Biosensors for determination of glucose with glucose oxidase immobilized on an eggshell membrane

A glucose biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for glucose determination has been fabricated. Glucose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent. The glucose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a dissolved oxygen sensor. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution and the decrease in the oxygen level was monitored and related to the glucose concentration. The effect of glutaraldehyde concentration, pH, phosphate buffer concentration and temperature on the response of the glucose biosensor has been studied in detail. Common matrix interferents such as ethanol, d-fructose, citric acid, sodium benzoate, sucrose and l-ascorbic acid did not give significant interference. The resulting sensor exhibited a fast response (100 s), high sensitivity (8.3409 mg L⁻¹ oxygen depletion/mmol L⁻¹ glucose) and good storage stability (85.2% of its initial sensitivity after 4 months). The linear response is 1.0×10⁻⁵ to 1.3×10⁻³ mol L⁻¹ glucose. The glucose content in real samples such as commercial glucose injection preparations and wines was determined, and the results were comparable to the values obtained from a commercial glucose assay kit based on a spectrophotometric method.     

Quality and timeliness in medical laboratory testing

In terms of testing, modern laboratory medicine can be divided into centralized testing in central laboratories and point-of-care testing (POCT). Centralized laboratory medicine offers high-quality results, as guaranteed by the use of quality management programs and the excellence of the staff. POCT is performed by clinical staff, and so such testing has moved back closer to the patient. POCT has the advantage of shortening the turnaround time, which potentially benefits the patient. However, the clinical laboratory testing expertise of clinical staff is limited. Consequently, when deciding which components of laboratory testing must be conducted in central laboratories and which components as POCT (in relation to quality and timeliness), it will be medical necessity, medical utility, technological capabilities and costs that will have to be ascertained. Provided adequate quality can be guaranteed, POCT is preferable, considering its timeliness, when testing vital parameters. It is also preferred when the central laboratory cannot guarantee the delivery of results of short turn-around-time (STAT) markers within 60 or (even better) 30 min. POCT should not replace centralized medical laboratory testing in general, but it should be used in cases where positive effects on patient care have been clearly demonstrated.     

Investigation of the origin of the glucose response in a glucose oxidase|polyaniline system

The origin of the signal seen in response to glucose in a polyaniline|glucose oxidase system is explored by immittance spectroscopy, by comparing data from an equivalent circuit model and the parameters obtained from a solution of the faradaic branch of the frequency dispersion for a coupled chemical—electrochemical reaction mechanism. It was shown that an RC subcircuit in the equivalent circuit model was sensitive to peroxide concentration, and the interaction of peroxide with polyaniline at potentials where it either oxidised or reduced the polyaniline was discussed. This information was used to compare the data obtained in a bulk and entrapped glucose oxidaselglucose system, and it was seen that the origin of the response could not be fully attributed to peroxide interaction in the latter case. Under anaerobic conditions with entrapped enzyme, it was proposed that a complex between the gluconolactone product of the enzyme reaction and the polymer leads to a more conducting polymer, with inherent charge compensation, and this results in the observed enhanced current signal.     

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h⁻¹) and ease of automation.     

Mediator-free amperometric determination of glucose based on direct electron transfer between glucose oxidase and an oxidized boron-doped diamond electrode

A mediator-free glucose biosensor, termed a “third-generation biosensor,” was fabricated by immobilizing glucose oxidase (GOD) directly onto an oxidized boron-doped diamond (BDD) electrode. The surface of the oxidized BDD electrode possesses carboxyl groups (as shown by Raman spectra) which covalently cross-link with GOD through glutaraldehyde. Glucose was determined in the absence of a mediator used to transfer electrons between the electrode and enzyme. O₂ has no effect on the electron transfer. The effects of experimental variables (applied potential, pH and cross-link time) were investigated in order to optimize the analytical performance of the amperometric detection method. The resulting biosensor exhibited fast amperometric response (less than 5 s) to glucose. The biosensor provided a linear response to glucose over the range 6.67×10⁻⁵ to 2×10⁻³ mol/L, with a detection limit of 2.31×10⁻⁵ mol/L. The lifetime, reproducibility and measurement repeatability were evaluated and satisfactory results were obtained.     

Dual nanocatalysts co-decorated three-dimensional,laser-induced graphene hybrid nanomaterials integrated with a smartphone portable electrochemical system for point-of-care non-enzymatic glucose diagnosis

Non-enzymatic biosensors based on various nanomaterials with large surface-volume ratios and high catalytic efficiencies have been proposed to compensate for the non-stability and high cost of enzymatic biosensors. However, the construction of a stable, highly sensitive, flexible, three-dimensional (3D), microstructured, non-enzymatic biosensor integrated with a smartphone-based portable system has been challenging. Herein, highly conductive laser-induced graphene (LIG) array with a honeycomb-like 3D microstructure co-decorated with copper(I) oxide and gold nanocatalysts was developed via simple and green electro-deposition and chemical reduction approaches for a miniaturized electrochemical flexible non-enzymatic biosensor. SEM, XRD, Raman and XPS analyzations indicated that the Cu₂O and Au nanocatalysts co-decorated three-dimensional, laser-induced graphene hybrid nanomaterials were developed successfully. The signal of the biosensor was improved by more than 10 fold compared to the LIG alone due to the co-decorated with copper(I) oxide and gold nanocatalysts. The fabricated electrochemical biochip was integrated with a smartphone-based microstation for glucose monitoring, presenting a larger linear interval of 1–20 mM with an excellent sensitivity of 236 μA/mM/cm² and a relatively low detection limit of 0.31 μM. Noticeably, the biochip could measure blood sugar on curved surfaces and still deliver stable sensing signals after being bent back-and-forth 25 times. The novel biosensor is a potentially valuable flexible electronic device. The hybrid nanomaterials developed in this work may be applicable to other biosensing, catalytic, and energy devices (supercapacitors and batteries).     

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.     

葡萄糖氧化酶-辣根过氧化物酶-葡萄糖-愈创木酚-荧光反应体系测定血糖

利用愈创木酚的荧光特性,将其导入葡萄糖-葡萄糖氧化酶(GOD)-辣根过氧化物酶(HRP)的荧光猝灭反应体系,并与荧光毛细分析技术结合,开发了一种能测定微量葡萄糖的新方法.本方法的的最佳测定条件为:反应时间15 min,反应温度30C,GOD和HRP的浓度分别为4500U/L、2500U/L,愈创木酚浓度为150μmol...     

Multicommutated flow system for the determination of glucose in honey with immobilized glucose oxidase reactor and spectrophotometric detection

A new automated method for the determination of glucose in honey is proposed. The method is based on multicommutated flow analysis (MCFA) and employs an immobilized glucose oxidase reactor and spectrophotometric detection at 505 nm of the red quinoneimine formed (Trinder's method).The calibration curve obeyed a second order equation in the range 0-0.14 g L⁻¹ (h = −2.2199 C² + 1.3741C + 0.0077, r² = 0.9991, where h is the peak height (absorbance) and C the concentration in g L⁻¹). The method was validated analyzing eight commercial samples, both by the AOAC 954.11 and 977.20 official methods. According to Student's t-test of mean values, at the confidence level of 95% the results obtained with the proposed method were in agreement with those obtained by the official methods. Precision (s_r(%), n = 10) was 3% and the sampling frequency of the system was 20 samples h⁻¹.     

Simultaneous detection of lactate and glucose by integrated printed circuit board based array sensing chip

An integrated printed circuit board (PCB) based array sensing chip was developed to simultaneously detect lactate and glucose in mouse serum. The novelty of the chip relies on a concept demonstration of inexpensive high-throughput electronic biochip, a chip design for high signal to noise ratio and high sensitivity by construction of positively charged chitosan/redox polymer Polyvinylimidazole-Os (PVI-Os)/carbon nanotube (CNT) composite sensing platform, in which the positively charged chitosan/PVI-Os is mediator and electrostatically immobilizes the negatively charged enzyme, while CNTs function as an essential cross-linker to network PVI-Os and chitosan due to its negative charged nature. Additional electrodes on the chip with the same sensing layer but without enzymes were prepared to correct the interferences for high specificity. Low detection limits of 0.6 μM and 5 μM were achieved for lactate and glucose, respectively. This work could be extended to inexpensive array sensing chips with high sensitivity, good specificity and high reproducibility for various sensor applications.     

Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor

This paper describes a microquantitative method for glucose determination in situ of living cells in real-time. In this novel technique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injection lab-on-valve system (microSI-LOV). Glucose sensing is performed through the use of a two-step, NAD-linked enzymatic process. The course of the assay is monitored in real-time, by absorbance of NADH at 340 nm. The microsequential assay based on plug/nozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM. The design of the (microSI-LOV) system allows the assay to be carried out using only 40 microL of the enzyme reagent and 3 microL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopore beads. Rapid cellular glucose consumption, in this technique, is facilitated by a high cell density, which allows a large number of cells (10(4)-10(5)) to be retained in a very small volume (3 microL). In turn, this cell density results in the rapid depletion of glucose from the cell medium over short time periods (< 2 min). In conjunction with the assay development, the plug/nozzle design and its ramifications on mixing in general are presented and discussed.     

Simultaneous determination of glucose and sucrose by a glucose-sensing enzyme electrode combined with an invertase-attached cell

Glucose and sucrose are simultaneously determined by using a glucose-sensing enzyme electrode combined with a cell that contains immobilized invertase. The electrode current changes linearly with time for several minutes from ca. 1 min after the addition of a glucose-sucrose mixture. The concentration of sucrose (60 μM-6 mM) is determined from the rate of current change in the linear region, and that of glucose (5 μM-1 mM) is determined by extrapolating the straight current-time line to t=0.45 min and by measuring the intercept on the vertical (current) axis at t=0.45 min. The relative standard deviations are 1.8% for glucose and 3.7% for sucrose (n=10). More than 20 food samples can be analysed in 1 h.     

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.     

Evaluation of an amperometric glucose biosensor based on a ruthenium complex mediator of low redox potential

An amperometric glucose ring-disk biosensor based on a ruthenium complex mediator of low redox potential was fabricated and evaluated. This thin-layer radial flow microsensor (10 μl) with ring-disk working electrode displayed remarkable amperometric sensitivity. For Ru₃(μ₃-O)(AcO)₆(Py)₃(ClO₄) (Ru-Py), a trinuclear oxo-acetate bridged cluster, a reversible redox curve of low redox potential and narrow potential window (redox potentials were −0.190 and −0.106 V versus Ag/AgCl wire, respectively) was observed, which is comparable to many reported mediators such as ferrocene derivatives and other ruthenium complexes. The glucose and hydrogen peroxide assays were carried out with this complex-modified electrode Ru-Py-HRP-GOx/Nafion. The sensitivity was obtained 24 nA (15.4 mA M⁻¹ cm⁻²) for 10 μM glucose and 126 nA (160 mA M⁻¹ cm⁻²) for 5 μM H₂O₂, respectively with a working potential at 0 V versus Ag/AgCl. Ascorbic acid was studied as interference to the glucose assay. The application of 0 V potential versus Ag/AgCl did not avoid the occurrence of the oxidation of ascorbic acid, however, the pre-coating of ascorbate oxidase on the disk part of the ring-disk working electrode efficiently pre-oxidized the ascorbic acid and hence eliminated its interference on the glucose response. The practical reliability was also evaluated by assaying the dialysate from the prefrontal cortex of Wistar rats.     

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3σ).     

Amperometric determination of bonded glucose with an MnO(2) and glucose oxidase bulk-modified screen-printed electrode using flow-injection analysis

A screen-printed amperometric biosensor based on carbon ink double bulk-modified with MnO₂ as a mediator and glucose oxidase as a biocomponent was investigated for its ability to serve as a detector for bonded glucose in different compounds, such as cellobiose, saccharose, (-)-4-nitrophenyl-β-d-glucopyranoside, as well as in beer samples by flow-injection analysis (FIA). The biosensor could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.5) and exhibited good reproducibility and stability. Bonded glucose was released with glucosidase in solution, and the free glucose was detected with the modified screen-printed electrode (SPE). The release of glucose by the aid of glucosidase from cellobiose, saccharose and (-)-4-nitrophenyl-β-d-glucopyranoside in solution showed that stoichiometric quantities of free glucose could be monitored in all three cases.The linear range of the amperometric response of the biosensor in the FIA-mode flow rate 0.2 mL min⁻¹, injection volume 0.25 mL, operation potential 0.48 V versus Ag/AgCl) extends from 11 to 13,900 μmol L⁻¹ glucose in free form. The limit of detection (3σ) is 1 μmol L⁻¹ glucose. A concentration of 100 μmol L⁻¹ yields a relative standard deviation of approximately 7% with five injections. These values correspond to the same concentrations of bonded glucose supposed that it is liberated quantitatively (incubation for 2 h with glucosidase).Bonded glucose could be determined in beer samples using the same assay. The results corresponded very well with the reference procedure.     

Biotinylated alginate immobilization matrix in the construction of an amperometric biosensor: application for the determination of glucose

The use of biotinylated alginate as an immobilization matrix of enzymes on the surface of the amperometric transducer is described herein. The model used is that of the well-established glucose detection. Several types of immobilization protocols were tested. In the exception of one protocol, biotin labeled glucose oxidase was shown to first require conjugation with avidin, before its immobilization onto a biotin-alginate gel matrix. The response of the biosensors to incremental additions of glucose, was measured by potentiostating the modified electrodes at 0.6 V/SCE. The permeability of the modified electrodes was thereafter measured by using rotating disk electrode (RDE) voltammetry with ferrocenemonocarboxylic acid as the electroactive probe.     

A new electrochemiluminescent sensing system for glucose based on the electrochemiluminescent reaction of bis-[3,4,6-trichloro-2-(pentyloxycarbonyl)-phenyl] oxalate

In this paper, the electrochemiluminescence (ECL) behavior of bis-[3,4,6-trichloro-2-(pentyloxycarbonyl)-phenyl] oxalate (BTPPO) at glassy carbon electrode (GCE) in phosphate buffer solution in the presence of hydrogen peroxide has been investigated when linear sweep voltammetry was applied. The optimum chemical conditions and electrochemical parameters for this ECL system have been investigated in detail. Under the optimum conditions, it was found that the concentration of BTPPO was linear with the ECL intensity in the range of 3.0 × l0⁻⁶ to 3.0 × 10⁻⁴ mol/L, and the detection limit (S/N = 3) for BTPPO was 1.0 × 10⁻⁷ mol/L. The possible mechanism for ECL of BTPPO at the GCE in the presence of hydrogen peroxide was also discussed. Furthermore, based on the fact that glucose oxidase can react with glucose to produce hydrogen peroxide, a new ECL sensing system of BTPPO has been developed for detection of glucose. The enhanced ECL intensity has a linear relationship with the concentration of glucose in the range of 1.0 × l0⁻⁴ to 1.0 × 10⁻³ mol/L, and the detection limit for glucose is found to be 5.0 × 10⁻⁵ mol/L (S/N = 3).     

Implementation of ISO 9001 in a medical testing laboratory

 The Department of Clinical Chemistry and Molecular Genetics, within the Institute of Clinical Pathology and Medical Research at Westmead Hospital, is a medical testing laboratory operating within the public sector health system of New South Wales, Australia. It provides acute-care pathology services to Westmead Hospital (a 900-bed tertiary referral university teaching hospital) as well as to three district hospitals and three rural hospitals. In addition to these core clinical chemistry services, it offers approximately 150 specialised biochemistry, pharmacology, toxicology, trace metal and molecular genetics assays as a reference laboratory service. In 1993, the Department became Australia's first medical testing laboratory to be registered to ISO 9001-1987/AS3901-1987. In 1995, this certification was extended to AS/NZS ISO 9001-1994. We are currently preparing for further accreditation to ISO/IEC Guide 25-1990, with additional supplementary requirements for medical testing. This paper describes the Quality System that the Department developed and which has been successfully maintained and extended since original certification. Important features of the Quality System are: 1. Primary design of the Quality System to meet medical and customer needs, with subsequent addition of required ISO elements. 2. Use of national Quality Award criteria to identify key business processes. 3. Development of integrated technical non-conformance, customer complaint, staff suggestion, and quality system corrective action procedures. 4. Implementation without external resources. Our conclusions are that ISO 9000 Quality Systems can be applied to medical testing laboratories, and can be implemented with minimum resource costs. Improvements in technical and service quality and business performance have resulted from this process. However, implementation of ISO 9000 at the level of individual Departments is not ideal. Greater improvements are possible when this process is undertaken at the level of the entire organisation. Received: 9 September 1996 Accepted: 5 October 1996