On line continuous monitoring of blood lactate in men by a wearable device based upon an enzymatic amperometric lactate sensor

A wearable device for the continuous measurement of lactate in the blood was constructed by the combination of continuous blood sampling employing a double lumen catheter with an amperometric lactate sensor. In vitro, the lactate sensor turned out to have a linear concentration range between 0 and 15 mmol/l. The response time of the sensor itself amounted to 100 sec, whereas the lag time for blood sampling amounted to 2·2 min. In vivo, the lactate sensor was successfully used for the detection of changes of the blood lactate concentration following strenuous exercise in 7 healthy volunteers, in two cases up to 22 h. In conclusion, the technique of continuous blood sampling by the use of a double lumen catheter in combination with the amperometric lactate sensor is feasible and simplifies frequent blood lactate estimations.     

Ureteral catheter system for renal function studies in conscious rabbits

To overcome the problem of obstruction of ureteral catheters with blood clots, a ureteral catheter was constructed which had a double lumen. Irrigation of blood from the catheter was accomplished by perfusion of heparinized saline through a small inner catheter. In renal function studies performed in 12 rabbits using this system, no instances of catheter obstruction occurred.     

Maturation and regulation of the motility of spermatozoa in the epididymis of the tammar wallaby (Macropus eugenii).

Demembranated spermatozoa from the rete testis developed vigorous flagellation when reactivated with ATP, but showed no forward progression such as that seen in samples from the cauda epididymidis. The proportion of spermatozoa that were reactivated was smaller for samples from the rete testis than from the cauda epididymidis. Studies in vitro of undiluted micropuncture samples from the epididymis indicated that the activity of spermatozoa is suppressed as they develop the capacity for motility. However, as spermatozoa spontaneously became activated during the collection or subsequent incubation of undiluted samples, it was concluded that the suppressive action is labile. The activity of spermatozoa in vitro was examined in diluted samples from the cauda epididymidis. A concentration of 2.5 mmol extracellular calcium/l was better than lower concentrations. Diluents at pH 5.5 completely inhibited sperm motility when they contained 20 mmol lactate/l (but not glutamate) and the effect was reversed by readjusting the diluent to pH 7.4. However, lactate was not considered to suppress sperm motility in situ, as the plasma from the cauda epididymidis contained only 2.7 +/- 0.5 mmol lactate/l. There was no effect of sodium concentration (1 and 115 mmol/l), pH (5.5 and 7.4) or amiloride (0 and 1 mmol/l) on sperm motility, indicating that motility is not dependent on the concentration of sodium above 1 mmol/l or on a sodium-proton exchange system. The relative viscosity of plasma from the cauda epididymidis did not affect the motility of spermatozoa.     

Long-term in vivo experience of an electrochemical sensor using the potential step technique for measurement of mixed venous oxygen pressure

An implantable amperometric blood oxygen sensor was developed to improve rate adaptation of heart pacemakers. Two different working electrode materials in direct contact with the blood were tested, smooth glassy carbon and gold. Reference electrodes of Ag/AgCl and porous pyrolytic carbon were evaluated. A counter electrode being the titanium housing of the pulse generator was partly coated with carbon. An implantable pacemaker system with chronocoulometric oxygen detection was developed. Heart synchronous potential steps were periodically applied to the 7.5 mm² working electrode in the atrium. Both single and double potential step techniques were evaluated. The oxygen diffusion limited current was used to calculate the stimulation rate. Bench tests and studies on 31 animals were performed to evaluate long-term stability and biocompatibility. In five dogs, the AV node was destroyed by RF ablation to create a realistic animal model of a pacemaker patient. Sensor stability and response to exercise was followed up to a maximum implantation time of 4 years. Post-mortem examinations of the electrode surfaces and tissue response were performed. The results show that a gold electrode is more stable than glassy carbon. The Ag/AgCl reference was found not to be biocompatible, but activated carbon was stable enough for use as reference for the potentiostat. Double potential steps stabilize the sensor response in comparison to single steps. Blood protein adsorption on the gold surface decreased the oxygen transport but not the reaction efficacy. No adverse tissue reactions were observed.     

Influence of catheter design on lumen wall temperature distribution in intracoronary thermography

Intracoronary thermography is a currently used vulnerable plaque detection method. We studied how catheter design and catheter location influence the temperature readings, and thus its capacity to detect vulnerable plaques. Finite element calculations were performed on geometries representing the coronary artery, the vulnerable plaque and the catheter. Catheter material, diameter and location with respect to the plaque were varied. Both flow and no-flow situations were studied. Maximal lumen wall temperature difference without a catheter (DeltaT=0.12 degrees C, flow=75 cm(3) min(-1)) was considered the reference. Presence of a 1.0mm nitinol catheter right under the plaque increased DeltaT to 0.14 degrees C, whereas a 1.0 mm polyurethane catheter increased DeltaT to 0.51 degrees C. The location at which a thermosensitive element should be placed for most optimal temperature readings during a pullback was shown to lie at the catheter edge for the nitinol catheter and at 1.1mm from the catheter edge for the polyurethane catheter. Temperature readings decreased to background temperature when the catheter was in close proximity but not overlapping the plaque. DeltaT decreased approximately by 70% when a gap of 0.2 mm existed between the catheter and the lumen wall. Occlusion of blood flow increased DeltaT values in all cases, but most pronounced for nitinol catheters. A polyurethane catheter increased the temperature readings, since its heat conductivity is lower than that of blood, which makes it a very good choice for heat source detection. Catheter design can contribute to enhanced temperature readings and thus can enable more optimal vulnerable plaque detection.     

Micromachined sensor for lactate monitoring in saliva

A miniaturised sensor for continuous lactate measurement in saliva was developed and tested. The sensor was built using silicon microfabrication technologies. The size of the chip is 5.5 mmx6.4 mmx0.7 mm and features a working, a counter and an Iridium reference electrode. The chip has a cavity whose floor is perforated by fine pores. The cavity contains the enzyme lactate oxidase (LOD), which is immobilised in an agarose gel. Prior to the amperometric detection of the reaction product hydrogen peroxide at the working electrode, the analyte lactate has to pass the pores to reach the cavity with the lactate oxidase by diffusion. To test the silicon sensor, capillary blood and saliva samples were obtained during standardised ergometer tests. Salivary lactate concentrations were determined with the sensor and compared to photometrically derived data from a lab-automate. In addition the saliva data were compared to standard capillary blood lactate concentrations measured with a pocket photometer. Lactate concentration versus load graphs were plotted and compared visually showing very similar progressions. The novel approach enables a location independent, permanent real-time measurement of the lactate concentration during exercise.     

Preparation and characterization of heparinized multi-walled carbon nanotubes

The characteristics of heparinized multiwalled carbon nanotubes (MWNTs) were investigated in terms of the activated partial thromboplastin time (APTT) to verify the heparin activity, a carbazole assay was done to measure the content of the immobilized heparin, and the octanol-water partition coefficient was assessed to determine the lipophilicity. Two heparin-immobilized MWNTs were prepared to evaluate their differences. The first preparation method involved polymer-coated MWNTs with heparin indirectly center-point-attached. In the second approach, heparin was directly end-point-attached through its reducing end onto acid-treated MWNTs. The blood compatibility of MWNTs to which heparin was end-point-attached through its reducing end was greatly enhanced compared to that of the MWNTs onto which heparin was center-point-attached. The APTT and carbazole assay results demonstrated that heparinized MWNTs prepared through end-point attachment result in prolonged plasma-based anticoagulant activity. The blood compatibility of MWNTs heparinized by end-point attachment was not decreased up to the fourth pasteurization. Heparinized MWNTs were also studied using octanol-water partition, which should be useful for exploring heparinized MWNTs as drug carriers including delivery systems. The results of octanol/water partition on the design of heparinized MWNTs prepared by end-point attachment with a specific binding can facilitate the design of drug delivery carriers with high blood compatibility.     

Amperometric glucose biosensor with extended concentration range utilizing complexation effect of borate.

Borate buffer strongly decreases amperometric response of a glucose oxidase linked pO2 or H2O2 sensing electrode, extending substantially its linear calibration range. With increasing pH and concentration of the buffer the upper limit for glucose can be varied between 1 and 30 mmol l-1 glucose. The effect of borate ion is explained by the rapid complexation of glucose decreasing the equilibrium concentration of free beta-anomer, the specific substrate of glucose oxidase. The high loading of cross-linked enzyme inside the sensor membrane is necessary for the measurement to ensure an almost constant response factor (delta i per 1 mmol l-1) between pH 5 and 10. Analysis in stirred solution and in a flow-through system has been employed for the measurement of elevated glucose levels in heparinized human blood or plasma samples.     

Chronic double lumen intravenous cannulation of the unrestrained rat

Chronic double lumen intravenous cannulation was performed on the rat. The cannulation system consisted of a pulley assembly, protective tube, mounting post, and a double lumen cannula. Using a double lumen expanded catheter, a two-channel connection was made with the rat's vascular system through a single entry into the external jugular vein. The cannulation system permitted free animal movement in a large cage and simultaneous measurement of behavioral and physiologic variables. The procedure minimized stress and provided a simple alternative to other systems using two cannulas.     

Optical-transparent and flexible glucose sensor with ITO electrode

The glucose sensor was constructed by immobilizing glucose oxidase (GOD) with glutaraldehyde solution onto the sensitive area of the transparent oxygen electrode. The oxygen electrode was fabricated by sealing KCl electrolyte solution including the Indium-Tin Oxide (ITO)-electrode with both metal-weldable film and gas-permeable membrane coated with Ag/AgCl electrode. The sensor behavior was evaluated using standard glucose solutions in a batch measurement system with a computer-controlled potentiostat at a reduction potential of -900 mV. The sensor device has flexible structure and good optical transparency (less than 0.6 abs) at the visible wavelength from 400 to 700 nm. The sensor was possible to be used for measuring glucose from 0.06 to 1.24 mmol/l (correlation coefficient: 0.999), including the reported concentration of tear glucose in normal (0.14 mmol/l), with good reproducibility.     

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.     

Biosensor based on Langmuir–Blodgett films of poly(3-hexyl thiophene) for detection of galactose in human blood

An amperometric biosensor was developed to estimate galactose in human blood serum. Monolayers of poly(3-hexyl thiophene) were placed on glass plates coated with indium tin oxide formed by dispensing a mixed solution of stearic acid in chloroform on to a water sub-phase. Galactose oxidase was mixed with poly(3-hexyl thiophene)/stearic acid in chloroform and dispensed on to the air-water interface of Langmuir-Blodgett trough. These monolayers were transferred on to glass plates which were used as working electrodes with platinum as a reference electrode. The amperometric galactose biosensor thus fabricated had a linear response from 0.05 to 0.5 g galactose l(-1) in blood serum. The normal level in blood is < 0.05 g galactose l(-1) in adults and 0-0.2 g galactose l(-1) in infants. In case of galactosemia, this increases to above 0.2 g galactose l(-1) in infants.     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

Microbial sensor for aspartame

Summary A microbial amperometric sensor using immobilized Bacillus subtilis cells was developed for the determination of the dipeptide sweetener aspartame (l-aspartyl-l-phenylalaninemethylester). From 0.07 to 0.6 mmol/l aspartame, a linear dependence of the initial current change (i.e., change in respiration rate) was obtained. The sensitivity for aspartame was one order of magnitude higher than for its amino acid constituents. The microbial sensor was stable for 8 weeks.     

Affinity apheresis for treatment of bacteremia caused by Staphylococcus aureus and/or methicillin-resistant S. aureus (MRSA)

Staphylococcus aureus (SA) bacteremia is associated with high mortality, and often results in metastatic infections. The methicillin-resistant SA (MRSA) is an urgent health care issue, as nosocomial infections with these bacteria represent limited treatment alternatives. Samples of whole blood containing challenge inoculums of SA and MRSA strains were passed through columns packed with surface-heparinized polyethylene beads. The bound bacteria were eluted and quantitatively determined by culturing and by real-time PCR. Significant amounts of both SA and MRSA adhered to the heparinized beads (more than 65% of inoculated bacteria). After rinsing with buffer at high ionic strength, viable bacteria or bacterial DNA were eluted from the columns, indicating that the binding was specific. The conclusions that can be made from these experiments are that, as earlier reported in the literature, the high affinity of SA to heparin is retained in whole blood, and MRSA in whole blood binds to heparin with similar or higher affinity than SA. It should be possible to lower the amount of SA and/or MRSA from the blood of infected patients to levels that could be taken care of by the immune system. In previous studies, we have shown that passing blood from septic patients over beads coated with end-point-attached, biologically active heparin is a useful technique for regulating the levels of heparin-binding cytokine. These findings in combination with the present findings indicate the possibility of creating an apheresis technology for treatment of sepsis caused by SA and/or MRSA.     

Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood

BACKGROUND: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. METHODS: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha). RESULTS: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. CONCLUSION: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood.     

Effects of cholecystokinin on appetite and pyloric motility during physiological hyperglycemia

Recent studies suggest that the interaction between small intestinal nutrient stimulation and the blood glucose concentration is important in the regulation of gastric motility and appetite. The purpose of this study was to determine whether the effects of cholecystokinin octapeptide (CCK-8) on antropyloric motility and appetite are influenced by changes in the blood glucose concentration within the normal postprandial range. Seven healthy volunteers were studied on 4 separate days. A catheter incorporating a sleeve sensor was positioned across the pylorus, and the blood glucose was stabilized at either 4 mmol/l (2 days) or 8 mmol/l (2 days). After the desired blood glucose had been maintained for 90 min, an intravenous infusion of either CCK-8 (2 ng. kg(-1). min(-1)) or saline (control) was given for 60 min. Thirty minutes after the infusion began, the catheter was removed and subjects drank 400 ml of water with guar gum before being offered a buffet meal. The amount of food consumed (kcal) was quantified. The order of the studies was randomized and single-blinded. There were fewer antral waves at a blood glucose of 8 than at 4 mmol/l during the 90-min period before the infusions (P<0.05) and during the first 30 min of CCK-8 or saline infusion (P = 0.07). CCK-8 suppressed antral waves (P<0.05), stimulated isolated pyloric pressure waves (IPPWs) (P<0.01), and increased basal pyloric pressure (P<0.005) compared with control. During administration of CCK-8, basal pyloric pressure (P<0.01), but not the number of IPPWs, was greater at a blood glucose of 8 mmol/l than at 4 mmol/l. CCK-8 suppressed the energy intake at the buffet meal (P<0.01), with no significant difference between the two blood glucose concentrations. We conclude that the acute effect of exogenous CCK-8 on basal pyloric pressure, but not appetite, is modulated by physiological changes in the blood glucose concentration.     

Nano-Au/cMWCNT Modified <Emphasis Type="Italic">speB</Emphasis> Gene Specific Amperometric Sensor for Rapidly Detecting <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> causing Rheumatic Heart Disease

A specific 5′ NH₂ labeled DNA probe of speB gene was immobilized onto the gold nanoparticles/carboxylated multi walled carbon nanotubes (Nano-Au/cMWCNT) screen printed electrode using EDC/NHS cross linking chemistry. This was followed by hybridization with 0.5–50 ng/6 µl of single stranded genomic DNA Streptococcus pyogenes infected patient throat swab samples. Electrochemical amperometric assay was deciphered by using cyclic voltammetry (CV) with methylene blue a redox indicator. The sensor had a sensitivity of 104.7 µA cm^?2 ng^?1 using CV with a R² of 0.907 and 0.01 ng/6 µl as the limit of detection (LOD). The modified electrode surface morphology was characterized using scanning electron microscopy. The stability of the electrode was seen at 4 °C for 180 days having 6% loss in the initial current. The sensor is speB gene specific and can detect the pathogen within 30 min.     

Attenuated hepatosplanchnic uptake of lactate during intense exercise in humans.

We evaluated whether the increase in blood lactate with intense exercise is influenced by a low hepatosplanchnic blood flow as assessed by indocyanine green dye elimination and blood sampling from an artery and the hepatic vein in eight men. The hepatosplanchnic blood flow decreased from a resting value of 1.6 +/- 0.1 to 0.7 +/- 0.1 (SE) l/min during exercise. Yet the hepatosplanchnic O2 uptake increased from 67 +/- 3 to 93 +/- 13 ml/min, and the output of glucose increased from 1.1 +/- 0.1 to 2.1 +/- 0.3 mmol/min (P < 0.05). Even at the lowest hepatosplanchnic venous hemoglobin O2 saturation during exercise of 6%, the average concentration of glucose in arterial blood was maintained close to the resting level (5.2 +/- 0.2 vs. 5.5 +/- 0.2 mmol/l), whereas the difference between arterial and hepatic venous blood glucose increased to a maximum of 22 mmol/l. In arterial blood, the concentration of lactate increased from 1.1 +/- 0.2 to 6.0 +/- 1.0 mmol/l, and the hepatosplanchnic uptake of lactate was elevated from 0.4 +/- 0.06 to 1.0 +/- 0.05 mmol/min during exercise (P < 0.05). However, when the hepatosplanchnic venous hemoglobin O2 saturation became low, the arterial and hepatosplanchnic venous blood lactate difference approached zero. Even with a marked reduction in its blood flow, exercise did not challenge the ability of the liver to maintain blood glucose homeostasis. However, it appeared that the contribution of the Cori cycle decreased, and the accumulation of lactate in blood became influenced by the reduced hepatosplanchnic blood flow.     

Rapid method for evaluating intra-erythrocyte Na+ activity

The measurements of intracellular "Na+ activity" was performed in 10 ml of heparinized venous blood. First the blood was three times washed in isotonic magnesium chloride solution (114 mmol/l). Thereby the buffy coat was removed. Then the microhematrocrit was taken for packet cell volume determination. After the erythrocytes were lysed by ultrasound. Sodium "Na+ Activity" is measured in the hemolysate by Ion-Selective electrode. With this method all "pipetting" operations are eliminated and for the "Na+ activity" determination was used ion-selective electrode with an indirect measurements, which is less influenced by the matrix. Reference intervals determined for a healthy population were 7.3 +/- 0.6 mmol/l.