Determination of Glucose in Diluted Blood with a Thermal Flow Injection Analysis Biosensor

Abstract

The glucose concentration in diluted whole blood has been measured, using a miniaturized thermal biosensor based on the enzyme thermistor principle. The biosensor is a small flow injection system. A sample volume of 20μl is injected into a flow of 50μl/min. The heat produced when the sample passes the enzyme column is measured with a thermistor connected to a Wheatstone bridge. The enzyme column contains glucose oxidase and catalase co-immobilized on a solid support material. Samples of whole blood usually cause problems in flow-systems. The blood cells tend to block the enzyme column and the back pressure increases. We have tested a superporous agarose material as enzyme support material using tenfold diluted samples of whole human blood. The blood was collected from the finger-tip and diluted with buffer containing an anticoagulant and sodium fluoride. The number of samples possible to inject and the accuracy compared to the Boehringer Mannheim Reflolux have been determined. At least 100 ten-fold diluted blood samples could be injected on a micro-column of superporous agarose. The obtained glucose concentration correlated well with the one obtained with the reference instrument.     

Glucose Biosensor Based on Oxygen Electrode Part IV: In Vivo Evaluation of the Rechargeable Glucose Sensor

Abstract

A glucose monitoring system consisting of a pair of amperometric sensors: a glucose biosensor based on oxygen electrode and an oxygen sensor, two miniature potentiostats, an instrumentation amplifier and a data logger has been developed. The glucose sensor has linear response to the glucose concentration in vitro at 37°C up to 26 mM (480 mg/dL) in the phosphate buffer solution (pH 7.4), and linear range up to 21 mM (380 mg/dL) in undiluted bovine plasma. The system was evaluated in vivo with the sensors subcutaneously implanted in healthy mongrel dogs. During the implantation the system output was continuously recorded. The results of short-term subcutaneous implantation of the integrated system demonstrated good agreement between the glucose concentration measured by the biosensor and that obtained using standard glucose determination methods. The delay-time between the tissue glucose level (measured by the biosensor) and the blood glucose level (obtained by standard methodology) was 3 to 10 minutes. During the chronic implantation the biosensor was refilled in vivo. Rejuvenation of the sensor response after refilling was observed demonstrating the potential of such sensors for long-term implantation.     

Determination of Damaged Starch in Wheat Flour Using an Electrochemical Bienzyme Maltose Probe

Abstract

The development of an electrochemical biosensor based on a bienzyme maltose probe and a third enzyme α-amylase in solution is reported for the rapid and inexpensive determination of damaged starch. Analytical parameters, such as probe stability, pH, temperature and response time, were optimised. Damaged starch was measured in the range of 5 × 10^?6 - 5 × 10^?4 mol/L as maltose produced by the enzymatic reaction and the detection limit was calculated according with the free maltose and/or glucose in the sample. The damaged starch was determined in different wheat flours, and the data significantly correlated with those obtained using a reference procedure (r² = 0.994; P ≤ 0.0001). In addition the results showed a comparable precision (CV < 5%). This method is rapid, inexpensive and friendly for unskilled operators.     

Continuous Lactate Measurement that Combines a Portable Ultrafiltration Storage Device with an Enzyme Sensor

ABSTRACT|A simple portable continuous L-lactate monitoring system combining elegant ultrafiltration sample collection and storage with a lactate biosensor has been developed.|The sensor has a detection range from 0.05mM to 30mM lactate dependent on different assignments of the PCS hydrogel complex layer.|The sensor shows excellent performance on sensitivity and validity as well as operational stability over one month.|Samples containing different concentrations of lactate were collected in six hours with an ultrafiltration probe and stored in a 6m long PEEK tube of 125μm in internal diameter.|The ultraslow flow rate of 100 to 350nl/min was controlled by adjusting the restriction in the portable plastic syringe pump and a constant flow can be maintained over 24 hours.|The collected samples were then detected after reversing the flow with a lactate sensor.

The total weight of the collection system is only 8g, allowing free movement of the person being tested.     

Determination of kynurenic acid levels in rat brain microdialysis samples and changes in kynurenic acid levels induced by N‐acetylaspartic acid

The levels of kynurenic acid, an endogenous antagonist of α₇ nicotinic acetylcholine and N‐methyl‐D ‐aspartate receptors, were measured in microdialysis samples obtained from the prefrontal cortices of rats using column‐switching high‐performance liquid chromatography with fluorescence detection. When the perfusate was constantly infused at a rate of 1.0 μ/min, the in vitro recovery of kynurenic acid through the dialysis membrane was approximately 20.4%, and the precision was within 1.31%. Endogenous kynurenic acid in the microdialysis sample was clearly detected using column‐switching high‐performance liquid chromatography. As an application study, N‐acetyl‐L ‐aspartic acid, an endogenous metabolite and precursor of N‐acetyl‐L ‐aspartyl‐L ‐glutamic acid, which is an agonist of metabotropic glutamate receptors, was infused for 120 min through the microdialysis probe. The kynurenic acid level significantly increased during the infusion of N‐acetyl‐L ‐aspartic acid, suggesting that kynurenic acid might have some association with N‐acetyl‐L ‐aspartic acid in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Glucose Biosensor Based on Oxygen Electrode. Part II: Long-Term Operational Stability of the Rechargeable Glucose Biosensor

Abstract

A potentially implantable glucose biosensor for measuring blood or tissue glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and immobilized glucose oxidase enzyme, in which the immobilized enzyme can be replaced (the sensor recharged) without surgical removal of the sensor from the patient. Recharging of the sensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. A special technique for immobilization of the enzyme on Ultra-Low Temperature Isotropic (ULTI) carbon powder held in a liquid suspension has been developed.

In vitro studies of the sensors show stable performance during several recharge cycles over a period of 3 months of continuous operation.

Diffusion membranes which ensure linear dependence of the sensor response on glucose concentration have been developed. These membranes comprise silastic latex-rubber coatings over a microporous polycarbonate membrane. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations (up to 16 mM), covering hypoglycemic, normoglycemic and hyperglycemic conditions.

The experimental results confirm the suitability of the sensors for in vitro measurements in undiluted human sera.     

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.     

Amperometric Biosensor for Hypoxanthine Based on Immobilized Xanthine Oxidase on Iron (III) Meso‐tetraphenylporphyrin Nanoparticles Modified Glassy Carbon Electrode

Abstract

The preparation and performance of hypoxanthine (Hx) electrochemical biosensor, which was based on iron (III) meso‐tetraphenylporphyrin (FeTPP) nanoparticles (NPs), is reported in this work. FeTPP NPs prepared by mixing solvent techniques with diameters ca. 25~45 nm and were used as a mediator. The XOD/FeTPPNP/GC electrode exhibited good amperometric signal for Hx. Based on the consumption of dissolved oxygen during the oxidation process of Hx catalyzed by the immobilized XOD, the biosensor could detect the concentration of Hx up to 0.34 mM with a detection limit of 1.0 µM. The usefulness of this biosensor for the analysis of real sample was also demonstrated by determining Hx in rat brain dialysate coupled with microdialysis.     

A novel electrochemiluminescence glucose biosensor based on platinum nanoflowers/graphene oxide/glucose oxidase modified glassy carbon electrode

A novel electrochemiluminescence (ECL) biosensor based on platinum nanoflowers (PtNFs)/graphene oxide (GO)/glucose oxidase (GODx) was discovered for glucose detection. PtNFs/GO was synthesized using a nontoxic, rapid, one-pot and template-free method and characterized by transmission electron microscopy (TEM) and high-resolution TEM techniques. The as-prepared PtNFs/GO with clean surface and multiporous structure was used to assemble GODx to form a glucose biosensor. Based on ECL results, the PtNFs/GO/GODx film-modified electrode displayed a high electrocatalytic activity towards the oxidation of glucose, which generated hydrogen peroxide (H₂O₂) to react with the luminol radicals thus enhanced the luminol ECL. Under the optimized conditions, two linear regions of ECL intensity to glucose concentration were valid in the range from 5 to 80 μmol/L (r?=?0.9957) and 80 to 1,000 μmol/L (r?=?0.9909) with a detection limit (S/N?=?3) of 2.8 μmol/L. In order to verify the reliability, the thus-fabricated biosensor was applied to determine the glucose concentration in glucose injection, glucose functional drink, and blood serum. The results indicated that the proposed biosensor presented good characteristics in terms of high sensitivity and good reproducibility for glucose determination, promising the applicability of this sensor in practical analysis.     

Development of a Novel Silver Nanoparticles-Enhanced Screen-Printed Amperometric Glucose Biosensor

Abstract

A disposable glucose biosensor is developed by immobilizing glucose oxidase into silver nanoparticles-doped silica sol-gel and polyvinyl alcohol hybrid film on a Prussian blue-modified screen-printed electrode. The silver nanoparticles-enhanced biosensor shows a linear amperometric response to glucose from 1.25 × 10^?5 to 2.56 × 10^?3 with a sensitivity of 20.09 mA M^?1 cm^?2, which is almost double that of the biosensors without silver nanoparticles. The immobilized glucose oxidase retained 91% of its original activity after 30 days of storage in phosphate buffer (pH 6.9; 0.1 M) at 4°C. Blood glucose in a rabbit serum sample was successfully measured with the biosensor.     

Rapid Preconcentration for Liquid Chromatography–Mass Spectrometry Assay of Trace Level Neuropeptides

Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MSⁿ) has proven to be effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75 μm inner diameter (i.d.) capillary column packed with 10 μm reversed phase particles for increasing the throughput in CLC-MSⁿ based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column has reduced analysis time from ～30 min to 3.8 min for 5 μL sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A_1-8 in 5 μL sample. The use of isotope-labeled internal standard lowered peptide signal variation to less than 5 %. This method was validated for in vivo detection of Leu and Met enkephalin with microdialysate collected from rat globus pallidus. The improvement in speed and stability makes CLC-MSⁿ measurement of neuropeptides in vivo more practical.

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.     

A Novel Potentiometric Glucose Biosensor Based on Polyaniline Semiconductor Films

Abstract

Application of polyaniline semiconductor films to potentiometric biosensor development provides certain advantages comparing with the known systems. Using self-doped polyaniline instead of common polymer as pH transducer the stable potentiometric response of 70 mV/pH was obtained. Taking as an example glucose biosensor we showed that polyaniline based electrode possessed three-four fold increased potential shift than glucose-sensitive field-effect transistor did. One can increase the sensitivity of potentiometric biosensor using thick ion-exchange membranes (in our case Nafion) in order to concentrate product near electrode surface. Such sensor possessed higher response time.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

Trends of flow injection sample pretreatment approaching the new millennium

The important role of flow injection (FI) techniques for the automation, acceleration and miniaturization of solution handling in sample pretreatment as well as some recent trends in the development of the field are discussed, illustrated mainly by recent achievements in the author’s laboratory, including: (a) sample pretreatment for vapor generation and electrothermal AAS based on sequential injection (SI) techniques, with low reagent consumption and enhanced ruggedness; (b) combination of FI and SI sample pretreatment (filtration, dialysis, gas diffusion, column sorption) with capillary electrophoresis (CE) giving enhanced reproducibility and efficiency; (c) application of on-line microdialysis in in vivo monitoring of blood glucose in test animals and (d) application of on-line microdialysis and solvent extraction in continuous monitoring of drug dissolution processes with high resolution of process events. Future perspectives of FI sample pretreatment are discussed, emphasizing the improvement in ruggedness of the equipment and methods, the combination and synchronization of different means for liquid propulsion, and the development of miniaturized systems.     

Continuous Amperometric Determination of Glucose Using an Immobilized Enzyme Reactor in Combination with an Immersible Dialysis Probe

Abstract

Glucose was continuously determined by reaction in a packed-bed enzyme reactor containing glucose oxidase and catalase. Oxygen consumption was measured amperometrically with a polarographic Clark electrode. Glucose was sampled through a dialysis probe immersed in the solution to be measured. An extension of the normal range for the enzyme was achieved by modulating the flow rate through the dialysis probe and a linear response was obtained in the range of 1.0-60 mM glucose. The correlation between the glucose transfer and the membrane area of the dialysis probe was also studied. Six different membranes were used, all showing variations in the adhesion of yeast cells.     

Application to Gel Termination Chromatography of Micro High Performance Liquid Chromatography

Abstract

The raicroscale liquid chromatographic tecnnique was applied to gel perraination chromatography. As the results, the miniaturized GPC column requires smaller amounts of sample, packing materials and carrier solvent than those required with the ordinary GPC column, and it would be applicable to preparatory experiments to select the operation conditions for fractionation by GPC.     

基于芬顿反应和硫磺素T构建免标记荧光传感器用于葡萄糖的检测

基于芬顿反应和硫磺素T(ThT)构建新奇的免标记荧光传感器用于葡萄糖的检测。当无葡萄糖存在时,ThT诱导富G-DNA探针形成G-四链体/ThT复合物,ThT的荧光强度显著增强;当葡萄糖存在时,葡萄糖氧化酶催化葡萄糖产生H2 O2,在Fe^2+催化的芬顿反应作用下,H2 O2转化为羟基自由基(·OH),·OH引发DNA的氧化损伤导致富G-DNA探针裂解为短寡核苷酸片段而丧失形成G-四链体/ThT的能力,ThT的荧光强度显著降低,从而实现对葡萄糖的检测。在优化的检测条件下,G-四链体/ThT荧光强度变化和葡萄糖浓度在0.5~45μmol/L的范围内呈现较好的线性关系(R^2=0.99268),检出限为0.1μmol/L。利用本法对葡萄糖加标的血液样品进行分析,葡萄糖的回收率为90.7%~118.3%,相对标准偏差为1.7%~5.8%,方法可用于血糖检测。     

Integrated Microcentrifuge Carbon Entrapped Glucose Oxidase Poly (N-Isopropylacrylamide) (pNIPAm) Microgels for Glucose Amperometric Detection

This study demonstrates a miniaturized integrated glucose biosensor based on a carbon microbeads entrapped by glucose oxidase (GOx) immobilized on poly (N-isopropylacrylamide) (pNIPAm) microgels. Determined by the Lowry protein assay, the pNIPAm microgel possesses a high enzyme loading capacity of 31?mg/g. The pNIPAm GOx loaded on the microgel was found to maintain a high activity of approximately 0.140?U determined using the 4-aminoantipyrine colorimetric method. The integrated microelectrochemical cell was constructed using a microcentrifuge vial housing packed with (1:1, w/w) carbon entrapped by pNIPAm GOx microgels, which played the dual role of the microbioreactor and the working electrode. The microcentrifuge vial cover was used as a miniaturized reference electrode and an auxiliary electrode holder. The device can work as biosensor, effectively converting glucose to H₂O₂, with subsequent amperometric detection at an applied potential of ?0.4?V. The microelectrochemical biosensor was used to detect glucose in wide linear range from 30?µM to 8.0?mM, a low detection limit of 10?µM, a good linear regression coefficient (R²) of 0.994, and a calibration sensitivity of 0.0388?µA/mM. The surface coverage of active GOx, electron transfer rate constant (ks), and Michaelis–Menten constant (K_M^app) of the immobilized GOx were 4.0?×?10^?11?mol/cm², 5.4?s^?1, and 0.086?mM, respectively. To demonstrate the applicability and robustness of the biosensor for analysis of high sample matrix environment, glucose was analyzed in root beer. The microelectrochemical device was demonstrated for analysis of small sample (<50?µL), while affording high precision and fast signal measurement (≤5?s).