A Microdialysis Probe Coupled with A Miniaturized Thermal Glucose Sensor for In Vivo Monitoring

Abstract

A miniaturized thermal flow injection analysis biosensor has been coupled with a microdialysis probe for continuous subcutaneous glucose monitoring. Thermal biosensors are based on the principle of measuring the heat evolved during enzyme catalysed reactions. The system presented here consists of a miniaturized thermal biosensor with a small column containing coimmibolized glucose oxidase and catalase. The analysis buffer passes through the column at a flow rate of 60μL/min via an 1μL sample loop which is connected to a microdialysis probe.

Invitro results showed constant permeability of the probe and stability of the biosensor response during 24 hours. The response time was 85 sec giving a sample rate of 42 samples/hour.

During a load experiment, the glucose profile in a healthy volunteer was followed both in the subcutaneous tissue and blood using the microdialysis set-up proposed and comparing to blood glucose analyser.     

A Split-Flow Enzyme Thermistor

Abstract

The split-flow system is comprised of two identical micro-columns, one of which contains an immobilized enzyme preparation, the other an inert support material.

The heat produced in each column on introduction of a sample is measured with thermistors placed in these columns. The use of a reference column virtually eliminates the influence on the measurements of artifactual signals as unspecific heat, i.e., heat not produced by the enzymic reaction. The performance of the split-flow enzyme thermistor at a variety of pH's, ionic strengths or viscosities associated with the sample has been investigated and compared with previously described alternative enzyme thermistor arrangements. In this comparative study glucose at a concentration of 5 · 10^?4 M was used throughout. On passage through the imnobilized glucose oxidase preparation this solution gave rise to a heat change At of about 0.01°C. The insensitivity of the system described herein towards such variations makes it particularly suitable for the analysis of metabolities present in crude solutions such as urine and skim-milk.     

Improved Blood Compatibility at a Glucose Enzyme Electrode Used for Ejctraoorpokeal Monitoring

Abstract

A glucose enzyme electrode based on glucose oxidase has been used in an extracorporeal flow system which allows standardization during monitoring. The detection limit for glucose was 0.02 mmol/l, and the linear range, which extended to 5 mmol/l glucose, allowed measurement of whole blood after dilution. Restandardization following whole blood exposure led to a loss of sensitivity which was attributed to coating of electrode membranes by blood constituents. The problem was largely overcome through use of silane-treated membranes and the operation of electrodes in a narrow channel flow cell.     

Determination by the enzyme thermistor of cellobiose formed on degradation of cellulose

A calorimetric assay procedure for the determination of cellobiose has been developed. The cellobiose is hydrolyzed by β-glucosidase and the glucose formed is measured calorimetrically by an enzyme thermistor containing co-immobilized glucose oxidase and catalase. The system was optimized with regard to the arrangement of the enzymes, the pH-dependence of the separate enzymic steps, and of the total system. By placing the β-glucosidase in a precolumn that could be switched in and out of the flow through the enzyme thermistor, both cellobiose and glucose present in the sample could be determined. The performance with standard solutions and with crude samples from cellulose degradation experiments was investigated.     

Glucose Biosensor Based on Oxygen Electrode Part III: Long-Term Performance of the Glucose Biosensor in Blood Plasma at Body Temperature

Abstract

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and Glucose Oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Glucose diffusion membranes from polycarbonate (PC) and from polytetrafluorethylene (PTFE) coated with silastic are used.

Sensors were evaluated continuously operating in phosphate buffer solution and in undiluted blood plasma at body temperature. Calibration curves of the sensors were periodically obtained. The sensors show stable performance during at least 1200 hours of operation without refilling of the enzyme. The PTFE membrane demonstrates high mechanical stability and is little effected by long-term operation in undiluted blood plasma.     

Direct Determination of Glucose in Blood Serum Using Trinder's Reaction

Abstract

A very simple flow injection analysis system for direct determination of glucose in blood serum based on Trinder's reaction is described. The sera samples (15 μl) can be injected directly to the system without the deproteinization or the use of a dialyzer.

Calibration curves are linear in the range 50–400 mg/dl. The sampling frequency is 60 samples per hour. Results obtained by the proposed procedure are compared with those acquired at a local hospital using their routine glucose procedure also based on Trinder's reaction. It is shown that a better mix between sample and reagents is achieved using the single bead string reactor (SBSR).     

Measurement of Glucose Concentration in Blood Plasma Based on a Wireless Magnetoelastic Biosensor

Abstract

A wireless magnetoelastic glucose biosensor in blood plasma is described, based on using a mass sensitive magnetoelastic sensor as transducer. The glucose biosensor was fabricated by coating the ribbon‐like, magnetoelastic sensor with a pH sensitive polymer and a biolayer of glucose oxidase (GOx) and catalase. The pH response polymer swells or shrinks, thereby changing sensor mass loading, respectively, in response to increase or decrease of pH values. The GOx–catalyzed oxidation of the glucose in blood plasma produces gluconic acid, resulting in the pH sensitive polymer shrinking, which in turn decreases the sensor mass loading. The results show that the proposed magnetoelastic glucose biosensor can be successfully applied to determine the concentration of glucose in blood plasma. At glucose concentration range of 2.5–20.0 mmol/l, the biosensor responses are reversible and linear, with a detection limit of 1.2 mmol/l. Since no physical connections between the sensor and the monitoring instruments are required, this proposed biosensor can potentially be applied to in vivo and in situ measurement of glucose concentration in physiological fluids.     

Determination of Urinary Glucose by a Flow Injection Analysis Amperometric Biosensor and Ion-Exchange Chromatography

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 ±2.4 RU μM^-1 (RU or relative unit is defined as 2.86 μV at the detection output) for glucose with a minimum detection level of 10 μM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25–30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.     

The enzyme thermistor—A realistic biosensor concept. A critical review

This review describes principles and features of thermal biosensors and microbiosensors in flow injection analysis. Examples are given that illustrate the great versatility and excellent operational stability offered by thermal biosensors. The examples are mostly from work with the original type of enzyme thermistor operating with an enzyme column, but there will also be work described involving miniaturised devices including thermal lab-on-chip constructions and other types of sensing materials, such as MIPs (molecularly imprinted polymers) for both affinity and catalytic reactions. Several recently presented thermal biosensor concepts are reviewed including a thermal–electrochemical hybrid sensor for lactose based on immobilised cellobiose dehydrogenase. Another recent method is the determination of fructose using a fructose-6-phosphate kinase column. Operation with complex sample matrices such as blood, plasma and milk and how to avoid non-specific temperature effects are considered.     

Determination of Serum Urea with an Enzyme Thermistor Using Immobilized Urease

Abstract

The application of an enzyme thermistor device in a simple and accurate procedure for the determination of serum urea is described. The enzyme thermistor measures the heat produced when urea is passed through a small column containing immobilized urease. The stability and sensitivity as well as the performance with clinical serum samples of the system is evaluated. Advantages are the simplicity, the low enzyme cost and the insensitivity to the optical properties of the sample and interfering substances, which may affect the commonly used assay procedures.     

Development of an amperometric biosensor based on acetylcholine esterase covalently bound to a new support material

A new type of amperometric biosensor based on immobilised acetylcholine esterase was designed and constructed. The enzyme was immobilised on a flow-through working electrode, which was prepared from reticulated vitreous carbon (RVC) or from a composite material consisting of RVC and superporous agarose. The sensor was operated in FIA mode using acetylthiocholine as a substrate. The sensor responded to inhibitors such as paraoxon-10(-9) mol was detected by the sensor in a non-optimised configuration. The practical lifetime of the sensor was at least 1 month.     

Fabrication of superporous agarose beads for protein adsorption: Effect of CaCO3 granules content

Agarose gels were fabricated by water-in-oil emulsification with the addition of CaCO₃ granules at 8–16 wt%. Thus agarose beads of different superporosities were produced after dissolving the solid porogen. The superporous agarose (SA) and homogeneous agarose gels were double cross-linked and modified with diethylaminoethyl chloride to produce anion exchangers. We have proposed to use a superporous replica (porous titania microspheres) to examine the superporous structure and pore size distribution of the soft gel. The replica was prepared with the agarose gel entrapping CaCO₃ granules by a sol–gel-templating method. It was found that the superpores created by CaCO₃ granules were uniformly distributed and ranged from 0.95 μm to 1.33 μm. The physical properties of the gels were significantly affected by the porogen content. Importantly, by increasing the solid porogen to 12 wt%, the bed permeability and effective porosity increased about 48% and 33%, respectively. Further increase in the porogen to 16 wt% led to a decrease of the mechanical strength. With increasing superpores in the beads, the dynamic adsorption capacity of the packed columns increased obviously at 305–916 cm/h. Besides, the column efficiency changed less with increasing flow velocity up to 1200 cm/h. It was concluded that the use of 12 wt% CaCO₃ granules in agarose solution was beneficial for the fabrication of the SA gel with good mechanical stability and promising performance for protein chromatography.     

壳聚糖固定化葡萄糖氧化酶生物传感器测定葡萄糖的含量

应用壳聚糖将葡萄糖氧化酶固定于鸡蛋膜上,结合氧电极制得葡萄糖传感器.实验表明,壳聚糖比戊二醛能更好地固定葡萄糖氧化酶,最佳条件为壳聚糖浓度0.3%、固定化酶量0.8 mg、 pH 7.0、缓冲溶液浓度300 mmol/L和温度25 ℃.本葡萄糖传感器的线性范围为0.016～1.10 mmol/L;检出限为8.0 μmol/L(S/N=3), 响应时间<60 s,有很好的稳定性,寿命>3个月.同一个传感器重复使用以及同方法制作的不同传感器之间都有很好的重现性,RSD分别为2.5%(n=10)和4.7%(n=4).实际样品中可能存在的烟酰胺、 VB6、 VB12、 VE、Ca2+、 Mg2+、 K+和Zn2+等对葡萄糖的测定不产生干扰.本传感器已成功地应用于市售饮料中葡萄糖含量的测定.     

Superporous agarose beads as a hydrophobic interaction chromatography support

Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231–240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106–180 μm) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927).     

Automated direct determination of chromium in blood and urine by electrothermal atomic absorption spectrometry

A simple direct procedure for the determination of chromium in whole blood and urine by graphite-furnace atomic absorption spectrometry is described. Whole blood samples are diluted with 0.1% Triton-X solution before injection, whereas urine samples are injected directly. Calibration is done by direct comparison against matrix-matched standards. Between-run precision is 5.4% at 154 nmol l^?1 for urine and 3.6% at 142 nmol l^?1 for blood. The detection limits are 3.8 nmol l^?1 for urine and 11.5 nmol l^?1 for blood, each for a 20μl sample. The calibration range extends up to 770 nmol l^?1 for both blood and urine. This allows the determination of chromium in both occupationally exposed and unexposed groups. The graphite-furnace conditions for each matrix are similar. Elimination fo sample pretreatment minimizes the risk of contamination and allows a rapid sample throughput of 50–60 samples per day. The methods described are particularly suited for the screening and surveying of populations occupationally exposed to chromium.     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.     

Prussian blue modified glassy carbon electrodes-study on operational stability and its application as a sucrose biosensor

Stabilisation of electrochemically deposited Prussian blue (PB) films on glassy carbon (GC) electrodes has been investigated and an enhancement in the stability of the PB films is reported if the electrodes are treated with tetrabutylammonium toluene-4-sulfonate (TTS) in the electrochemical activation step following the electrodeposition. A multi-enzyme PB based biosensor for sucrose detection was made in order to demonstrate that PB films can be coupled with an oxidase system. A tri-enzyme system, comprising glucose oxidase, mutarotase and invertase, was crosslinked with glutaraldehyde and bovine albumin serum on the PB modified glassy carbon electrode. The deposited PB operated as an electrocatalyst for electrochemical reduction of hydrogen peroxide, the final product of the enzyme reaction sequence. The electrochemical response was studied using flow injection analysis for the determination of sucrose, glucose and H₂O₂. The optimal concentrations of the immobilisation mixture was standardised as 8 U of glucose oxidase, 8 U of mutarotase, 16 U of invertase, 0.5% glutaraldehyde (0.025 μl) and 0.5% BSA (0.025 mg) in a final volume of 5 μl applied at the electrode surface (0.066 cm²). The biosensor exhibited a linear response for sucrose (4-800 μM), glucose (2-800 μM) and H₂O₂ (1-800 μM) and the detection limit was 4.5, 1.5 and 0.5 μM for sucrose, glucose and H₂O₂, respectively. The sample throughput was ca. 60 samples h⁻¹. An increase in the operational and storage stability of the sucrose biosensor was also noted when the PB modified electrodes were conditioned in phosphate buffer containing 0.05 M TTS during the preparation of the PB films.     

Activation‐Based Catalase Enzyme Electrode and its Usage for Glucose Determination

Abstract

In this study, a novel type amperometric biosensor, which is based on the activation of catalase enzyme by glucose, was developed and used for the sensitive determination of glucose. For the preparation of the biosensor catalase enzyme was immobilized in gelatin by using cross‐linking agent glutaraldehyde and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Glucose was used as an activator for the catalase enzyme and determination of glucose is based on the assay of the differences on the catalase activity of the biosensor on the oxygenmeter in the absence and the presence of glucose in the reaction medium. The responses of the activation based catalase biosensor have a linear relation to glucose concentrations and good measurement correlation between 0.5 and 5.0 µM with 2 min response time. In the optimization studies of the biosensor the most suitable catalase amount were found as 1324 U cm^?2 and also phosphate buffer (pH: 7.0; 50 mM) and 35°C were obtained as the optimum working conditions. For the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response, reproducibility and operational stability were performed.     

A blood-assisted optical biosensor for automatic glucose determination

A new approach for glucose determination in blood based on the spectroscopic properties of blood hemoglobin (Hb) is presented. The biosensor consists of a glucose oxidase (GOx) entrapped polyacrylamide (PAA) film placed in a flow cell. Blood is simply diluted with bidistilled water (150:1, v:v) and injected into the carrier solution. When reaching the PAA film, the blood glucose reacts with the GOx and the resulting H₂O₂ reacts with the blood Hb. This produces an absorbance change in this compound. The GOx-PAA film can be used at least 100 times. Lateral reactions of H₂O₂ with other blood constituents are easily blocked (by azide addition). The linear response range can be fitted between 20 and 1200 mg dL⁻¹ glucose (R.S.D. 4%, 77 mg dL⁻¹). In addition to the use of untreated blood, two important analytical aspects of the method are: (1) the analyte concentration can be obtained by an absolute calibration method; and (2) the signal is not dependent on the oxygen concentration.A mathematical model relating the Hb absorbance variation during the reaction with the glucose concentration has been developed to provide theoretical support and to predict its application to other compounds after changing the GOx by another enzyme. The method has been applied to direct glucose determination in 10 blood samples, and a correlation coefficient higher than 0.98 was obtained after comparing the results with those determined by an automatic analyzer. As well as sharing some of the advantages of disposable amperometric biosensors, the most significant feature of this approach is its reversibility.     

Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimmed milk. Four aminoglycosides (gentamicin, neomycine, kanamycin and a streptomycin derivative) were immobilised onto the sensor surface of a biosensor chip (CM5) in the four Fcs of the biosensor system by amine coupling. In the Biacore, milk (reconstituted from skimmed milk powder) was 10 times diluted with a mixture of the four specific antibodies and injected through the four serially connected Fcs (1 min at a flow rate of 20 μl min⁻¹). The responses measured just prior to the injection (20 μl at a flow rate of 20 μl min⁻¹) of the regeneration solution (0.2 M NaOH + 20% acetonitril) were indicative for the presence or absence of the aminoglycosides in reconstituted milk. The limits of detection were between 15 and 60 ng ml⁻¹, which was far below the maximum residue limits (MRLs) (varying from 100 to 500 ng ml⁻¹) and the total run time between samples was 7 min.