A contact lens with embedded sensor for monitoring tear glucose level

We report the design, construction, and testing of a contact lens with an integrated amperometric glucose sensor, proposing the possibility of in situ human health monitoring simply by wearing a contact lens. The glucose sensor was constructed by creating microstructures on a polymer substrate, which was subsequently shaped into a contact lens. Titania sol-gel film was applied to immobilize glucose oxidase, and Nafion? was used to decrease several potential interferences (ascorbic acid, lactate, and urea) present in the tear film. The sensor exhibits a fast response (20s), a high sensitivity (240 μA cm(-2) mM(-1)) and a good reproducibility after testing a number of sensors. It shows good linearity for the typical range of glucose concentrations in the tear film (0.1-0.6 mM), and acceptable accuracy in the presence of interfering agents. The sensor can attain a minimum detection of less than 0.01 mM glucose.     

A novel wireless glucose sensor employing direct electron transfer principle based enzyme fuel cell

In this paper we present a novel wireless glucose biosensing system employing direct electron transfer principle based enzyme fuel cell. Using the glucose dehydrogenase complex, which is composed of a catalytic subunit containing FAD, the cytochrome c subunit that harbors heme c as the electron transfer subunit, and chaperone-like subunit, a direct electron transfer-type glucose enzyme fuel cell was constructed. The enzyme glucose fuel cell generated electric power, and the open-circuit voltage showed glucose concentration dependence, which suggests potential applications for this glucose-sensing system. We constructed a miniaturized "all-in-one" glucose enzyme fuel cell, which represents a compartmentless fuel that is based on the direct electron transfer principle. This involved the combination of a wireless transmitter system and a simple and miniaturized continuous glucose monitoring system, which operated continuously for about 3 days with stable response. This is the first demonstration of an enzyme-based direct electron transfer-type enzyme fuel cell and fuel cell-type glucose sensor which can be utilized as a subcutaneously implantable system for continuous glucose monitoring.     

Boronic acid fluorescent sensors for monosaccharide signaling based on the 6-methoxyquinolinium heterocyclic nucleus: progress toward noninvasive and continuous glucose monitoring

The synthesis, characterization, and spectral properties of strategically designed boronic acid containing fluorescent sensors, o-, m-, p-BMOQBA, for the potential detection of tear glucose concentrations when immobilized in plastic disposable contact lenses is described. The new probes, BMOQBAs, consist of the 6-methoxyquinolinium nucleus as a fluorescent indicator, and the boronic acid moiety as a glucose chelating group. A control compound BMOQ, which has no boronic acid group and therefore does not bind monosaccharides has also been prepared. In this paper, we show that structural design considerations of the new probes have afforded for their compatibility within the lenses, with reduced probe sugar-bound pK(a) favorable with the mildly acidic lens environment. In addition, the new probes are readily water soluble, have high quantum yields, and can be prepared by a simple one-step synthetic procedure.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Engineering of ligand specificity of periplasmic binding protein for glucose sensing

A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K (d) value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K(d) value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K(d) value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP.     

Multiple hypothalamic circuits sense and regulate glucose levels

The hypothalamus monitors body energy status in part through specialized glucose sensing neurons that comprise both glucose-excited and glucose-inhibited cells. Here we discuss recent work on the elucidation of neurochemical identities and physiological significance of these hypothalamic cells, including caveats resulting from the currently imprecise functional and molecular definitions of glucose sensing and differences in glucose-sensing responses obtained with different experimental techniques. We discuss the recently observed adaptive glucose-sensing responses of orexin/hypocretin-containing neurons, which allow these cells to sense changes in glucose levels rather than its absolute concentration, as well as the glucose-sensing abilities of melanin-concentrating hormone, neuropeptide Y, and proopiomelanocortin-containing neurons and the recent data on the role of ventromedial hypothalamic steroidogenic factor-1 (SF-1)/glutamate-containing cells in glucose homeostasis. We propose a model where orexin/hypocretin and SF-1/glutamate neurons cooperate in stimulating the sympathetic outflow to the liver and pancreas to increase blood glucose, which in turn provides negative feedback inhibition to these cells. Orexin/hypocretin neurons also stimulate feeding and reward seeking and are activated by hunger and stress, thereby providing a potential link between glucose sensing and goal-oriented behavior. The cell-type-specific neuromodulatory actions of glucose in several neurochemically distinct hypothalamic circuits are thus likely to be involved in coordinating higher brain function and behavior with autonomic adjustments in blood glucose levels.     

Mathematical Modeling of Interacting Glucose-Sensing Mechanisms and Electrical Activity Underlying Glucagon-Like Peptide 1 Secretion

Intestinal L-cells sense glucose and other nutrients, and in response release glucagon-like peptide 1 (GLP-1), peptide YY and other hormones with anti-diabetic and weight-reducing effects. The stimulus-secretion pathway in L-cells is still poorly understood, although it is known that GLP-1 secreting cells use sodium-glucose co-transporters (SGLT) and ATP-sensitive K⁺-channels (K(ATP)-channels) to sense intestinal glucose levels. Electrical activity then transduces glucose sensing to Ca²⁺-stimulated exocytosis. This particular glucose-sensing arrangement with glucose triggering both a depolarizing SGLT current as well as leading to closure of the hyperpolarizing K(ATP) current is of more general interest for our understanding of glucose-sensing cells. To dissect the interactions of these two glucose-sensing mechanisms, we build a mathematical model of electrical activity underlying GLP-1 secretion. Two sets of model parameters are presented: one set represents primary mouse colonic L-cells; the other set is based on data from the GLP-1 secreting GLUTag cell line. The model is then used to obtain insight into the differences in glucose-sensing between primary L-cells and GLUTag cells. Our results illuminate how the two glucose-sensing mechanisms interact, and suggest that the depolarizing effect of SGLT currents is modulated by K(ATP)-channel activity. Based on our simulations, we propose that primary L-cells encode the glucose signal as changes in action potential amplitude, whereas GLUTag cells rely mainly on frequency modulation. The model should be useful for further basic, pharmacological and theoretical investigations of the cellular signals underlying endogenous GLP-1 and peptide YY release.     

In vivo glucose monitoring: the clinical reality and the promise

Glucose monitoring is an essential component of modern diabetes management. Three in vivo glucose sensors are now available for clinical use: a subcutaneously implanted amperometric enzyme electrode, a reverse iontophoresis system and a microdialysis-based device. Improvements in glucose-sensing technology continue to be sought, e.g. wired enzyme technology, viscometric affinity sensing and totally implanted glucose sensors. Non-invasive glucose sensing is the ultimate goal of glucose monitoring, but the most investigated approach, near-infrared (NIR) spectroscopy, is presently too imprecise for clinical application. Fluorescence-based glucose sensing offers several advantages and we are investigating strategies which include NIR-based fluorescence resonance energy transfer using concanavalin A/dextran; changes in the intrinsic fluorescence of hexokinase encapsulated in sol-gel; and non-invasive glucose monitoring of cells by measuring glucose-related changes in NADP(H).     

Glucose-sensing mechanisms in pancreatic beta-cells

The appropriate secretion of insulin from pancreatic beta-cells is critically important to the maintenance of energy homeostasis. The beta-cells must sense and respond suitably to postprandial increases of blood glucose, and perturbation of glucose-sensing in these cells can lead to hypoglycaemia or hyperglycaemias and ultimately diabetes. Here, we review beta-cell glucose-sensing with a particular focus on the regulation of cellular excitability and exocytosis. We examine in turn: (i) the generation of metabolic signalling molecules; (ii) the regulation of beta-cell membrane potential; and (iii) insulin granule dynamics and exocytosis. We further discuss the role of well known and putative candidate metabolic signals as regulators of insulin secretion.     

Glucose-sensing neurons of the hypothalamus

Specialized subgroups of hypothalamic neurons exhibit specific excitatory or inhibitory electrical responses to changes in extracellular levels of glucose. Glucose-excited neurons were traditionally assumed to employ a 'beta-cell' glucose-sensing strategy, where glucose elevates cytosolic ATP, which closes KATP channels containing Kir6.2 subunits, causing depolarization and increased excitability. Recent findings indicate that although elements of this canonical model are functional in some hypothalamic cells, this pathway is not universally essential for excitation of glucose-sensing neurons by glucose. Thus glucose-induced excitation of arcuate nucleus neurons was recently reported in mice lacking Kir6.2, and no significant increases in cytosolic ATP levels could be detected in hypothalamic neurons after changes in extracellular glucose. Possible alternative glucose-sensing strategies include electrogenic glucose entry, glucose-induced release of glial lactate, and extracellular glucose receptors. Glucose-induced electrical inhibition is much less understood than excitation, and has been proposed to involve reduction in the depolarizing activity of the Na+/K+ pump, or activation of a hyperpolarizing Cl- current. Investigations of neurotransmitter identities of glucose-sensing neurons are beginning to provide detailed information about their physiological roles. In the mouse lateral hypothalamus, orexin/hypocretin neurons (which promote wakefulness, locomotor activity and foraging) are glucose-inhibited, whereas melanin-concentrating hormone neurons (which promote sleep and energy conservation) are glucose-excited. In the hypothalamic arcuate nucleus, excitatory actions of glucose on anorexigenic POMC neurons in mice have been reported, while the appetite-promoting NPY neurons may be directly inhibited by glucose. These results stress the fundamental importance of hypothalamic glucose-sensing neurons in orchestrating sleep-wake cycles, energy expenditure and feeding behaviour.     

Self monitoring of glucose by people with diabetes: evidence based practice.

The inappropriate use of self monitoring of glucose is wasteful of NHS resources and can cause psychological harm. Although a few patients find that self monitoring enables them to understand and take control of their diabetes, many people with diabetes are performing inaccurate or unnecessary tests. There is no convincing evidence that self monitoring improves glycaemic control, nor that blood testing is necessarily better than urine testing. It may be appropriate for some patients not to monitor their own glucose but to rely instead on regular laboratory estimations of glycaemic control. Glucose self monitoring should be performed only when it serves an identified purpose. It is widely assumed that glucose self monitoring, preferably of blood glucose concentrations, is desirable or even essential for everyone with diabetes. It is common for patients who have previously tested their urine, or have done no glucose monitoring at home, to be taught to measure their blood glucose when they are admitted to hospital. In the community too, patients are often encouraged to monitor their blood glucose, and newly diagnosed patients of all ages are usually taught to measure their blood glucose concentrations. Self monitoring can sometimes be useful, but evidence is mounting that its indiscriminate use is of questionable value. In 1995, Pounds 42.6 million was spent on home monitoring of glucose in the United Kingdom (Intercontinental Medical Statistics, personal communication). Is this enormous cost justified? Is blood testing necessarily better than urine testing? Is glucose self monitoring always necessary, or is it sometimes a waste of time and money? Are recommendations for self monitoring based on sound evidence?     

Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.     

The construction of a glucose-sensing luciferase

A novel luminescence-based glucose-sensing molecule was created by combining a galactose-/glucose-binding protein (GGBP) with luciferase. The glucose-sensing luciferase (GlcLuc) was constructed using a GGBP fused with a large domain and a small domain of Firefly luciferase (Lluc and Sluc). The luminescence intensity-based analysis with E. coli recombinant protein showed that the GlcLuc had luciferase activity in glucose or galactose in a concentration-dependent manner (K_d = 3.9 μM for glucose and 11 μM for galactose), and that the increase in the activity saturated within one minute after the injection of the ligands. These results indicated that the conformation change of the GGBP moiety following the ligand binding effectively induced the reconstitution of the GGBP-fused split luciferase. The Asp459Asn mutation, which was expected to lead to a glucose specific binding ability, was then introduced into the GlcLuc. The GlcLuc mutant showed the luciferase activity increasing only with the increase of glucose concentration, but not with that of galactose. Our results demonstrate that the GGBP fused with a split luciferase, which is reconstituted rapidly and specifically in the presence of glucose, provides a novel glucose-sensing system based on luminescence and may also contribute to the construction of luminescence-based sensing molecules for other substrates using other PBPs.     

Effect of nutrient limitation on adhesion characteristics of Pseudomonas aeruginosa

Pseudomonas aeruginosa causes a variety of diseases in humans including lung and ocular infections. Infections of the cornea are usually associated with wearing contact lenses and can result in loss of vision. This study aimed to determine the effect of carbon or nitrogen limitation on the adhesion to contact lenses of a strain of Ps. aeruginosa isolated from contact lens-related corneal inflammation. Cells were grown in a continuous culture apparatus in varying levels of glucose or ammonia to effect nutrient limitation. Adhesion to contact lenses was measured as total counts and viable counts. The cell surface hydrophobicity and charge were measured using adhesion to surface-modified Sepharose. Changes in lipopolysaccharide were determined using 1D SDS-PAGE and changes in cell-surface proteins were measured using 2D gel electrophoresis. The more the cultures were nitrogen limited, the greater the increase in adhesion to unworn hydrogel contact lenses 0.3 x 10(3) - 2.2 x 10(3) cells/mm2 on Etafilon A lenses. Cells that were carbon limited showed a greater increase in adhesion to contact lenses when the lenses had been coated in artificial tears. It appeared that lipopolysaccharide may have been involved in the constitutive adhesion to unworn lenses that occurred during C-limitation, whereas changes in the outer membrane proteins contributed to the increased adhesion under nitrogen limitation, or the change in adhesion that occurred to carbon-limited cells using contact lenses coated in artificial tears. Nine cell-surface proteins appeared during nitrogen limitation with kDa/pI of 75/4.8, 4.9, 5.0; 62/5.6; 89/6.5; 38/6.4; 28/1.5; 18/6.4; 12/4.5. Any or all of these may have been involved in the increased adhesion and further experiments are underway to examine this possibility.     

Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells

Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c); glucose evoked an immediate elevation of [Ca²⁺]_c, which was followed by a decrease in [Ca²⁺]_c, and after a certain lag period it induced large oscillatory elevations of [Ca²⁺]_c. Initial rapid peak and subsequent reduction of [Ca²⁺]_c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca²⁺, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]_c). The elevation of [cAMP]_c was blocked by transduction of the dominant-negative G_s, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca²⁺]_c and [cAMP]_c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

The physiology and pathophysiology of the neural control of the counterregulatory response

Despite significant technological and pharmacological advancements, insulin replacement therapy fails to adequately replicate β-cell function, and so glucose control in type 1 diabetes mellitus (T1D) is frequently erratic, leading to periods of hypoglycemia. Moreover, the counterregulatory response (CRR) to falling blood glucose is impaired in diabetes, leading to an increased risk of severe hypoglycemia. It is now clear that the brain plays a significant role in the development of defective glucose counterregulation and impaired hypoglycemia awareness in diabetes. In this review, the basic intracellular glucose-sensing mechanisms are discussed, as well as the neural networks that respond to and coordinate the body's response to a hypoglycemic challenge. Subsequently, we discuss how the body responds to repeated hypoglycemia and how these adaptations may explain, at least in part, the development of impaired glucose counterregulation in diabetes.     

The effects of non-ionic polymeric surfactants on the cleaning of biofouled hydrogel materials

Block co-polymer surfactants have been used for cleaning hydrogel medical devices that contact the body (eg contact lenses) because of their biocompatibility. This work examined the relationship between concentration and detergency of two non-ionic polymeric surfactants (Pluronic F127 and Triton X-100) for cleaning protein soil, with anionic surfactants (sodium dodecyl sulfate and sodium laureth sulfate) as positive controls. Surface plasmon resonance was used to quantify removal of simulated tear soil from self-assembled monolayer surfaces, and a microplate format was used to study the removal of fluorescently labeled soil proteins from contact lenses. While detergency increased as a function of concentration for anionic surfactants, it decreased with concentration for the two polymeric surfactants. The fact that the protein detergency of some non-ionic polymeric surfactants did not increase with concentration above the critical micelle concentration could have implications for optimizing the tradeoff between detergency and biocompatibility.     

Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83?±?0.61 vs 0.77?±?0.20, p?=?0.81) or proline rich protein-4 (0.11?±?0.04 vs 0.15?±?0.12, p?=?0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9?±?9.01, 0.84?±?0.50 or 2.06?±?1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88?±?0.13, 0.50?±?0.10 or 0.27?±?0.23, respectively) (p?相似文献