Performance characteristic studies of glucose biosensors modified by (3-mercaptopropyl)trimethoxysilane sol–gel and non-conducting polyaniline

The materials (3-mercaptopropyl)trimethoxysilane (MPTMS) sol–gel and non-conducting polyaniline (PAn) were prepared for coating on two different amperometric biosensors fabricated by the immobilization of glucose oxidase (GOx) in conducting polyaniline (PA) as permselective membranes. A few performance characteristics of the MPTMS and the PAn-modified glucose sensors are comparable. In detecting 1 mM glucose, the biosensors’ response times were 11 and 12 s, respectively, and results from the repeating measurements (N = 10) showed that R.S.D. = 2.09 and 1.70%, respectively. The detection linearity of the two biosensors was up to 12.5 mM with R = 0.9989 and 0.9969. When they were used to detect 0.5 mM of ascorbic acid (AA), uric acid (UA), and acetaminophen at an anodic potential of 0.4 V, current signals were either insignificant or invisible. The PAn-coated glucose sensor exhibited superior sensitivity at 18.91 μA/(mM cm²), and a maximum current density of 3.70 mA/cm². GOx in the MPTMS-modified biosensor presented a stronger affinity to glucose with K_m = 48.87. More importantly, the stability of this biosensor is superior to that of the PAn-modified biosensor and has lasted for almost 5 months.     

Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

Development of a bienzyme system based on sugar–lectin biospecific interactions for amperometric determination of phenols and aromatic amines

Bienzyme electrode with horseradish peroxidase (HRP) and glucose oxidase (GOD) multilayers was constructed based on sugar–lectin biospecific interactions for amperometric determination of phenolic compounds and aromatic amines. Atomic force microscopy (AFM) was applied to monitor the uniform layer-by-layer assembly of concanavalin A (Con A) and HRP or GOD on polyelectrolyte precursor film-modified Au electrode. Substituted phenolic compounds and aromatic amines could be determined with in situ generation of H₂O₂ by GOD-catalyzed oxidation of glucose. The parameters of the biosensor including the number of assembled HRP and GOD layer, and the concentrations of glucose were optimized. The linear range for the determination of catechol and p-phenyldiamine was 6.0–60.0 μmol L⁻¹ and 7.6–68.4 μmol L⁻¹ with detection limit of 0.9 μmol L⁻¹ and 0.4 μmol L⁻¹, respectively. The biosensor possessed high sensitivity and fast response for phenolic compounds and 95% of the maximum response could be reached in about 3 s. Glucose, ascorbic acid, tartaric acid, citric acid and starch exhibited no interference for the detection. The biosensor presented high stability due to the design for in situ generation of H₂O₂ with bienzyme system.     

Fabrication of novel chitosan nanofiber/gold nanoparticles composite towards improved performance for a cholesterol sensor

A novel amperometric cholesterol biosensor was fabricated by the immobilization of ChOx (cholesterol oxidase) onto the chitosan nanofibers/gold nanoparticles (designated as CSNFs/AuNPs) composite network (NW). The fabrication involves preparation of chitosan nanofibers (CSNFs) and subsequent electrochemical loading of gold nanoparticles (AuNPs). Field emission scanning electron microscopy (FE-SEM) was used to investigate the morphology of CSNFs (sizes in the range of ∼50-100 nm) and spherical AuNPs. Cyclic voltammetry, hydrodynamic voltammetry and amperometry were used to examine the performance of CSNF-AuNPs/ChOx biosensor. The CSNF-AuNPs/ChOx biosensor exhibited a wide linear response to cholesterol (concentration range of 1-45 μM), good sensitivity (1.02 μA/μM), low response time (∼5 s) and excellent long term stability. The combined existence of AuNPs within CSNFs NW provides the excellent performance of the biosensor towards the electrochemical detection of cholesterol.     

Dispersion of single-walled carbon nanotubes in poly(diallyldimethylammonium chloride) for preparation of a glucose biosensor

Poly(diallyldimethylammonium chloride) (PDDA) was chosen to disperse single-walled carbon nanotubes (SWCNTs). The optimal conditions to prepare stable PDDA–SWCNTs aqueous dispersions were presented. Then, the positively charged PDDA–SWCNTs composite and negatively charged glucose oxidase (GOD) were employed to fabricate multilayer films on platinum (Pt) electrodes by layer-by-layer self-assembly technique. The consecutive growth of the multilayer films was confirmed by quartz crystal microbalance. Electrochemical measurements were used to study the properties of the proposed biosensor. Results demonstrated that SWCNTs were evenly dispersed within the PDDA films and efficiently improved the conductivity of the resulting films. Among the biosensors, the one based on seven layers of multilayer films got the best performance. It showed wide linear range of 0.05–12 mM, high sensitivity of 63.84 μA/(mM cm²), low detection limit of about 4 μM and small value of the apparent Michaelis–Menten constant, 8.46 mM. In addition, the biosensor also exhibited good suppression of interference and long-term operational stability. This protocol could be used to immobilize other enzymes to construct a range of biosensors.     

Preparation of Ag–Au nanoparticle and its application to glucose biosensor

In this paper, Ag–Au nanoparticles are produced in sodium-bis(2-ethylhexyl)-sulfosuccinate (AOT)–cyclohexane reverse micelle system. The properties of the obtained nanoparticles are characterized with transmission electron microscope (TEM) and UV–vis absorption spectrophotometer. Glucose biosensors have been formed with glucose oxidase (GOx) immobilized in Ag–Au sol. GOx are simply mixed with Ag–Au nanoparticles and crosslinked with a polyvinyl butyral (PVB) medium by glutaraldehyde. Then a platinum electrode is coated with the mixture. The effects of the various molar ratios of Ag–Au particles with respect to the current response and the stability of the GOx electrodes are studied. The experimental results indicate the current response of the enzyme electrode containing Ag–Au sol increase from 0.32 to 19 μA cm⁻² in the solution of 10 mM β-d-glucose. In our study, the stability of enzyme electrodes is also enhanced.     

A hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin in Hb–Ag sol films

Hemoglobin (Hb) was used as a template to fabricate hemoglobin–silver (Hb–Ag) sol in which the hemoglobin showed direct electrochemistry on a glass carbon (GC) electrode. Ultraviolet–visible (UV–vis) spectra and reflectance absorption infrared (RAIR) spectra suggested that hemoglobin in Hb–Ag sol retained its native secondary structure. Scanning electron microscopy (SEM) demonstrated that the morphology of the Hb film was much different from the Hb–Ag sol film. The Hb–Ag film proved to exhibit a good electrocatalytic activity for the reduction of hydrogen peroxide. Based on this, a novel amperometric hydrogen peroxide biosensor was developed, which showed a sensitive response to the reduction of H₂O₂ without any electron mediator. Under optimum conditions, the biosensor responded linearly to H₂O₂ in the concentration range of 1 × 10⁻⁶ to 2.5 × 10⁻² M with detection limit of 1 × 10⁻⁷ M at 3σ. Moreover, the studied biosensor exhibited high sensibility, good reproducibility, and long-term stability.     

Fabrication of polypyrrole–glucose oxidase biosensor based on multilayered interdigitated ultramicroelectrode array with containing trenches

A miniature glucose biosensor has been developed based on electropolymerization of polypyrrole–glucose oxidase on a multilayered gold interdigitated ultramicroelectrode array with containing trenches. The basal band ultramicroelectrode with a functional width of 362 nm is fabricated using multilayered materials and conventional photolithographic techniques. The electrode surface is inside the containing trenches, the depth of which is larger than 1.5 μm. High quality electrodes with uniform geometries are characterized by microscopy and electrochemical techniques. The corrosion resistance is investigated on exposure to the normal saline, which indicates that the electrodes are adequate for acute experiments lasting a few hours. Fabricated by electropolymerization, the glucose oxidase/polypyrrole (GOx/PPy) biosensors can be used for detecting glucose concentration in the linear range of 0–10 mmol/L, with a sensitivity of 13.4 nA/(mmol L) and a correlation coefficient of 0.998, respectively.     

A disposable amperometric biosensor for determining total cholesterol in whole blood

A disposable amperometric biosensor for detecting the total cholesterol was fabricated which comprises a sensing electrode and a reference electrode in simultaneous contact with an integrated reagent layer. The integrated reagent layer formed by coating a working ink containing cholesterol esterase, cholesterol dehydrogenase, coenzyme, redox mediator, surfactant, stabilizer, filler and at least one aqueous thickening agent. The biosensor showed the linearity for 50-500 mg/dL cholesterol acetate. The minimum detection limit of the cholesterol was 50 mg/dL. The effects of temperature and the stability of immobilized enzymes were also studied. A good correlation was found among cholesterol values obtained by commercial colorimetric test strip and clinical/laboratory methods. The biosensor showed an acceptable reproducibility, good stability and low interferences.     

Determination of reduced glutathione using an amperometric carbon paste electrode chemically modified with TTF–TCNQ

The development of an amperometric sensor for the determination of reduced glutathione (GSH) is described. The sensor is based on tetrathiafulvalene–tetracyanoquinodimethane (TTF–TCNQ) incorporated into the graphite powder/Nujol oil matrix. The electrooxidation of GSH was monitored amperometrically at 200 mV versus SCE (saturated calomel electrode). The amperometric response of the sensor was linearly proportional to the GSH concentration between 20 and 300 μmol l⁻¹, in 0.1 mol l⁻¹ phosphate buffer (pH 8.0), containing 0.1 mol l⁻¹ KCl and 0.5 mmol l⁻¹ Na₂H₂EDTA, as supporting electrolyte.

The detection limit, considering signal/noise ratio equal three, was 4.2 μmol l⁻¹ for GSH and the repeatability obtained as relative standard deviation was of 5.1% for a series of 10 successive measurements.     

Amperometric biosensor for formic acid in air

The possibility of developing a simple, inexpensive and specific personal passive “real-time” air sampler incorporating a biosensor for formic acid was investigated. The sensor is based on the enzymatic reaction between formic acid and formate dehydrogenase (FDH) with nicotinamide adenine dinucleotide (NAD⁺) as a co-factor and Meldola's blue as mediator. An effective way to immobilise the enzyme, co-factor and Meldola's blue on screen-printed, disposable, electrodes was found to be in a mixture of glycerol and phosphate buffer covered with a gas-permeable membrane. Steady-state current was reached after 4–15 min and the limit of detection was calculated to be below 1 mg/m³. However, the response decreased by 50% after storage at −15°C for 1 day.     

全血L􀀁乳酸􀀂生物传感器

全血L-广乳酸生物传感器适用于快速方便地测量全血中L-乳酸的浓度。它包括印有一对碳电极的基片，由L-乳酸氧化酶和作为电子传递剂的铁氰化钾构成的反应层和塑料薄膜形成的反应腔。将血样(2μL)滴在反应腔中，然后在电极两端施加一个300mV的恒电压，50s后读取电流值，即可得到对应的L-乳酸浓度。该传感器的线性范围为0-15mol。保存120d后，响应没有明显衰减。该传感器的测量结果和美国强生VITROS250 SYSTEM全自动干化学分析仪的测量结果有良好的相关性(r=0．9809)，该传感器适合大规模生产。     

一种安培型尿酸生物传感器的初步研究

利用铁氰化钾作为电化学反应的媒介体,将尿酸氧化酶固定在羧甲基纤维素处理的碳电极表面,制成了一种新型的尿酸安培传感器.该传感器在恒电位0.3 V和尿酸氧化酶的催化作用下,使被检测物--尿酸--氧化,铁氰化钾还原,在电极表面产生灵敏的氧化-还原峰,然后利用安培法即可对尿酸进行间接测定.10秒内即可达到输出稳态电流,尿酸的测定范围为1 mg/dL～20 mg/dL,线性范围较好,拟合系数为0.9723.同时该传感器对尿酸的测定避免了常规电化学传感器测定中样品所含大量的易氧化物质带来的干扰.     

一种葡萄糖氧化酶安培传感器研究

利用铁氰化钾作为电化学反应的媒介体,将葡萄糖氧化酶固定在羧甲基纤维素处理的碳电极表面,制成了一种新型的葡萄糖安培传感器.该传感器在恒电位0.4 V和葡萄糖氧化酶的催化作用下,使被检测物--葡萄糖--氧化,铁氰化钾还原,在电极表面产生灵敏氧化-还原峰,利用安培法可对葡萄糖进行间接测定.葡萄糖的测定范围为2.7～27 mmol/L,线性范围较好,拟合系数为0.997 8,灵敏度较当前葡萄糖传感器有明显的提高,11 s内即可达到输出稳态电流.同时该传感器对葡萄糖的测定避免了常规电化学传感器测定中样品所含大量的易氧化物质--如抗坏血酸和尿酸--带来的干扰.     

Self-containing reactant biochips for the electrochemiluminescent determination of glucose, lactate and choline

A multi-parametric biochip for glucose, lactate and choline has been developed based on luminol/hydrogen peroxide electrochemiluminescence (ECL). The sensing layers developed were composed of enzyme-bound beads co-entrapped in a photopolymer with luminol-charged beads and spotted at the surface of a glassy carbon electrode (GCE). Lactate oxidase, choline oxidase and luminol were immobilised via electrostatic interactions with DEAE (diethylaminoethyl)-Sepharose whereas glucose oxidase was immobilised, after modification with histidine, by chelation on nickel-charged IDA (imidodiacetic acid)-Sepharose. The luminol immobilisation enabled the achievement of micro-biosensors, present at the surface of the same GCE and free of lateral contamination between each spot by the other’s reaction product. After optimisation of the applied potential, the integration time and the luminol charge in the sensing layer, the electrochemiluminescent H₂O₂ sensor exhibited a detection limit of 2 μM and a working range from 2 μM to 0.5 mM.

The multi-biosensor enabled the concomitant detection of glucose, lactate and choline in the ranges 20 μM–2 mM, 2 μM–0.2 mM and 2 μM–0.2 mM, respectively. Glucose and lactate measurements in complex matrix such as human serum, were in good agreement with the reference methods, without internal calibration of the sample, demonstrating the absence of matrix interference with the present analytical system.     

Characteristics of a label-free piezoelectric immunosensor detecting Pseudomonas aeruginosa

A label-free immunosensor system detecting a psychrophylic bacterium, Pseudomonas aeruginosa was developed as follows. Four types of anti-P. aeruginosa antibody were individually chemisorbed onto one-side gold electrodes of piezoelectric quartz crystals according to a thiolated antibody coupling procedure initiated with a thiol-cleavable heterobifunctional cross-linker, sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate. A flow-type biosensor system was operated optimally at 0.2 M sodium potassium phosphate, pH 7.2 with a minimal matrix effect and the selected flow rate for it was 0.155 ml/min. A biosensor response was detected by measuring a steady-state resonant frequency shift after the response time around 8 min. The frequency shifts obtained were quite specific according to the antibody types and P. aeruginosa strains. The biosensor responses to varying concentrations of the P. aeruginosa cells ranging from 1.3×10⁷ to 1.3×10⁸ CFU/ml were determined as 17–176 Hz and a linear calibration curve (r=0.942) was obtained by plotting the responses in a double-logarithmic scale. The selectivity of the biosensor between P. aeruginosa and Xanthomonas spp. which both belong to the aerobic pseudomonads was, however, not so good owing to the property of the antibody used. The sensor chip could be reused at least seven times without an appreciable decrease in sensitivity.     

Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on nanocrystal gold–carbon paste electrodes

The preparation and performance of a xanthine oxidase (XOD) biosensor, based on a carbon paste electrode (CPE) modified with electrodeposited gold nanoparticles (nAu), for the amperometric determination of hypoxanthine (Hx) is reported. Different XOD biosensor configurations were evaluated and compared with electrodes constructed by immobilizing XOD onto unmodified CPE and with biosensors prepared using glassy carbon electrodes and gold disk electrodes modified with electrodeposited gold. The XOD–nAu–CPE in which the enzyme was immobilized by cross-linking with glutaraldehyde (GA) and BSA exhibited the highest amperometric signal for Hx. Although Hx detection is usually carried out at potential values of around +600 mV versus Ag/AgCl, the GA–BSA–XOD–nAu–CPE allowed this detection to be carried out at 0.00 V, thus minimizing potential interferences from electrochemically oxidizable substances such as ascorbic acid. Experimental variables concerning the biosensor preparation were optimized. Calibration plots for Hx were constructed with the biosensor operating at +600 mV and at 0.00 V. The detection limit for Hx, 2.2 × 10⁻⁷ mol l⁻¹, obtained using the latter potential value is similar to the best detection limits reported in the literature with other biosensor designs working at much more extreme potentials. The usefulness of the biosensor for the analysis of real samples was demonstrated by determining Hx in sardines and chicken meat.     

Direct electrochemistry of glucose oxidase on screen-printed electrodes through one-step enzyme immobilization process with silica sol–gel/polyvinyl alcohol hybrid film

A one-step enzyme immobilization process with silica sol–gel/polyvinyl alcohol was described to achieve direct electrochemistry of glucose oxidase on screen-printed electrodes. The immobilized glucose oxidase displays a couple of stable and well-defined redox peaks with an electron transfer rate constant of 8.38 s⁻¹ and a formal potential of −460 mV (versus SCE) in phosphate buffer (0.05 M, pH 7.0) at a scan rate of 300 mV s⁻¹. The results suggested that conformation and bioactivity of glucose oxidase could be retained efficiently using the proposed immobilization method and the porous structure of screen-printed electrode surface was helpful for electron communication of glucose oxidase and the electrode. Furthermore, the modified electrode was used as a glucose biosensor, exhibiting a linear response to glucose concentration ranging from 0 to 4.13 mM and a sensitivity of 3.47 μA mM⁻¹ cm⁻² at an applied potential of −0.5 V. The detection limit of the biosensor is 9.8 μM, based on a signal-to-noise ratio of 3. The present work provided a promising strategy for fabricating a novel and disposable mediatorless glucose biosensor, which could be mass-produced through further development.     

Development of a molecular imprinting thick film electrochemical sensor for cholesterol detection

A combined molecular imprinting and thick film electrochemical sensor for cholesterol concentration detection had been developed. The ferro-ferric cyanide coupled redox reaction was used as the means to quantify the cholesterol presented in the test medium. This electrochemical sensor employed a modified gold working electrode, a platinum counter electrode and an Ag/AgCl reference electrode. The alkanethiol was used to form the self-assembled monolayer (SAM) on the gold working electrode. The SAM was then used with the cholesterol as the template forming the molecular imprinting layer. This sensor prototype could detect cholesterol concentrations between 66 and 700 nM and only a 1 μL of the sample volume was required.     

Perchlorate selective membrane electrodes based on synthesized platinum(II) complexes for low-level concentration measurements

Three synthesized platinum(II) complexes, [PtR₂(NN)] (R = Me, p-MeC₆H₄ and p-MeOC₆H₄; NN = 2,2′-bipyridyl), were studied to characterize their ability as an anion carrier in a PVC membrane electrode. The polymeric membrane electrodes (PME) and also coated glassy carbon electrodes (CGCE) prepared with [Pt(p-MeOC₆H₄)₂(NN)] showed excellent response characteristics to perchlorate ions. The electrodes exhibited Nernstian responses to ClO₄⁻ ions over a wide concentration range from 5 × 10⁻⁷ to 4.0 × 10⁻¹ M for PME and 1.5 × 10⁻⁷ to 2.7 × 10⁻¹ M for CGCE with low detection limits (4.0 × 10⁻⁷ M for PME and 1.0 × 10⁻⁷ M for CGCE). The electrodes possess fast response time, satisfactory reproducibility, appropriate lifetime and, most importantly, good selectivity toward ClO₄⁻ relative to a variety of other common anions. The potentiometric response of the electrodes is independent of the pH of the test solution in the pH range 2.5–9.5. The proposed sensors were used in potentiometric determination of perchlorate ions in mineral water, urine samples and also samples containing interfering anions. The interaction of the ionophore with perchlorate ions was shown by UV–vis spectroscopy.