A Double-Needle Gold-Silver Electrodes Continuous Glucose Monitoring Device

ObjectivesDiabetes is a serious, long-term disease and the use of continuous glucose monitoring sensors can reduce reliance on other painful invasive blood testing methods such as the finger blood glucose test. According to our work, a low-cost continuous glucose sensor has been developed based on electrochemical measurement techniques.MaterialsThe sensor is based on a two needles system; a gold and a silver electrode are integrated into a circular shaped electronic printed circuit board (PCB). The sensing part is based on biological electrochemical measurements. Glucose oxidase (Gox) was used as the active sensing element and ferrocene (Fc) as a mediator. Simple and low-cost coating methods were used; these methods are self-assembled monolayers and deep coating. This will reduce the final cost of the sensor as no expensive technique was used. The electrical subsystem contains a low-noise and low-power trans-impedance front-end as well as a single-chip low-power Bluetooth microcontroller with a 12-bit Analog-to-Digital Converter (ADC).ResultsThe sensor was tested in various concentrations of glucose. As a result of initial in vitro experiments, detailed analytical performance metrics are presented. The device has consistently shown a sensitivity of 3.059 mV/(mg/dl) reading with a linear range of 0-400 mg/dl.ConclusionThe proposed study shows promising results for glucose detection. Thus, this type of sensor can be used for different analyzes targeting biological applications after further investigations and analysis.     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

Enzyme immunosensors based on electropolymerized polytyramine modified electrodes

Highly sensitive amperometric enzyme immunosensors for human immunoglobulin G (IgG) were prepared on the basis of electrogenerated polytyramine (PTy, tyramine = p-(2-aminoethyl)-phenol) modified electrodes. Properties of PTy films changed depending on electrolysis conditions. On the basis of the found properties of the films, an effective IgG sensor was prepared: a PTy film was formed first from an acid solution on a Pt electrode, and the surface was further covered with a PTy film from an alkaline methanol solution to give a PTy doubly coated electrode on which anti-IgG was then immobilized. This electrode provided a large surface area with little non-specific adsorption of proteins. By means of the competitive enzyme immunoassay technique using glucose oxidase (GOD) labeled IgG conjugates, IgG was determined in the concentration range of c. 10 pg/ml-1 mg/ml from the oxidation current of H2O2 generated by the enzyme (GOD) reaction using the above IgG sensor. Also, an anti-IgG immobilized electrode, prepared by using a Pt electrode singly covered with a PTy film from an alkaline methanol solution, acted as an effective IgG sensor with a detection limit for IgG of c. 100 pg/ml.     

Effect of obesity on erythrocyte count and hemoglobin levels in Libyan diabetic patients

Fasting blood glucose, erythrocyte count and hemoglobin levels of obese and nonobese Libyan diabetic women were determined. The mean values of fasting blood glucose, erythrocyte count and hemoglobin of obese diabetic women were 209.55 +/- 8.85 mg/dl, 4.986 +/- 0.04 X 10(6)/mm3 and 14.51 +/- 0.18 g/dl. The respective values for nonobese diabetic women were 243.47 +/- 12.56 mg/dl, 4.865 +/- 0.06 X 10(6)/mm3 and 14.31 +/- 0.19 g/dl. The mean values of the three variables of obese patients were significantly different from those of the nonobese patients. Statistically significant correlations were found between fasting blood glucose levels and erythrocyte count, and hemoglobin levels in both obese and nonobese patients. The levels of erythrocyte count and hemoglobin of obese patients were higher than those of their nonobese counterparts. This elevation was attributed to the effect of obesity. It is suggested that regulation of body weight should be considered an essential step in the management of diabetes.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

Development of biosensor based on immobilized L-glutamate oxidase for determination of monosodium glutamate in food

A monosodium glutamate (MSG) biosensor with immobilized L-glutamate oxidase (L-GLOD) has been developed and studied for analysis of MSG in sauces, soup etc. The immobilized enzymatic membrane was attached with oxygen electrode with a push cap system. The detection limit of the sensor was 1 mg/dl and the standard curve was found to be linear upto 20 mg/dl. Response time of the sensor was 2 min. Cross-linking with glutaraldehyde in presence of Bovine Serum Albumin (BSA) as a spacer molecule has been used for immobilization. Optimization of the sensor was done with an increase in L-GLOD concentration (6.3-31.5 IU) and also with increase in loading volume of enzyme solution (5-20 microl). Optimization of pH and temperature was also studied. The permeability of O2 through different membrane was studied with and without immobilized L-GLOD. The enzymatic membrane was used for over 20 measurements and stability of the membrane was observed.     

Rechargeable glucose electrodes for long-term implantation

A unique enzyme electrode was designed using glucose oxidase immobilized on fine graphite powder. The graphite-enzyme is in a fluid state which enables recharging of the system when the enzyme activity decreases, therefore allowing the system to have a long lifetime in a diabetic patient. The sensor was tested using glucose concentration in the range of 20–300 mg d⁻¹. The electrode construction includes a hydrophobic membrane and a platinum electrode for detection of hydrogen peroxide. The signal output is large with minimal noise when tested in buffer. The sensor has been performing at ambient temperature for 4 months when stored overnight at 4° C.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

Highly selective amperometric glucose microdevice derived from diffusion layer gap electrode

Although most of enzyme catalytic reactions are specific, the amperometric detection of the enzymatic reaction products is largely nonselective. How to improve the detection selectivity of the enzyme-based electrochemical biosensors has to be considered in the sensor fabrication procedures. Herein, a highly selective amperometric glucose biosensor based on the concept of diffusion layer gap electrode pair which we previously proposed was designed. In this biosensor, a gold tube coated with a conductive layer of glucose oxidase/Nafion/graphite was used to create an interference-free region in its diffusion layer by electrochemically oxidizing the interfering electroactive species at proper potentials. A Pt probe electrode was located in this diffusion layer of the tube electrode to selectively detect hydrogen peroxide generated from the enzyme catalytic oxidation of glucose in the presence of oxygen in the solution. In practical performance of the microdevice, parameters influencing the interference-removing efficiency, including the tip-tube opening distance, the tube electrode potential and the electrolyzing time had been investigated systematically. Results showed that glucose detection free from interferents could be achieved at the electrolyzing time of 30s, the tip-tube opening distance of 3mm and the tube electrode potential of 0.4V. The electrochemical response showed linear dependence on the concentration of glucose in the range of 1 x 10(-5) to 4 x 10(-3) M (the correlation coefficient: 0.9936, without interferents; 0.9995, with interferents). In addition, the effectiveness of this device was confirmed by numerical simulation using a model system of a solution containing interferents. The simulated results showed good agreement with the experimental data.     

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.     

Insulin glargine maintains equivalent glycemic control and better lipometabolic control than NPH insulin in type 1 diabetes patients who missed a meal.

Our goal was to investigate blood glucose and lipometabolism control in type 1 diabetes patients who missed breakfast and the accompanying insulin injection of NPH insulin (NPH) or insulin glargine (glargine) as part of a basal-bolus regimen. This was a multi-center, open-label, controlled study in adults (> or =18 years) with HbA (1c)< or =11.5% on insulin therapy with NPH as basal insulin. Patients were randomized to receive prandial insulin plus either bedtime glargine (n=28) or NPH (n=32). Insulin was titrated to target fasting blood glucose levels 80-130 mg/dl at 06:00-07:00. Patients had no intake of insulin or food between 22:00 and 12:00 the next day. The change in blood glucose levels (07:00-11:00) was similar (27.5 mg/dl vs. 35.4 mg/dl), but the mean blood glucose level was higher with glargine vs. NPH at 22:00 (158.2 mg/dl vs. 130.2 mg/dl). During the period without insulin or food intake, blood glucose decreased with glargine (-25.8 mg/dl) and increased with NPH (+9.1 mg/dl; p=0.0284). Nonesterified fatty acid (07:00 and 09:00-12:00) and beta-hydroxybutyrate (07:00 and 10:00-12:00) levels were lower with glargine vs. NPH (both p<0.05). For patients who miss a morning meal, glargine is associated with maintained glycemic and lipometabolic control compared with NPH insulin.     

Decreased tissue guanylate cyclase activity in glycosuric Djungarian hamsters (Phodopus sungorus) that is correctable with insulin

Twelve hyperglycemic, glycosuric, and ketonuric Djungarian hamsters with average blood glucose concentrations of 295+-32 mg/dl were compared to twelve non-glycosuric, but ketonuric Djungarian hamsters with average blood glucose concentrations of 88+-11 mg/dl with regards to their cyclic nucleotide metabolism. The glycosuric Djungarian hamsters had decreased guanylate cyclase (E.C.4.6.1.2.) activity in vitro and cyclic GMP levels in vivo in liver, lung, kidney, colon, heart, spleen, and pancreas that was approximately 50% of the guanylate cyclase activity in these same tissues of non-glycosuric Djungarian hamsters. The decreased tissue guanylate cyclase activity and cyclic GMP levels in the glycosuric animals could be restored to the level of non-glycosuric Djungarian hamsters with 100 U regular insulin, but not with 50 or 10 U of regular insulin. Fifty and 100 U of regular insulin also increased the level of guanylate cyclase activity in the non-glycosuric (control) animals. There was no change in adenylate cyclase (E.C.4.6.1.1.) activity but there were increased cyclic AMP levels in the glycosuric when compared to the non-glycosuric Djungarian hamsters that were correctable with 100 U of insulin. We conclude that guanylate cyclase activity is decreased in the peripheral tissues of glycosuric Djungarian hamsters as compared to non-glycosuric Djungarian hamsters and that insulin modulates this enzyme.     

骨髓间充质细胞联合PDMS支架构建移植胰岛微环境的实验研究

目的为了提高移植胰岛的活性和功能,构建适合移植胰岛生存的微环境。 方法采用聚二甲基硅氧烷（PDMS）和氯化钠晶体构建三维支架,联合骨髓间充质细胞（MSCs）、纤维蛋白和胰岛共同构建迷你"人工胰腺"。采用链脲佐菌素（STZ）诱导的糖尿病大鼠移植模型评价效果,将"人工胰腺"移植到糖尿病大鼠大网膜内,对照组行假手术,术后隔天监测移植大鼠血糖水平;数据采用t检验和曼-惠特尼U检验。 结果用PDMS构建的三维巨孔支架,支架内可见大量不规则孔洞空间。胰岛和MSCs可成功装载入支架内,HE染色结果显示,支架孔内存在胰岛,胰岛周围包绕有MSCs。糖尿病大鼠大网膜内移植结果显示,移植后各时间点（1,3,5,7 d）,"人工胰腺"移植组糖尿病大鼠血糖水平分别为（278.70±86.06）mg/ dl、（323.50±44.29）mg/ dl、（283.30±74.00）mg/dl、（304.80±13.33）mg/dl,较假手术对照组（606.00±52.40）mg/dl、（589.70±55.78）mg/dl、（615.00±54.84）mg/dl、（630.30±48.17）mg/ dl均降低,差异具有统计学意义（t = 7.96、9.15、8.82,U = 0.00,P均< 0.01）。 结论MSCs联合PDMS三维支架构建的微环境,可为移植胰岛提供生存的环境,为临床开展胰岛移植提供新的策略。     

Nutrient and waste product concentration differences in upper and lower body arteries of fetal sheep

Well oxygenated blood returning from the placenta is preferentially shunted into the left side of the fetal heart and the ascending aorta. This results in higher oxygen saturation in arterial blood supplying the fetal upper body than in blood supplying the lower body. Since the placenta is also the site of nutrient and waste exchange, we evaluated differences in arterial concentrations of nutrients and waste products in fetal upper and lower body. Studies were carried out on ten, chronically catheterized, third trimester, fetal sheep. Blood samples, drawn simultaneously from the carotid and femoral arteries, were analyzed for glucose, oxygen saturation, oxygen content, total amino acids, lactate, urea nitrogen, and hydrogen ion concentration. Carotid arterial blood had higher levels of glucose (1.4 +/- 0.1 mg/dl (SEM); P less than 0.001), of alpha-amino nitrogen (0.4 +/- 0.1 mg/dl, equivalent to amino acid concentration difference of 2.5 mg/dl, P less than 0.025), of oxygen saturation (9.9 +/- 0.5%, P less than 0.001), and of oxygen content (1.0 +/- 0.1 ml/dl; P less than 0.001). Carotid values exceeded femoral by an average of 10% for glucose, 4% for amino nitrogen, 29% for oxygen saturation and 23% for oxygen content. Carotid arterial blood had lower urea nitrogen, (-0.5 +/- 0.2 mg/dl; P less than 0.05) and hydrogen ion (-1.1 +/- 0.1 nM/L; P less than 0.001) concentrations, but these differences averaged only 2% between vessels. Lactate concentration in the carotid and femoral arteries was the same. Fetal glucose and oxygen levels were closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-diabetic effect of ginsenoside Re in ob/ob mice

We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188+/-9.2 and 180+/-10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229+/-9.5 and 235+/-13.4 mg/dl, respectively). The EC(70) of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198+/-13.1 with ginsenoside treatment vs. 253+/-20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.     

Relationship between the severity of experimental diabetes and altered lung phospholipid metabolism

Glucose intolerance was induced in rats by iv infusion of streptozotocin (STZ) in doses of 30, 40, 50, and 100 mg/kg. Serum glucose concentrations were elevated versus controls and weight gains were reduced in a dose-dependent fashion up to 50 mg/kg. Urine outputs and blood urea nitrogen (BUN) values were higher than control values in the animals treated with 40 and 50 mg/kg and serum albumin concentrations were decreased after infusion with 50 mg STZ/kg. Lung phosphatidylcholine (PC) concentrations and dry-to-wet weight ratios were unchanged by STZ treatment, while lung protein and disaturated phosphatidylcholine (DSPC) concentrations were depressed in the 50-mg/kg group. Animals surviving treatment with 100 mg/kg demonstrated increased fasting blood glucose levels, BUN values, and 48-hr urine outputs, and decreased lung protein levels. However, these alterations were less than those found in the 50-mg/kg animals. Pulmonary concentrations of PC, DSPC, and lung dry-to-wet weight ratios were unchanged. It was found advantageous to express the results relative to fasting blood glucose levels. This demonstrated that urine output and BUN values increased and weight gain decreased with rising glucose concentrations, but serum albumin decreased only in moderate and severe hyperglycemia. Fasting glucose concentrations greater than 400 mg/dl were associated with reduced lung DSPC and protein levels, while pulmonary PC and dry-to-wet weight ratios demonstrated no change with increasing hyperglycemia.     

Influence of sampling conditions on blood chemistry values of Adriatic sturgeon Acipenser naccarii (Bonaparte, 1836)

Data on the blood chemistry of a chondrostean fish, the Adriatic sturgeon ( Acipenser naccarii ), are reported as measured with different sampling procedures, and as related to rearing conditions and age. Serum Cortisol, glucose, osmolality, Na⁺, Cl^-, Ca²⁺ and total protein concentrations were measured. Reference values for the blood chemistry of farmed sturgeon were measured on samples from resting undisturbed animals collected via a chronic indwelling catheter in the dorsal aorta that was implanted under anaesthesia. Following 24h recovery from catheterization, serum Cortisol, glucose and osmolality levels were 9.4 ng/ml, 58.8 mg/dl and 261.4 mOsm/kg, respectively. Furthermore, blood samples collected with the chronic indwelling catheters indicated that the surgical procedure of cannulation caused a stress response, with physiological changes that followed a pattern like that described in teleosts. Cortisol, glucose and osmolality were more sensitive to stress than the other variables measured. Sampling by cardiac puncture tended to be associated with elevated serum Cortisol levels in older, larger sturgeon, but not in young fish. Greater capture, confinement and handling stress in older, larger, sturgeon may have been responsible for this and other age-related differences in blood chemistry values measured following cardiac puncture. Within the same age class, both rearing conditions and temperature affected Cortisol, sodium and total protein concentrations significantly. Anaesthesia did not appear to reduce the degree of stress associated with cardiac puncture but altered serum ion concentrations.     

The development of an implantable electrochemical glucose sensor: response to glucose in bovine serum ultrafiltrate.

A glucose sensor based on organic oxidation reactions at a platinum electrode is being developed as the key component for an implantable artificial beta cell for diabetic patients. Sensitivity of a membrane-covered platinum electrode to changing glucose concentration in bovine serum ultrafiltrate has been demonstrated, with current response as high as 60 microamperes per 100 mg/dl change in glucose concentration. Reproducibility of measurements probably will depend on satisfactory mechanical assembly of the membrane-electrode combination. An approach to selectivity based on multiple current measurements is suggested.     

Monitoring of low concentrations of glucose in fermentation broth

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.     

Urea biosensor based on PANi(urease)-Nafion/Au composite electrode

The polyaniline (PANi)-Nafion composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion/Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 microA in 60 mg dl(-1) NH4Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl(-1) LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 microA in the 60 mg dl(-1) NH4Cl buffer solution when the cycle number for preparing PANi-Nafion/Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion/Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 microA (mg dl(-1))(-1)cm(-2), as well as 6 and 0.3 mg dl(-1), respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion network, respectively.