Use of charged membranes to control interference by body chemicals in a glucose biosensor

The prerequisite for the continuous in vivo monitoring of glucose concentration is the development of an implantable glucose sensor with long-term stability. A new enzyme electrode concept featuring fluid-state glucose oxidase modified carbon powder along with a cross-linked glucose oxidase enzyme layer has been developed. The glucose sensor incorporating this enzyme electrode has been tested in vitro at 37°C. It has a lifetime of three months after which it can be recharged with fresh enzyme. The next step in the characterization of this sensor is its in vitro behaviour in the presence of interfering substances commonly encountered in human blood. Here we report such a study of the sensor. The glucose diffusion membranes used were polycarbonate membranes. We used standard polycarbonate membranes (membranes treated with polyvinylpyrrolidone or PVP), PVP-free polycarbonate membranes, and standard polycarbonate membranes coated with positively and negatively charged hydrogel layers. The sensors showed a response to glucose concentrations <300 mg dL⁻¹, both in pure phosphate buffer and in the presence of interferences. The influence of ascorbic acid, bilirubin, creatinine, L-cystine, glycine, uric acid and urea on the amperometric signal of the sensor was investigated. The polycarbonate membrane coated with the negatively charged hydrogel layer provided good protection for the enzyme electrode, especially in the presence of ascorbic acid and uric acid.     

Design of a self-cleaning thermoresponsive nanocomposite hydrogel membrane for implantable biosensors

Following implantation of a biosensor, adhesion of proteins and cells and eventual fibrous encapsulation will limit analyte diffusion and impair sensor performance. A thermoresponsive nanocomposite hydrogel was developed as a self-cleaning biosensor membrane to minimize the effect of the host response and its utility for an optical glucose sensor, demonstrated here. It was previously reported that thermoresponsive nanocomposite hydrogels prepared from photopolymerization of an aqueous solution of N-isopropylacrylamide (NIPAAm) and polysiloxane colloidal nanoparticles released adhered cells with thermal cycling. However, poly(N-isopropylacrylamide) hydrogels exhibit a volume phase transition temperature (VPTT) of ～33–34 °C, which is below body temperature. Thus, the hydrogel would be in a collapsed state in vivo, which would ultimately limit diffusion of the target analyte (e.g., glucose) to the encapsulated sensor. In this study, the VPTT of the nanocomposite hydrogel was increased by introducing N-vinylpyrrolidone (NVP) as a comonomer, so that the hydrogel was in the swollen state in vivo. This thermoresponsive nanocomposite hydrogel was prepared by the photopolymerization of an aqueous solution of NIPAAm, NVP, and polysiloxane colloidal nanoparticles. In addition to a VPTT a few degrees above body temperature, the hydrogel also exhibited good mechanical strength, glucose diffusion, and in vitro cell release upon thermal cycling. Thus, this nanocomposite hydrogel may be useful as a biosensor membrane to minimize biofouling and extend the lifetime and efficiency of implantable glucose sensors and other biosensors.     

Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel

Abstraet-A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37°C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37°C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70 000 with diffusion ratios, D/D₀ (ratio of diffusion in the gel to diffusion in water), of 0.20 ± 0.04 and 0.35±0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.     

Mediation of in vivo glucose sensor inflammatory response via nitric oxide release

In vivo glucose sensor nitric oxide (NO) release is a means of mediating the inflammatory response that may cause sensor/tissue interactions and degraded sensor performance. The NO release (NOr) sensors were prepared by doping the outer polymeric membrane coating of previously reported needle-type electrochemical sensors with suitable lipophilic diazeniumdiolate species. The Clarke error grid correlation of sensor glycemia estimates versus blood glucose measured in Sprague-Dawley rats yielded 99.7% of the points for NOr sensors and 96.3% of points for the control within zones A and B (clinically acceptable) on Day 1, with a similar correlation for Day 3. Histological examination of the implant site demonstrated that the inflammatory response was significantly decreased for 100% of the NOr sensors at 24 h. The NOr sensors also showed a reduced run-in time of minutes versus hours for control sensors. NO evolution does increase protein nitration in tissue surrounding the sensor, which may be linked to the suppression of inflammation. This study further emphasizes the importance of NO as an electroactive species that can potentially interfere with glucose (peroxide) detection. The NOr sensor offers a viable option for in vivo glucose sensor development.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. VIII. Adult and fetal guinea pig (Cavia procellus)

The diffusional water permeability (P _d) of adult, pregnant female and fetal guinea-pig red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were around 5.0 × 10^?3 cm/s at 15 °C, 5.3 × 10-3 cm/s at 20 °C, 6.6 × 10^?3 cm/s at 25 °C, 7.5 × 10^?3 cm/s at 30 °C and 8.6 × 10^?3 cm/s at 37 °C with no significant differences between adult, pregnant female and fetal RBCs. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 10 min at 37 °C with 0.1 mm PCMBS. The values of maximal inhibition ranged from 70%–77% at 15–30 °C to 57%–63% at 37 °C in the case of adult and from 64%–67% at 15–30 °C to 51% at 37 °C in the case of fetal RBCs. The basal permeability to water was estimated at 1.1 × 10^?3 cm/s at 15 °C ,1.3 × 10^?3 cm/s at 20 °C, 1.6 × 10^?3 cm/s at 25 °C, 2.2 × 10^?3 cm/s at 30 °C and 3.2 × 10^?3 cm/s at 37 °C for adult and slightly higher values for fetal guinea pig RBCs as 1.6 × 10^?3 cm/s at 15 °C, 2.0 × 10^?3 cm/s at 20 °C, 2.4 × 10^?3 cm/s at 25 °C, 2.6 × 10^?3 cm/s at 30 °C and 4.2 × 10^?3 cm/s at 37 °C. The activation energy of water diffusion was around 22 kJ/ mol, with no significant differences between the adult pregnant female and fetal RBCs, and increased to about 40 kJ/mol in the case of adult and pregnant RBCs and 34 kJ/mol for fetal RBCs after incubation with PCMBS in conditions of maximal inhibition of water diffusion. The membrane polypeptide electrophoretic pattern of adult and fetal guinea-pig RBCs was compared with its human counterpart. The guinea-pig membrane contained higher amounts of spectrin (band 1 and 2), whereas the proteins in bands 4.1, 4.2 and 6 were present in lower amounts. Considerable differences in polypeptides migrating in the region of bands 7 and 8 and in front of them were apparent between the two sources of RBC membranes where some bands were present only in the guinea-pig RBC membranes. The adult guinea-pig membranes contained smaller amounts of proteins migrating in band 4.5 and lacked band 8.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. IX. Australian feral chicken and domestic chicken (Gallus domesticus)

The diffusional water permeability (P _d) of Australian feral chicken and Australian and European domestic chicken red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique. The values of P _d were around 1.7 × 10^?3 cm/s at 15°C, 2.0 × 10^?3 cm/s at 20°C, 2.5 × 10^?3 cm/s at 25°C, 3.7 × 10^?3 cm/s at 30°C, 4.3 × 10^?3 cm/s at 37°C, and 6.1 × 10^?3 cm/s at 42°C, with no significant differences between the three strains of chicken. There was no effect of p-chloromercuribenzene sulphonate on water diffusion. The activation energy of water diffusion was around 37 kJ/mol for all strains of chicken. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of chicken introduced to Australia (and that membrane proteins play little role in the diffusion of water across chicken RBC membrane).     

Parylene-on-oil packaging for long-term implantable pressure sensors

This paper reports and analyzes the feasibility study of a parylene-on-oil encapsulation packaging method of pressure sensors targeted for long-term implantation. Commercial barometric digital-output pressure sensors are enclosed in silicone oil and then encapsulated in situ with parylene-C or –D (PA-C, PA-D) chemical vapor deposition. Experimentally, sensors encapsulated with 30,000 cSt silicone oil and 27 μm PA-D show good performance for 6 weeks in 77 °C saline with >99 % of original sensitivity, corresponding to an extrapolated lifetime of around 21 months in 37 °C saline. This work shows that, with proper designs, such a packaging method can preserve the original pressure sensor sensitivity without offset, validated throughout accelerated lifetime tests. In experiments, wires on the prototypes are used for external electronics but it is found that they contributed to early failures, which would be absent in real wireless versions, indicating a potential for even longer lifetimes. Finally, a verified model is presented to predict the pressure sensor sensitivity of parylene-on-oil packaging with and without the presence of a bubble in the oil.     

Evanescent field-fiber loop ringdown glucose sensor

Evanescent field-fiber loop ringdown (EF-FLRD) is a relatively new hybrid sensing technique which combines a versatile sensing mechanism with a sensitivity-enhanced ringdown detection scheme. An array of low cost, fast response, and high sensitivity biosensors based on the EF-FLRD technique can be developed. In this work, new fiber loop ringdown glucose sensors using refractive index-difference evanescent field attenuation effect as a sensing mechanism are described. The sensor head consists of either a section of partially-etched bare single mode fiber or a section of the etched fiber with glucose oxidase (GOD) immobilized on the etched fiber surface. Effects of the sensor head, with and without the immobilized GOD, on the sensor's performance are comparatively examined. The sensors' responses to standard glucose solutions and synthetic urines in different glucose concentrations ranging from 50 mg/dl to 10 g/dl are studied. The sensors, with or without the immobilized GOD, showed a linear response to glucose concentrations in the range of 100 mg/dl to 1 g/dl, but a nonlinear response in the higher glucose concentration ranging from 1 to 10 g/dl. The detection sensitivities of the sensors for the glucose solutions and artificial urine samples are 75 and 50 mg/dl respectively, and the sampling rate of the sensors is 10 to 100 Hz. Estimated theoretical detection sensitivity of the EF-FLRD glucose sensors is 10 mg/dl, which is approximately 17 times lower than the glucose renal threshold concentration.     

Multifunctional Biocompatible Membrane and Its Application to Fabricate A Miniaturized Glucose Sensor with Potential for Use In Vivo

A multifunctional membrane with biocompatibility, diffusion-limiting effect, and the ability to curtail the responses of an H₂O₂ electrode to ascorbate and urate was prepared. It was composed of MB, AB, and CTA, where MB is the copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butylmethacrylate (BMA), AB is the copolymer of acrylamide-2-methylpropane sulfonic acid (AMPS) and BMA, CTA is cellulose triacetate. Investigation of the biocompatibility of this membrane showed that, compared with CTA, relatively few platelets bound to it. The membrane was coated onto the working electrode of a needle-type glucose sensor on which immobilized glucose oxidase membrane has been coated. The sensor did not respond to ascorbate and urate at their concentration normally encountered in blood. Its response was not inhibited by metal ions in blood at usual concentration. The sensor exhibited superior thermostability in addition to a rapid response (<90 seconds in batch operation), good reproducibility (RE<5%), good stability (more than 36 hours continuously in heparinized whole blood), and a wide dynamic range (5–650 mg/dl glucose). The sensor was used to determine glucose in serum. The data obtained from the sensor showed good agreement with that from a clinical autoanalyzer (R=0.973). This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel (Camelus dromedarius) and alpaca (Lama pacos)

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10^?3 cm/s at 25°C, 5.4×10^?3 cm/s at 30°C, 6.6×10^?3 cm/s at 37°C and 7.7×10^?3 cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10^?3 cm/s and 9.0×10^?3 cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10^?3 cm/s at 25°C, 1.7×10^?3 cm/s at 30°C and of camel RBC as 1.8×10^?3 cm/s at 25°C and 3.0×10^?3 cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Zwitterionic carboxybetaine polymer surfaces and their resistance to long-term biofilm formation

In this work, we report a systematic study of zwitterionic poly(carboxybetaine methacrylate) (pCBMA) grafted from glass surfaces via atom transfer radical polymerization (ATRP) for their resistance to long-term bacterial biofilm formation. Results show that pCBMA-grafted surfaces are highly resistant to non-specific protein adsorption (fibrinogen and undiluted blood plasma) at 25, 30 and 37 °C. Long-term (over 24 h) colonization of two bacterial strains (Pseudomonas aeruginosa PAO1 and Pseudomonas putida strain 239) on pCBMA surface was studied using a parallel flow cell at 25, 30 and 37 °C. Uncoated glass cover slips were chosen as the positive reference. Results show that pCBMA coatings reduced long-term biofilm formation of P. aeruginosa up to 240 h by 95% at 25 °C and for 64 h by 93% at 37 °C, and suppressed P. putida biofilm accumulation up to 192 h by 95% at 30 °C, with respect to the glass reference. The ability of pCBMA coatings to resist non-specific protein adsorption and significantly retard bacterial biofilm formation makes it a very promising material for biomedical and industrial applications.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species. X. Camel ( Camelus dromedarius ) and alpaca ( Lama pacos )

The diffusional water permeability (P _d) of camel and alpaca red blood cells (RBCs) was measured by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition withp-chloromercuribenzene sulphonate (PCMBS). The values ofP _d were, in the case of alpaca RBC≈4.6×10⁻³ cm/s at 25°C, 5.4×10⁻³ cm/s at 30°C, 6.6×10⁻³ cm/s at 37°C and 7.7×10⁻³ cm/s at 42°C. In case of camel RBC the values ofP _d where ≈4.2×10⁻³ cm/s and 9.0×10⁻³ cm/s at 42°C. Systematic studies on the effects of PCMBS on water diffusion in camel RBC indicated that the maximal inhibition was reached in 45 min with 1–2 mm PCMBS. The values of maximal inhibition were around 47% at 25°C and 68% at 30°C for alpaca RBC and around 62% at 25°C and 56% at 37°C for camel RBC. The basal permeability to water of alpaca RBC was estimated at around 2.6×10⁻³ cm/s at 25°C, 1.7×10⁻³ cm/s at 30°C and of camel RBC as 1.8×10⁻³ cm/s at 25°C and 3.0×10⁻³ cm/s at 37°C. The values of the activation energy of water diffusion (E _{a, d}) were around 23 kJ/mol for camel and 34 kJ/mol for alpaca RBC. This suggests that in addition to the number of transport channels other features of the pathways might be important for defining the temperature dependence of the water permeability.     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XI. Horses Introduced to Australia and European Horses ( Equus caballus )

The diffusional water permeability (P _d) of red blood cells (RBCs) from horses introduced to Australia and from European horses was measured by a Mn²⁺ doping nuclear magnetic resonance (NMR) technique. The values of P _d were ∼3.5 × 10⁻³ cm/s at 25°C, 4.1 × 10⁻³ cm/s at 30°C, 5.6 × 10⁻³ cm/s at 37°C and 6.3 × 10⁻³ cm/s at 42°C with no significant differences between the two strains of horse. Systematic studies on the effect of p-chloromercuribenzene sulphonate (PCMBS) on water diffusion indicated that the maximal inhibition (60%) was reached in 60 min at 22°C with 1 mm PCMBS. The basal permeability to water was estimated at 1.2 × 10⁻³ cm/s at 25°C, 1.7 × 10⁻³ cm/s at 30°C, 2.0 × 10⁻³ cm/s at 37°C and 2.7 × 10⁻³ cm/s at 42°C. The activation energy (E _a,d) of water diffusion was ∼25 kJ/mol and increased to ∼39 kJ/mol after incubation with PCMBS, in conditions of maximal inhibition of water diffusion. The membrane protein electrophoretic pattern of horse RBCs was compared with its human counterpart. The horse RBC membranes had lower amounts of the proteins migrating as bands 4.1 and 4.2 and higher amounts of the protein migrating as band 4.9, and band 6 (glyceraldehyde-3-phosphate dehydrogenase) was undetectable. A noteworthy feature was the appearance of considerable differences in protein migration distances in the region of bands 7 and 9, between horse and human membranes.     

Condensation polymerization of 2,2′-diiodobiphenyl-4,4′-dicarbonyl dichloride with some mono- and dihydroxy aromatic compounds

Several potentially thermostable polyesters 3a–1 were prepared by Schotten-Baumann condensation of 2,2′-diiodo-4,4′-biphenyldicarbonyl dichloride ( 1 ) with several aromatic hydroxy compounds 2a–1 . The reactivity of 1 towards various phenols was tested and the spectra of the resulting model compounds 4a–d were compared with those of the polymers. With some exceptions ( 3h and 3c ) the polyesters were found to be soluble in organic solvents. Their inherent viscosities vary between 0,1 and 0,45 dl · g^?1 (in CHCl₃ at 25°C). The values of the inherent viscosity were in the range of 0,06–0,45 dl/g (c = 0,2 g/dl in CHCl₃ at 25°C). The melting points of the polymers were found to be higher than 300°C.     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XII. Dog (<Emphasis Type="BoldItalic">Canis familiaris</Emphasis>) and Cat (<Emphasis Type="BoldItalic">Felis domestica</Emphasis>)

The diffusional water permeability (P _d ) of dog and cat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulphonate (PCMBS). The values of P _d were in the case of cat RBC ∼3.0 × 10⁻³ cm/s at 15 °C, 3.5 × 10⁻³ cm/s at 20 °C, 4.2 × 10⁻³ cm/s at 25 °C, 4.4 × 10⁻³ cm/s at 30 °C and 5.9 × 10⁻³ cm/s at 38 °C. In case of dog RBC the values of P _d were higher ∼3.8 × 10⁻³ cm/s at 15 °C, 4.6 × 10⁻³ cm/s at 20 °C, 5.0 × 10⁻³ cm/s at 25 °C, 5.9 × 10⁻³ cm/s at 30 °C and 7.9 × 10⁻³ cm/s at 37 °C. Systematic studies of the effect of PCMBS on water diffusion indicated that in the case of dog RBCs the maximal inhibition was reached in 15–30 min with 1 mm PCMBS, whereas in the case of cat RBCs in 60 min with 1 mm PCMBS or in 30 min with 2 mm PCMBS. The values of maximal inhibition in the case of cat RBC were in the range of 55–60% at 15 °C, 60–68% at 20 °C and 25 °C, 50–60% at 30 °C and 50–55% at 37 °C. In the case of dog RBC the corresponding values were higher, 75–80% at 15 °C, 70–80% at 20 °C and 25 °C, 65–70% at 30 °C and 55–60% at 37 °C. The basal permeability to water was estimated to be ∼1 × 10⁻³ cm/s −2 × 10⁻³ cm/s in the range of temperatures of 25–37 °C. The activation energy of water diffusion E _a,d was ∼19 kJ/mol for the dog RBC and ∼23 kJ/mol for the cat RBC. After incubation with PCMBS the values of E _a,d increased, reaching 40 kJ/mol in conditions of maximal inhibition of water exchange. The membrane polypeptide electrophoretic pattern of dog and cat RBCs has been compared with its human counterpart. Dog and cat RBCs contained higher amounts of spectrin (band 1 and 2) and lower amounts of bands 4.4, 4.2, band 5 and band 7 compared to human RBCs. Band 4.9 was decreased only in the cat RBCs, whereas band 6 was decreased only in the dog RBCs. Correspondence and offprint requests to: Gheorghe Benga, Department of Cell and Molecular Biology, ‘Iuliu Haţieganu’ University of Medicine and Pharmacy, 6 Pasteur St, 3400 Cluj-Napoca, Romania. Tel:/Fax: 40–64–194373; e-mail: GBenga@personal.ro; gbenga@umfcluj.ro     

Layer‐By‐Layer Modified Superparamagnetic Iron Oxide Nanoparticles with Stimuli‐Responsive Drug Release Properties

Superparamagnetic iron oxide nanoparticles (SPIONs) are synthesized through ultrasound based coprecipitation method. SPIONs are coated with poly(2‐isopropyl‐2‐oxazoline) (PIPOX) and tannic acid (TA) in a layer‐by‐layer (LbL) fashion at pH 4 and 25 °C. PIPOX/TA coated SPIONs are then loaded with doxorubicin (DOX) at pH 7.5 and 25 °C. DOX release from LbL‐coated SPIONs is examined at pH 7.5 and pH 6 at 25 °C, 37 °C, and 42 °C. LbL‐coated SPIONs exhibit dual responsive behavior and release the greatest amount of DOX at pH 6 and 42 °C. Increasing layer number decreases the colloidal stability and saturation magnetization. Superparamagnetic behavior of SPIONs retains after coating. Overall, this study shows an alternative strategy to modify the surface of SPIONs with a dual responsive polymer coating which is capable of releasing DOX at moderately acidic pH of 6 within a physiologically related temperature range. Besides, it generates fundamental knowledge for further development of SPIONs‐based drug carriers.     

Micromachined oxygen gas sensors for microscopic energy consumption measurement systems

Although the indirect calorimeter is a useful tool, its size and expense mean that it is seldom used in hospitals. Furthermore, its flow-through measurement technique dilutes respiratory variations, so they can only be detected with some form of high-precision instrumentation. This study employs MEMS techniques to develop an oxygen sensor as one part of a microscopic energy consumption measurement system, which measures respiration dynamics in a real time manner. The oxygen sensor comprises a polysilicon resistor and a Li-doped (2 wt%) tin-oxide sensing film attached to a thermally isolated silicon-nitride membrane. The power consumption of the sensor is less than 25 mW at an operating temperature of 150°C. Furthermore, it measures oxygen concentrations between 25 and 85% with a linear output response. These characteristics render the proposed sensor suitable for use within a microscopic energy consumption measurement system in either hospital or homecare environments.     

Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer

The instability of enzymatic glucose sensors has prevented the development of a practical artificial pancreas for diabetic patients. We therefore developed an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer (MIP). This sensor has the advantages of improved stability and a simplified manufacturing procedure. An adduct of glucose and 4-vinylphenylboronic acid (VPBA) was synthesized by esterification and was then purified. The copolymer of the glucose/VPBA adduct and methylene bisacrylamide was grafted onto an indium tin oxide electrode surface. Glucose was washed out from the copolymer to obtain an MIP layer. Cyclic voltammetry of ferrocyanide in aqueous solution was performed using an MIP-grafted electrode, and the effect of glucose on the anodic current intensity was evaluated. The anodic current intensity was sensitive to the glucose concentration, and the dynamic range (0–900 mg/dl) covered the typical range of diabetic blood glucose levels. The response time of the MIP-grafted electrode to a stepwise change in the glucose concentration was approximately 3–5 min. Thus, we can conclude that, by taking advantage of its gate effect, it is feasible to use an MIP-grafted electrode as a glucose sensor for monitoring blood sugar in diabetic patients.     

Treating allergic conjunctivitis using in situ polyelectrolyte gelling systems

We prepared 0.3% Carbopol/15.5% Pluronic and 0.1% Alginate/15.5% Pluronic solutions as the in situ gelling vehicles for ophthalmic drug delivery of cromolyn. The flow properties, in vitro release and in vivo pharmacological response of these polyelectrolyte solutions and of commercial eye drops were evaluated and compared. The polyelectrolyte solutions were free-flowing at pH 4.0 and 25°C, and they turned to strong gels at pH 7.4 and 37°C due to the in situ phase transition. The in vitro release and in vivo pharmacological response indicated that both vehicles retained cromolyn better than the commercial eye drops. The results demonstrated that both systems are usable as in situ gelling vehicles to increase ocular therapeutic efficacy in treating allergic conjunctivitis.     

A comparison of shortening and Z line degradation in post-mortem bovine,porcine, and rabbit muscle

Ultrastructural changes in bovine, porcine, and rabbit muscle have been studied during the first 24 hours post-mortem. Samples were taken for phase and electron microscopy immediately after death, after 4, 8, and 24 hours of post-mortem storage at 2° and 37°C, and after 24 hours post-mortem at 16° and 25°C. The results show that two kinds of structural changes occur in muscle during the first 24 hours post-mortem: (a) a variable amount of shortening, this shortening occurring via a sliding of filaments in all species and at all post-mortem storage temperatures examined, and (b) degradation of the Z line, and at higher storage temperatures, of the M line also. Shortening of unrestrained muscle occurs soonest post-mortem at 37°C in all three species and is completed within four hours post-mortem in porcine and rabbit muscle and within eight hours post-mortem in bovine muscle. Post-mortem short-ening of unrestrained rabbit and porcine muscle is greatest at 37°C (sarcomere lengths of 1.5 μ); shortening of rabbit muscle is minimal at 2°C (sarcomere lenght of 1.7 μ), but shortening of porcine muscle is minimal at 25°C (sarcomere length of 1.8 μ) and is slightly greater at 2°C (sarcomere length of 1.6 μ) than at 16°C. Post-mortem shortening of bovine muscle is greatest at 2°C (sarcomere length of 1.3 μ), is minimal at 16–25°C (sarcomere length of 1.8 μ), and increases between 25–37°C (sarcomere length of 1.5 μ at 37°C). Sarcomere length measurements show that some variation occurs in the extent of post-mortem shortening within the same muscle. Z line degradation occurs sooner post-mortem and to a greater extent at storage temperatures of 25°C or above than at temperatures of 16°C or below. Also, bovine muscle Z lines are clearly more resistant to post-mortem degradation than porcine or rabbit muscle Z lines. Loss of fibrillar structure in porcine or rabbit muscle Z lines occurs during the first four hours post-mortem at 37°C, but eight hours of post-mortem storage at 37°C are required to cause loss of fibrillar structure of bovine muscle Z lines. After 24 hours at 25 or 37°C, Z lines of rabbit and porcine muscle are usually completely absent; M lines are also frequently absent in this muscle.