-gel)的新方法。通过对形成溶胶和凝胶化过程的控制，获得了制备超薄溶胶－凝胶膜的最佳条件，并对膜的性质进行了一系列表征。实验表明，所得溶胶－凝胶膜具有良好的结构特征。采用光度法、电化学方法可有效地对膜的厚度进行表征。     

A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine

A new amperometric biosensor based on urate oxidase-peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H₂O₂ from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 μM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 °C were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9×10⁻³ U cm⁻² for urate oxidase and 512.7 U cm⁻² for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out.     

Determination of fluoroquinolones in urine and serum by using high performance liquid chromatography and multiemission scan fluorimetric detection

A high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of four fluoroquinolones. The studied compounds have been enoxacin (ENO), norfloxacin (NOR), ofloxacin (OFLO) and enrofloxacin (ENRO). An isocratic elution method, using a mixture of tetrahydrofuran (8%) and phosphate buffer (pH 3.00, 30.0 mM, 92%) as mobile phase, has been developed. Fluorimetric detection, exciting at 277 nm, and multiemission scan (407 nm for ENO, 444 nm for both NOR and ENRO and 490 nm for OFLO) has been used. Detection limits of 500, 14.7, 25.2 and 15.0 ng mL⁻¹ for ENO, NOR, OFLO and ENRO, respectively, have been obtained. The proposed method has been satisfactorily applied to analyze NOR, OFLO and ENRO in human urine and serum samples.