Novel micromachined silicon sensor for continuous glucose monitoring

The construction and the application properties of a micro-machined silicon sensor for continuous glucose monitoring are presented. The sensor uses the conventional enzymatic conversion of glucose with amperometric detection of H(2)O(2). The innovation is the precise diffusion control of the analyte through a porous silicon membrane into a silicon etched cavity containing the immobilised enzyme. A variation of the number and size of the membrane pores allows to adjust the linear range of the sensor to the respective requirement. The sensor was tested in vitro as well as in clinical studies, being supplied with interstitial fluid. The cavity sensor was designed for a linear range between 0.5 and 20 mM. A signal response time of below 30 s and a signal stability exceeding 1 week is shown. By using a double cavity sensor falsification of the glucose signal by interfering substances can be compensated. In clinical trials the sensor measured continuously in interstitial fluid for up to 18 h without any signal drift and with good correlation to blood glucose reference values.     

Numerical modeling of fluid flow in solid tumors

A mathematical model of interstitial fluid flow is developed, based on the application of the governing equations for fluid flow, i.e., the conservation laws for mass and momentum, to physiological systems containing solid tumors. The discretized form of the governing equations, with appropriate boundary conditions, is developed for a predefined tumor geometry. The interstitial fluid pressure and velocity are calculated using a numerical method, element based finite volume. Simulations of interstitial fluid transport in a homogeneous solid tumor demonstrate that, in a uniformly perfused tumor, i.e., one with no necrotic region, because of the interstitial pressure distribution, the distribution of drug particles is non-uniform. Pressure distribution for different values of necrotic radii is examined and two new parameters, the critical tumor radius and critical necrotic radius, are defined. Simulation results show that: 1) tumor radii have a critical size. Below this size, the maximum interstitial fluid pressure is less than what is generally considered to be effective pressure (a parameter determined by vascular pressure, plasma osmotic pressure, and interstitial osmotic pressure). Above this size, the maximum interstitial fluid pressure is equal to effective pressure. As a consequence, drugs transport to the center of smaller tumors is much easier than transport to the center of a tumor whose radius is greater than the critical tumor radius; 2) there is a critical necrotic radius, below which the interstitial fluid pressure at the tumor center is at its maximum value. If the tumor radius is greater than the critical tumor radius, this maximum pressure is equal to effective pressure. Above this critical necrotic radius, the interstitial fluid pressure at the tumor center is below effective pressure. In specific ranges of these critical sizes, drug amount and therefore therapeutic effects are higher because the opposing force, interstitial fluid pressure, is low in these ranges.     

A viscosity-dependent affinity sensor for continuous monitoring of glucose in biological fluids

We present a viscometric affinity biosensor for continuous monitoring of glucose in biological fluids such as blood and plasma. The sensing principle of this chemico-mechanical sensor is based upon the viscosity variation of a sensitive fluid with glucose concentration. Basically, this device includes both an actuating and a sensing piezoelectric diaphragms as well as a flow-resistive microchannel. In order to confine the sensitive fluid and allow glucose diffusion into the sensor, a free-standing alumina nanoporous membrane is also used as size-selective interface. Measurements carried out at nominal temperatures of 25 and 37 °C reveal that this sensor topology exhibits a high resolution in the current range of physiological blood glucose concentrations, i.e. 2-20 mM. In addition, complete reversibility was also demonstrated for at least 3 days. Finally, measurements performed in human blood serum confirm that this sensor fulfils all basic requirements for a use in continuous glucose monitoring of biological fluids.     

Effects of vasomotion and venous pressure elevation on capillary-tissue fluid exchange across hetero-porous membrane

Flow autoregulation in the arteriolar network serves to maintain the capillary-tissue fluid balance by regulation of capillary pressure. In the present study, we have examined theoretically the effects of arteriolar vasomotion and venous pressure elevation on the capillary fluid exchange, the interstitial fluid pressure, and the interstitial osmotic pressure during capillary pressure regulation. We used Starling's hypothesis and extended it to include a consideration of a parallel hetero-porous pathway and to determine the effects of plasma protein filtration on interstitial fluid pressure and osmotic pressure. We have found that arteriolar vasomotion plays a primary role in protecting the capillary-tissue fluid balance during the elevation of capillary flow and that it is a secondary mechanism for the regulation of capillary arterial pressure.     

Evolution of osmotic pressure in solid tumors

The mechanical microenvironment of solid tumors includes both fluid and solid stresses. These stresses play a crucial role in cancer progression and treatment and have been analyzed rigorously both mathematically and experimentally. The magnitude and spatial distribution of osmotic pressures in tumors, however, cannot be measured experimentally and to our knowledge there is no mathematical model to calculate osmotic pressures in the tumor interstitial space. In this study, we developed a triphasic biomechanical model of tumor growth taking into account not only the solid and fluid phase of a tumor, but also the transport of cations and anions, as well as the fixed charges at the surface of the glycosaminoglycan chains. Our model predicts that the osmotic pressure is negligible compared to the interstitial fluid pressure for values of glycosaminoglycans (GAGs) taken from the literature for sarcomas, melanomas and adenocarcinomas. Furthermore, our results suggest that an increase in the hydraulic conductivity of the tumor, increases considerably the intratumoral concentration of free ions and thus, the osmotic pressure but it does not reach the levels of the interstitial fluid pressure.     

Elevated levels of colloid osmotic pressure in cecal contents of germfree animals.

Determinations of colloid osmotic pressure in the supernatant of germfree rat cecal contents indicated substantially elevated values in comparison to those of rat blood plasma or of conventional rat cecal supernatant. The germfree cecal supernatant, under conditions of similar total osmolality, was able to draw water at a sizable rate from a polyvinylpyrollidone solution whose colloid osmotic pressure was taken to be equivalent to that of interstitial fluid. It is suggested that the water absorption inhibition which was observed in the lower bowel of germfree rodents, is in part caused by the colloid osmotic pressure gradient which exists in these animals between the luminal contents and the tissue component.     

中医药防治腹膜纤维化的研究进展

腹膜透析是治疗终末期肾脏病较为有效的方法之一,但是腹膜长期暴露于非生理性的腹膜透析液中（低pH、高浓度葡萄糖以及高渗透压等）会致腹膜纤维化,从而导致腹膜结构和功能的丧失,这是患者放弃腹膜透析的原因之一。现代药理学实验证明,中医药对于腹膜纤维化的防治研究主要集中在腹膜间皮细胞的上皮间质转化方面,通过对细胞因子的作用,保护和／或改善腹膜间皮细胞的功能防治腹膜纤维化。     

Nonenzymatic continuous glucose monitoring in human whole blood using electrified nanoporous Pt

This paper describes the first devised method for the nonenzymatic and electrochemical glucose monitoring in 100% human whole blood and serum. The nanoporous Pt electrode allows for the selective amplification of glucose oxidation in the presence of electroactive interfering species without the need for enzymatic reaction. The outer membrane was particularly optimized to allow glucose molecules to be electrochemically detected against the numerous constituents of human blood. The proposed sensor provided reproducible amperometric responses to glucose in human serum and whole blood. Its sensitivity was maintained for at least 7h under constantly electrified conditions, and continued to work properly after 30 days of storage in human whole blood and serum. Unlike the enzyme-based glucose sensors, it was found to be minimally affected by thermal fluctuation, so as to remain successful even after steam sterilization at high temperature (134 °C) and pressure (0.22 Mpa). The unprecedented long-term stability and sterilization compatibility observed herein suggest a promising alternative to conventional enzymatic glucose sensors for many analytical and clinical applications, particularly for continuous glucose monitoring devices designed to potentially lead to a closed-loop artificial pancreas by combining them with an insulin pump.     

Isolation of rat trachea interstitial fluid and demonstration of local cytokine production in lipopolysaccharide-induced systemic inflammation.

Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate this fluid in rats by exposing excised trachea to G forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer 51Cr-labeled EDTA in fluid isolated at 239 g and plasma averaged 0.94 +/- 0.03 (n = 14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83% of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n = 5) was 18.8 +/- 0.6 mmHg with a corresponding pressure in plasma of 22 +/- 1.5 mmHg, whereas after overhydration (n = 5) these pressures fell to 9.8 +/- 0.4 and 11.9 +/- 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial fluid, and bronchoalveolar lavage fluid in LPS-induced inflammation. In control animals, low levels of IL-1 beta, IL-6, and TNF-alpha in serum, trachea interstitial fluid, and bronchoalveolar lavage fluid were detected. LPS resulted in a significantly higher concentration in IL-1 beta and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding, e.g., inflammatory-induced airway obstruction.     

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.     

On bone adaptation due to venous stasis

This paper addresses the question of whether or not interstitial fluid flow due to the blood circulation accounts for the observed periosteal bone formation associated with comprised venous return (venous stasis). Increased interstitial fluid flow induced by increased intramedullary pressure has been proposed to account for the periosteal response in venous stasis. To investigate the shear stresses acting on bone cell processes due to the blood circulation-driven interstitial fluid flow, a poroelastic model is extended to the situation in which the interstitial fluid flow in an osteon is driven by the pulsatile extravascular pressure in the osteonal canal as well as by the applied cyclic mechanical loading. Our results show that under normal conditions, the pulsatile extravascular pressure in the osteonal canal due to cardiac contraction (10mm Hg at 2Hz) and skeletal muscle contraction (30mm Hg at 1Hz) induce peak shear stresses on the osteocyte cell processes that are two orders of magnitude lower than those induced by physiological mechanical loading (100 microstrain at 1Hz). In venous stasis the induced peak shear stress is reduced further compared to the normal conditions because, although the mean intramedullary pressure is increased, the amplitude of its pulsatile component is decreased. These results suggest that the interstitial fluid flow is unlikely to cause the periosteal bone formation in venous stasis. However, the mean interstitial fluid pressure is found to increase in venous stasis, which may pressurize the periosteum and thus play a role in periosteal bone formation.     

A synthesis of interstitial fluid regulation and lymph formation.

The interstitial fluid spaces are filled with a mat of collagen fibers, and the interstices of this mat contain a mucopolysaccharide gel ground substance. Both the collagen fibers and the gel are elastic structures that can be expanded or compacted. In the expanded state the collagen fibers are pushed far apart and pockets of free fluid develop witin the gel. In the compacted state the elastic recoil of the compressed collagen fibers and gel reticular fibrillae seems to cause suction on the fluid within the tissue spaces, thus creating a subatmospheric pressure. Measurements of interstitial fluid pressure using a perforated capsule method indicate that this is normally slightly negative (subatmospheric) in most soft tissues. However, even very slight extra filtration of fluid into the tissue spaces increases the interstitial fluid pressure toward more positive values, which in turn increases lymph flow. The increased lymph flow then decreases the interstitial fluid volume and pressure back toward normal because of two mechanism, 1) direct removal of fluid from the tissue spaces in the lymph, and 2) removal of protein from the interstitial fluid in the lymph, thus decreasing the interstitial fluid colloid osmotic pressure and allowing more effective osmosis of fluid directly from the interstitial spaces back into the capillaries.     

Lowering of interstitial fluid pressure in rat submandibular gland: a novel mechanism in saliva secretion

The submandibular gland transports fluid at a high rate through the interstitial space during salivation, but the exact level of all forces governing transcapillary fluid transport has not been established. In this study, our aim was to measure the relation between interstitial fluid volume (V(i)) and interstitial fluid pressure (P(if)) in salivary glands during active secretion and after systemically induced passive changes in gland hydration. We tested whether interstitial fluid could be isolated by tissue centrifugation to enable measurement of interstitial fluid colloid osmotic pressure. During control conditions, V(i) averaged 0.23 ml/g wet wt (SD 0.014), with a corresponding mean P(if) measured with micropipettes of 3.0 mmHg (SD 1.3). After induction of secretion by pilocarpine, P(if) dropped by 3.8 mmHg (SD 1.5) whereas V(i) was unchanged. During dehydration and overhydration of up to 20% increase of V(i) above control, a linear relation was found between volume and pressure, resulting in a compliance (DeltaV(i)/DeltaP(if)) of 0.012 ml.g wet wt(-1).mmHg(-1). Interstitial fluid was isolated, and interstitial fluid colloid osmotic pressure averaged 10.4 mmHg (SD 1.2), which is 64% of the corresponding level in plasma. We conclude that P(if) drops during secretion and, thereby, increases the net transcapillary pressure gradient, a condition that favors fluid filtration and increases the amount of fluid available for secretion. The reduction in P(if) is most likely induced by contraction of myoepithelial cells and suggests an active and new role for these cells in salivary secretion. The relatively low interstitial compliance of the organ will enhance the effect of the myoepithelial cells on P(if) during reduced V(i).     

Osmosis and viscoelasticity both contribute to time-dependent behaviour of the intervertebral disc under compressive load: A caprine in vitro study

The mechanical behaviour of the intervertebral disc highly depends on the content and transport of interstitial fluid. It is unknown, however, to what extent the time-dependent behaviour can be attributed to osmosis. Here we investigate the effect of both mechanical and osmotic loading on water content, nucleus pressure and disc height. Eight goat intervertebral discs, immersed in physiological saline, were subjected to a compressive force with a pressure needle inserted in the nucleus. The loading protocol was: 10 N (6 h); 150 N (42 h); 10 N (24 h). Half-way the 150 N-phase (24 h), we eliminated the osmotic gradient by adding 26% poly-ethylene glycol to the surrounding fluid. For 62 additional discs, we determined the water content of both nucleus and annulus after 6, 24, 48, or 72 h. The compressive load was initially counterbalanced by the hydrostatic pressure in the nucleus. The load forced 4.3% of the water out of the nucleus, which reduced nucleus pressure by 44(±6)%. Reduction of the osmotic gradient disturbed the equilibrium disc height, and a significant loss of annulus water content was found. Remarkably, pressure and water content of the nucleus pulposus remained unchanged. This shows that annulus water content is important in the response to axial loading. After unloading, in the absence of an osmotic gradient, there was substantial viscoelastic recovery of 53(±11)% of the disc height, without a change in water content. However, for restoration of the nucleus pressure and for full restoration of disc height, restoration of the osmotic gradient was needed.     

Interstitial glucose concentration and glycemia: implications for continuous subcutaneous glucose monitoring

The changes in plasma glucose concentration and in interstitial glucose concentration, determined with a miniaturized subcutaneous glucose sensor, were investigated in anesthetized nondiabetic rats. Interstitial glucose was estimated through two different calibration procedures. First, after a glucose load, the magnitude of the increase in interstitial glucose, estimated through a one-point calibration procedure, was 70% of that in plasma glucose. We propose that this is due to the effect of endogenous insulin on peripheral glucose uptake. Second, during the spontaneous secondary decrease in plasma glucose after the glucose load, interstitial glucose decreased faster than plasma glucose, which may also be due to the effect of insulin on peripheral glucose uptake. Third, during insulin-induced hypoglycemia, the decrease in interstitial glucose was less marked than that of plasma glucose, suggesting that hypoglycemia suppressed transfer of glucose into the interstitial tissue; subsequently, interstitial glucose remained lower than plasma glucose during its return to basal value, suggesting that the stimulatory effect of insulin on peripheral glucose uptake was protracted. If these observations obtained in rats are relevant to human physiology, such discrepancies between plasma and interstitial glucose concentration may have major implications for the use of a subcutaneous glucose sensor in continuous blood glucose monitoring in diabetic patients.     

A new method for isolation of interstitial fluid from human solid tumors applied to proteomic analysis of ovarian carcinoma tissue

Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample's origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na(+), two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods.     

Osmotic and volume control of diuresis in conscious ducks (Anas platyrhynchos)

Summary In conscious Pekin ducks made diuretic either by infusing hyposmotic glucose solution or isosmotic saline, osmotic and volume effects on renal water excretion were investigated. As in mammals, antidiuresis mediated by enhanced release of antidiuretic hormone was induced by increasing carotid blood osmolality while a decrease augmented diuresis, indicating cerebral osmotic control of renal water excretion in birds.In contrast to the situation in mammals, a sensitive diuretic response to isosmotic volume expansion, corresponding to 1% of the extracellular volume, can be demonstrated, with intracarotid and intravenous application of the isosmotic saline infusion having identical effects.Volume loading with isosmotic saline produced a greater diuretic response than loading with the same amount of autologous blood, thereby indicating a major contribution of volume changes in the interstitial compartment to the control of renal water excretion. This corresponds to the importance of the interstitial fluid compartment for the control of salt gland activity in this species.Abbreviations AVP arginine vasopressin - ECF extracellular fluid - i.c., i.v. intracarotid, intravenous - ECFV ECF volume     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

Calibration of the viscometric glucose sensor before its use in physiological liquids--compensation for the colloid-osmotic effect

The function of the recently described viscometric affinity sensor (VAS), which measures glucose due to its strong effect on the viscosity of a sensitive liquid containing Concanavalin A (ConA) and dextran, was analysed for osmotic and colloid-osmotic effects on the glucose reading. The suction of low- and high-molecular weight osmotica on the membrane of the microdialysis fibre was measured using a membrane osmometer built from the microdialysis probe of the VAS. The reduction of the sensor read-out in blood plasma can be completely explained by a change in small osmotic volume fluxes through the dialysis membrane, which affect the ConA concentration and the viscosity after the flow of the sensitive liquid through the dialysis probe. The measuring error could be prevented by the presence of the polyethylene glycol 6000 at an isotonic concentration in the glucose standard solutions used for sensor calibration.     

Modeling of interstitial fluid movement in soft tissue under negative pressure – relevance to treatment of tissue swelling

Exact physiological mechanisms behind the potential positive treatment effects of pathological tissue swelling (edema), such as increased interstitial fluid flow, are poorly understood. Finite-element model was created and the model response was matched with the deformation data from the negative pressure (suction) measurements in human (N = 11) forearm. Two experimental suction protocols were simulated to evaluate their impact on interstitial fluid flow in soft tissues. Simulated continuous suction was up to 27 times more efficient in fluid transportation compared to the cyclic suction. The continuous suction that transports the interstitial fluid effectively may help to decrease soft tissue edema.