Performance enhancing genetic variants, oxygen uptake, heart rate, blood pressure and body mass index of elite high altitude mountaineers.

AIM: To analyse and compare the ACE (angiotensin-converting enzyme), ACTN3 (actinin-3) and AMPD1 (adenosine monophosphate deaminase 1) genetic variants, oxygen uptake (VO2max), heart rate (HR), blood pressure (BP) and body mass index (BMI) of elite high altitude mountaineers and average athletes. METHODS: Elite Bulgarian alpinists (n = 5) and control group of athletes (n = 72) were recruited. VO2max was measured using a treadmill graded protocol. HR, BP and BMI were recorded. Genotyping was done by polymerase chain reaction (PCR) amplification followed by agarose gel electrophoresis. Chi2-test and Fisher's exact test were used for statistical analysis. RESULTS: Alpinists showed significantly higher frequencies of 60% ACE I allele (p = 0.002), 50% ACTN3 X allele (p = 0.032) and 30% AMPD1 T allele (p = 0.003) compared to controls - 39%, 36%, 13%, respectively. ACE ID genotype prevalence and null DD genotype were observed in mountaineers. Higher absolute VO2max, but no differences in VO2max ml kg-1 min-1, HR, oxygen pulse, blood pressure and BMI were found. CONCLUSIONS: The ID genotype and higher frequencies of ACE I allele could contribute to successful high altitude ascents in mountaineers. The genetic make-up of the two mountaineers who made the summit of Mt Everest was distinctive, revealing ACE ID genotype, mutant ACTN3 XX and AMPD1 TT genotypes.

Engineering Au Nanoparticle Arrays on SiO2 Glass by Pulsed UV Laser Irradiation.

We study semi-regular arrays of Au nanoparticles (NP) obtained via UV laser irradiation of thin Au films on glass substrate. The NP structures are prepared from films of a thickness up to 60 nm produced by discharge sputtering or pulsed laser deposition, and annealed by nanosecond laser pulses at 266 or 308 nm, respectively, at fluencies in the range of 60-410 mJ/cm2. For the rare- and close-packed NP structures, consistent description of optical properties is derived from microscopic observation, measurements of the absorption, and Raman spectra, and modeling of the near-field intensity distributions. The absorption bands centered at 540-570 nm are ascribed to resonant absorption of the surface plasmons. For the band positions, half widths, and intensities, the dependence on the NP shape (partial spheres), size, size distribution, and also excitation energy is observed. The structures are characterized by markedly reduced dephasing times of ∼3 fs. It is shown, that laser annealing of thin Au films provides reliable and cost effective method for controlled preparation of semi-regular NP arrays favorable for photonic applications.

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7.

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.

Needle-type lactate biosensor.

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.

Aspects of CO2 laser engraving of printing cylinders.

Results of the experimental and theoretical investigations of CO(2) laser-engraved cylinders are presented. The processed surfaces of test samples are examined by a phase-stepping laser interferometer, digital microscope, and computer-controlled profilometer. Fourier analysis is made on the patterns parallel to the axis of the laser-scribed test ceramic cylinders. The problem of the visually observed banding is discussed.

Immunosensors: electrochemical sensing and other engineering approaches.

This article overviews the engineering approaches and the recent trends in the development of alternative immunoassay systems. A brief description of the main principles and limitations of conventional immunoassay is given. Immunosensing approaches overcoming these limitations are discussed. Alternatives to traditional immunoassay systems are discussed in terms of the enhancement of immunointeraction processes and in terms of the various detection principles. Applications of flow-injection techniques to the development of immunosensing systems are presented. Immunosensors are categorized based on the detection principle employed, as immunoelectrodes (electrochemical immunosensors), piezoelectric immunosensors, or as sensors based on optical detection of the immunointeraction. The discussion focuses on electrochemical immunosensors. In conclusion, the engineering issues involved in immunosensor development are outlined and trends towards practical applications are discussed.

Flow-through amperometric immunosensor: fast 'sandwich' scheme immunoassay.

A flow-through immunosensor based on a high-surface-area carbon immunoelectrode has been developed. Dispersed carbon material serves as a carrier for immobilized antibodies and at the same time as an electrode material. The 'sandwich' scheme of immunoassay has been used. Iodine formed as a result of the enzymatic oxidation of iodide by a peroxidase label has been detected amperometrically. The immunosensor consists of a disposable sensing element (immunocolumn) containing dispersed carbon material with immobilized antibodies which also acts as a working electrode. A current collector connects the working electrode to the measuring device. The electrochemical detection time of the peroxidase-labeled immuno-complex does not exceed several minutes. The overall time of analysis including flowing of analyte, flowing of antigen, washing and detection stages is as low as 22 min. This technique allows fast determination of rabbit IgG (used as a model analyte) with a low detection limit in the picomolar range and also the determination of human IgM in blood plasma with a low detection limit in the nanomolar range.

Implantation of a refillable glucose monitoring-telemetry device.

This study describes the components and short-term in vivo evaluation of an integrated implantable system consisting of an amperometric glucose biosensor, a miniature potentiostat, a FM signal transmitter, and a power supply. The device (dimensions: 5.0 x 7.0 x 1.5 cm) was implanted subcutaneously in healthy mongrel dogs. The biosensor performance was evaluated in vitro prior to implantation using standard solutions simulating the physiological environment. A linear response to glucose concentration was observed throughout the physiological and pathophysiological range (with an upper limit of 25 mM glucose, and a sensitivity of 0.5 microA/mM). 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 3-7 min. These results demonstrated the feasibility of data transmission by a telemetry system through the skin of a dog and allowed the commencement of chronic in vivo testing. During the chronic implantation the biosensor was refilled in vivo. A rejuvenation of the sensor's response after refilling was observed suggesting the potential of such sensors for long-term implantation.

A needle-type sensor for monitoring glucose in whole blood.

A new surface-process technology employing electrochemical fixation of a bioactive substance (enzyme and heparin) to a sensor electrode was developed to provide biocompatability and functionality. The fabrication process includes electroentrapment of glucose oxidase and heparin on a platinum electrode by using 1,3-phenylenediamine codeposition. Electrochemically grown 1,3-phenylenediamine was also used as the outer coating of the sensor's enzyme electrode in order to extend the linear range. The sensor shows a sensitivity of 3 nA/mM and a linear range from 40 to 400 mg/dL at 37 degrees C when tested in whole blood. This sensor is characterized by a fast response. The sensor shows a minimum change in its performance when stored inactive in buffer for 12 weeks. When tested at physiologic glucose levels, the sensor demonstrates satisfactory low interference from common interfering substances. This technology seems promising for the preparation of implantable intravascular biosensors.

Short-term canine implantation of a glucose monitoring-telemetry device.

In this study we report the development and short-term in vivo evaluation of an integrated implantable device consisting of an amperometric glucose biosensor, a miniature potentiostat, a FM signal transmitter, and power supply. The device (dimensions: 5.0 x 7.0 x 1.5 cm) was implanted under the skin of medium-size anaesthetized dog. The experimental set-up included several methods for data collection: analog recording via wired X-T chart recorders; data collection by wearable microprocessor--data logger, and remote data collection via antenna and receiver linked to a computer-based data acquisition system. The device (sensor) performance was evaluated in vitro prior to implantation, using different model solutions simulating the physiological environment. A linear response to glucose concentration was obtained up to 25 mM glucose, with a sensitivity of 0.5 microA/mM. The results of short-term subcutaneous implantation of the integrated device reveal adequate monitoring of an artificially-induced glycaemia. The delay-time was 3-7 minutes. These tests demonstrate the feasibility of data transmission by the telemetry system through the skin of a medium-sized dog and allow the commencement of chronic in vivo experimentation.

Glucose monitoring: state of the art and future possibilities.

This article reviews the development of glucose monitoring techniques and approaches during the last decade. The predominance of the electrochemical measuring principles reported in the literature makes them a focus of this work. Biosensors are still in the main stream of the research interest of most teams due to their high selectivity for glucose determination. Systematization and classification of the glucose monitoring principles and types of glucose sensors is shown. The review gives a brief description of the basic operational principles of the most popular types of glucose biosensors, providing an enhanced bibliography of the original works of the main groups in establishing or significantly contributing to the development of the particular type of glucose biosensor. Different design approaches are overviewed including needle-type sensors, sensors for chronical implantation and the combination of the glucose biosensors with microdialysis sampling technique. The authors approach for replacing of the spent enzyme and thus recharging the sensor in situ while implanted is widely discussed. This approach provides a way to increase the lifespan of the system and ultimately, it could lead to rare transcutaneous interventions for refilling of the implanted sensor.

A glucose biosensor based on an oxygen electrode: in-vitro performances in model buffer solution and in blood plasma.

An implantable biosensor for continuous monitoring of glucose levels in diabetic patients was developed. The biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in the form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Sensors with polycarbonate glucose-diffusion membranes coated with silastic were studied. Calibration curves of the sensors in phosphate buffer solution and in undiluted blood plasma at body temperature were obtained and compared. Sensors were tested for long-term performance in vitro in model phosphate buffer solution and in blood plasma at room and at body temperature. Reproducibility of the sensor responses in plasma at body temperature was demonstrated. During continuous operation at body temperature, the sensors have a lifetime of at least six weeks.

Glucose biosensors based on oxygen electrode with sandwich-type membranes.

A glucose biosensor based on an amperometric oxygen electrode has been developed. Polycarbonate and Silastic membranes were assembled (glued together) to form a multilayer sandwich glucose diffusion barrier. The effects of the glue layer composition and thickness of the Silastic membrane on sensor response parameters have been investigated in order to optimize the sensor. The parameters measured were the sensitivity, the concentration range of the linear dependence of the sensor response to glucose, and the long-term operation time. The sensors with the sandwich-type glucose diffusion membrane (Silastic membrane prepared from 20% Silastic suspension, glue layer prepared from polyurethane, 0.5 w/v % in ThF solution and standard polycarbonate membrane) demonstrated linearity of response up to 520 mg/dl glucose at 25 degrees C and up to 400 mg/dl at 37 degrees C. These sensor showed good reproducibility of response without significant interference effects (from 1 to 5% of the background current value). The long-term continuous operational time of the sensors was over 40 days at 37 degrees C, and over 60 days at 25 degrees C.

Effect of interference on the performance of glucose enzyme electrodes using Nafion coatings.

The negatively charged perfluorinated ionomer Nafion was used as a coating on hydrogen peroxide detecting platinum electrode as well as on a polycarbonate diffusion membrane in the construction of a glucose amperometric enzyme electrode. The current response of these electrodes to hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and glucose was studied and the coating procedure was optimized. It was confirmed that the Nafion coating prevents interference by anionic substances such as ascorbic acid and uric acid, and decreases acetaminophen interference. In this regard it was shown that coating the glucose diffusion membrane (polycarbonate) was more effective than coating the platinum wire itself, because of the prevention of the homogeneous redox reaction of the interference species with hydrogen peroxide, as well as the additional diffusional resistance to the glucose flux. The glucose levels in serum samples were estimated and the stability of the enzyme electrodes during continuous operation in serum was studied. An enzyme electrode with constant sensitivity of ca. 1 microA/mM and a linear range of up to 15 mM, unaffected by contact with serum, is reported.

Glucose biosensors with enzyme entrapped in polymer coating.

The pursuit of reliable biosensors for measuring glucose levels has been ongoing for decades. Their importance lies partly in the development of the implantable artificial pancrease, which can be used to deliver insulin to diabetics without the need to test glucose levels externally, with automatic delivery based on physiologic demand. Glucose sensors can also be used in short-term monitoring of glucose levels in hospitals and clinical laboratories. Three types of glucose biosensors were studied. All were based on a two-electrode system: an insulated platinum wire as a hydrogen peroxide electrode, and a silver wire twisted around the platinum wire as both a reference and a counter electrode. Each was coated with the enzyme glucose oxidase entrapped in a polymer matrix of cellulose acetate (CA) or poly 2-hydroxyethyl methacrylate (HEMA), then dip-coated by an additional polymer coating of polyvinylchloride (PVC), polyurethane (PU), or HEMA. The experiments were designed mainly to study the effectiveness of polymer coatings as diffusion-limiting membranes. The effect of each coating on the linear response to glucose concentration was examined. It was shown that additional (multiple) coatings can increase the linearity of the sensor response. The best results were obtained when the sensor was PVC-dip-coated three times. This preparation had a linear response up to 600 mg/DL glucose concentration. The sensors coated with PU and HEMA have linearity up to 280 and 240 mg/DL glucose concentrations, respectively. It was also shown that the coatings reduce interference from certain body chemicals.

Integrated implantable device for long-term glucose monitoring.

In this study we report the development of an integrated implantable device for glucose monitoring. The dimensions of the device (5.0 x 7.0 x 1.5 cm) allow implantation under the abdominal skin of a large animal for in vivo evaluation of sensor performance. The experimental set-up includes amperometric glucose biosensor, a miniature potentiostat, an FM signal transmitter, a power supply and an antenna and receiver linked to a computer-based data acquisition system. The device performance was evaluated in vitro using a ten-day continuous test and other long-term operation experiments. The biosensor was tested in different model solutions that simulated the physiological environment in which it will be ultimately used. A linear response to glucose concentration was obtained up to 25 mM glucose, with a sensitivity of less than 0.5 microA/mM. The ability of the biosensor to measure glucose levels in serum was also tested, and a good correlation demonstrated between glucose serum levels measured by routine technique and those measured using the biosensor (R2 = 0.993; slope = 0.996). Initial results obtained from the short-term subcutaneous implantation of the sensor demonstrate its potential for the monitoring of glucose concentration in vivo.

Biosensor for continuos glucose monitoring.

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. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.

Glucose biosensor based on carbon black strips.

Amperometric biosensors for the determination of beta-D-glucose have been constructed. They were based on a porous matrix of carbon blacks--'Ketjenblack' (KB) and 'Shawinigan black' (SB) wet-proofed with polytetrafluorethylene. Glucose-sensitive elements were prepared by subsequent adsorptional immobilization of 1,1'-dimethylferrocene (DMFc) and nickel-ocene (Nc) on 'Shawinigan black' or tetracyanoquinodimethane (TCNQ) on 'Ketjenblack' together with Penicillium chrysogenum glucose oxidase. Maximum surface concentrations of DMFc, Nc and TCNQ on carbon black electrodes were 95, 116 and 151 nmol cm-2. The biosensor based on KB and TCNQ (KB-TCNQ biosensor) could be used at a potential of 0.5 V (vs. Ag/AgCl reference electrode) in the concentration range up to 7 mM. This biosensor possessed an approximately ten times higher sensitivity than the ones based on SB and DMFc (SB-DMFc biosensor) and on SB and Nc (SB-Nc biosensor) which acted at 0.3 V and 0.05 V, respectively. The biosensors were suitable for practical use longer than one week.