Fluorescence-based glucose sensors

There is an urgent need to develop technology for continuous in vivo glucose monitoring in subjects with diabetes mellitus. Problems with existing devices based on electrochemistry have encouraged alternative approaches to glucose sensing in recent years, and those based on fluorescence intensity and lifetime have special advantages, including sensitivity and the potential for non-invasive measurement when near-infrared light is used. Several receptors have been employed to detect glucose in fluorescence sensors, and these include the lectin concanavalin A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein, and boronic acid derivatives (which bind the diols of sugars). Techniques include measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor either within a protein which undergoes glucose-induced changes in conformation or because of competitive displacement; measurement of glucose-induced changes in intrinsic fluorescence of enzymes (e.g. due to tryptophan residues in hexokinase) or extrinsic fluorophores (e.g. using environmentally sensitive fluorophores to signal protein conformation). Non-invasive glucose monitoring can be accomplished by measurement of cell autofluorescence due to NAD(P)H, and fluorescent markers of mitochondrial metabolism can signal changes in extracellular glucose concentration. Here we review the principles of operation, context and current status of the various approaches to fluorescence-based glucose sensing.     

Holographic glucose sensors

A novel holographic sensor system capable of detecting dynamic changes in glucose concentration has been developed. The hologram is recorded within a bio-compatible hydrogel matrix containing phenylboronic acid derivatives. On binding glucose, the colour of the hologram red-shifts to longer wavelengths as the hydrogel expands and this colour change is used to quantify glucose concentration. However, phenylboronic acids are non-selective and bind a wide variety of cis-diols. In blood, glucose is the only sugar found free at high concentration, whilst other sugars are typically found as part of glycoproteins and macromolecular structures. Although glycoproteins have been shown to have no effect on the sensor, phenylboronic acids can bind lactate much more readily than glucose. We have designed two polymer hydrogel systems to increase the selectivity of the sensor for glucose over lactate. The first involved the use of high concentrations of 3-acrylamidophenylboronic acid (3-APB) whilst the second system utilised 2-acrylamido-5-fluorophenylboronic acid (5-F-2-MAPB). Both systems displayed an increased selectivity to glucose over lactate at physiological pH and ionic strength and could be deployed as selective holographic sensors for glucose detection in physiological fluids.     

Carbon post-microarrays for glucose sensors

A novel design and fabrication method of glucose sensors based on high aspect ratio carbon post-microarrays is reported in this paper. Apart from the fact that carbon has a wide electrochemical stability window, a major advantage of using carbon post-microarrays as working electrodes for an amperometric glucose sensor is the large reactive surface per unit footprint substrate area, improving sensitivity of the glucose sensor. The carbon post-microarrays were fabricated by carbon-microelectromechanical systems (C-MEMS) technology. Immobilization of enzyme onto the carbon post-electrodes was carried out through co-deposition of glucose oxidase (GOx) and electrochemically polymerized polypyrrole (PPy). Sensing performance of the glucose sensors with different post-heights and various post-densities was tested and compared. The carbon post-glucose sensors show a linear range from 0.5 mM to 20 mM and a response time of about 20 s, which are comparable to the simulation result. Sensitivity per unit footprint substrate area as large as 2.02 mA/(mM cm²) is achieved with the 140 μm high (aspect ratio around 5:1) carbon post-samples, which is two times the sensitivity per unit footprint substrate area of the flat carbon films. This result is consistent with the hypothesis that the number of reaction sites scales with the reactive surface area of the sensor. Numerical simulation based on enzymatic reaction and glucose diffusion kinetics gives the optimum geometric design rules for the carbon post-glucose sensor. Glucose sensors with even higher sensitivity can be achieved utilizing higher carbon post-microarrays when technology evolution will permit it.     

Towards implantable glucose sensors: a review

The clinical treatment procedures to monitor and control diabetes mellitus include the use of mechanical devices. Of these devices, closed-loop devices are preferred to open loop devices. However, the development of such devices depends to a great extent on the availability of reliable, long-lasting glucose sensors. In this paper, the status of implantable glucose sensors is reviewed. Glucose sensors are classified and discussed under the following headings: Enzyme catalysed electrodes; metal catalysed sensors; affinity sensors; and coated wire sensors. The relative merits of each of the sensor types are presented and experimental results for both in vitro and in vivo studies are summarized.     

Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose‐responsive polymer‐based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15–31 mM (K_d), which match physiological glucose fluctuations. The dose‐responsive glucose‐mediated release of the novel insulins was demonstrated using glucamine‐derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%–122%) which is unusual for insulin LysB29 derivatives. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

In-vivo behaviour of hypodermically implanted microfabricated glucose sensors

The in-vivo behaviour of microfabricated GOD (glucose oxidase)/H2O2 glucose sensor implanted subcutaneously in normal anaesthetized rats has been studied. The sensor consists of a planar, three-electrode microcell, an enzyme membrane (glucose oxidase and bovine serum albumin cross-linked with glutaraldehyde) and an outer diffusion limiting polyurethane membrane. The sensor behaviour during hyperglycaemic (13.8 mM and 11.2 mM), euglycaemic (7.8 mM) and hypoglycaemic (3.5 mM) plateau levels was determined. The values of the in-vivo sensitivity (0.64 +/- 0.05 nA/mM) and background current (1.25 +/- 0.4 nA) were determined using a two-point calibration method and then used to calculate apparent subcutaneous glucose concentrations. The results show the presence of a good correlation between all the plasma glucose levels (G) and the apparent subcutaneous tissue concentrations (G'), with G' = 0.997.G - 0.066, r = 0.9782.     

Lithographically defined 3D nanoporous nonenzymatic glucose sensors

Nonenzymatic glucose oxidation is demonstrated on highly faceted palladium nanowflower-modified porous carbon electrodes fabricated by interference lithography. Varying electrodeposition parameters were used to control the final shape and morphology of the deposited nanoparticles on the 3D porous carbon which showed a 12 times increase in the electrochemically active surface area over analogous planar electrodes. Extremely fast amperometric glucose responses (achieving 95% of the steady state limiting current in less than 5s) with a linear range from 1 to 10mM and a detection limit of 10 μM were demonstrated. The unusual surface properties of the pyrolyzed photoresist films produced strongly adhered palladium crystal structures that were stable for hundreds of cycles towards glucose oxidation without noticeable current decay.     

Containment sensors for the determination of L-lactate and glucose

This paper reports some new results on enzyme based silicon containment sensors. For the first time an L-lactate sensor in containment technology is presented. Through optimization of the buffer system the stability of the lactate sensor was enhanced and the linear response of over 10 mM was achieved. The glucose sensor has also been optimized for a large linear measurement range exceeding 30 mM. A two-enzyme chip with glucose and lactate sensor elements which were integrated on one silicon chip is presented. The response behaviour of the two-enzyme chip was very similar to the single chip behaviour. No cross-talking effects could be observed. A fabrication process for mass-production is described.     

Sterilization of enzyme glucose sensors: problems and concepts

A useful method of enzyme glucose sensor sterilization has not only to ensure the needs of sterility assurance but has also to guarantee the functional stability of the sensors. The action of 2 or 3% alkalinized glutaraldehyde solution, as well as gamma irradiation with a dose of 25 kGy caused changes of the in vitro functionality and polymer material irritations, respectively. After a combined treatment by 0.6% hydrogen peroxide solution acting over 4 days with 7 kGy gamma irradiation only a slight loss of sensitivity must be registered. The combination of a specially designed universal homogeneous ultraviolet irradiation over 300 s with a 3 days lasting treatment by an inclusion compound of hydrogen peroxide with tensides in urea (0.15% effective hydrogen peroxide concentration) did not cause any influence on the glucose sensor function in vitro. With all methods tested here, a Bacillus subtilis spore reduction over 8 log(10) cycles from 10(6) to 10(-2) spores per test object on an average could be proved experimentally. In general, if non-thermal methods must be used it seems to be impossible to guarantee a sterility assurance level of 10(-6) as it is demanded by the pharmacopoeias. Consequently, effective concepts to produce sterile glucose biosensors for medical and biological applications should be based not only on final product treatments but should include germ reducing measures in every manufacturing step.     

The evolution of insulin resistance in muscle of the glucose infused rat

Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser⁴⁷³) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3β (Ser⁹) and AS160 (Thr⁶⁴²) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.     

Tissue response to subcutaneous implantation of glucose-oxidase-based glucose sensors in rats

Considerable progress in improved control of disturbed glucose metabolism can be expected by continuous glucose monitoring. The aim of the study was to evaluate in male Sprague-Dawley rats tissue response to implantation of a new amperometric glucose-oxidase-based glucose sensor (NTS) compared to a commercially available sensor system CGMS of MiniMed. Both sensors were tested under working conditions over a period of 3 days. Using NTS, glucose in interstitial fluid reflected glucose in arterial blood even in rapidly changing hyper- and hypoglycaemia whereas the CGMS did not detect the experimentally induced glucose changes adequately. Gene expression profiling was performed using Affymetrix chips. Acute phase response to injury by sensor application for a short time is indicated by down regulation of the increase in mRNA of proteases e.g. metallothionein-1alpha and matrix metalloprotease-3 at day 3. Improvement of anabolic situation is suggested by decrease in mRNA of insulin-like growth factor binding protein whereas increase of heme oxygenase and hypoxia-inducible factor may be related to defense mechanisms. Changes of mRNA together with slight fibrous capsule formation suggest good histocompatibility. Comparability of the patterns of changed mRNA in tissue surrounding SCGM with and without operating voltage as shown in dendrogram indicates no contribution of hydrogen peroxide to worsening biocompatibility. Beside established histological investigations of foreign body reaction weeks or months after implantation, gene expression profiling provides additional information to biocompatibility already early after implantation.     

The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast

Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor.     

Implantable glucose sensors: Choosing the appropriate sensing strategy

The special requirements for implantable glucose sensors which differ from laboratory analysers and in vitro probes include continuous operation without drift, compatibility with in vivo body conditions, electrical and toxicological safety and patient acceptability. We have studied the effect of oxygen tension, operating temperature and pH, and the stability of various potentially implantable amperometric glucose sensors so as to aid the choice of the technologies most suitable for in vivo application.     

Role of porosity in tuning the response range of microsphere-based glucose sensors

Luminescent microspheres encapsulating glucose oxidase have recently been developed as implantable glucose sensors. Previous work has shown that the response range and sensitivity can be tuned by varying the thickness and composition of transport-controlling nanofilm coatings. Nevertheless, the linear response range of these sensors falls significantly below the desired clinical range for in vivo monitoring. We report here an alternative means of tuning the response range by adjusting microsphere porosity. A reaction-diffusion model was first used to evaluate whether increased porosity would be expected to extend the response range by decreasing the flux of glucose relative to oxygen. Sensors exhibiting linear response (R(2)>0.90) up to 600 mg/dL were then experimentally demonstrated by using amine-functionalized mesoporous silica microspheres and polyelectrolyte nanofilm coatings. The model was then used for sensor design, which led to the prediction that sensors constructed from ～12 μm microspheres having an effective porosity between 0.005 and 0.01 and ～65 nm transport-limiting coatings would respond over the entire physiological glucose range (up to 600 mg/dL) with maximized sensitivity.     

A method for evaluating in vivo the functional characteristics of glucose sensors

A simple system for evaluating ex vivo the functional characteristics of glucose sensors was set up. Normal rats implanted with carotid and jugular catheters were connected under free-moving conditions to an extracorporeal circuit. Blood was allowed to circulate in contact with an enzyme glucose electrode. Glucose or insulin was infused intravenously at different rates to produce glycaemic alterations appropriate for sensor checking. Comparison of the changes in signal output with the corresponding variations in plasma glucose enabled in vivo evaluation of the performances of the sensor, i.e. of the linearity and of the speed of its response to glucose. This method, suitable for small laboratory animals, could therefore be used for the preliminary evaluation of glucose sensors, under in vivo conditions.     

Behavior reuse and virtual sensors in the evolution of complex behavior architectures

Summary In this paper we discuss some of the new work we have been carrying out with the objective of making evolutionarily obtained behavior based architectures and modules for autonomous robots more standardized and interchangeable. The architectures contemplated here are based on a multiple behavior structure where all of the modules, as well as their interconnections, are automatically obtained through evolutionary processes. The main objective of this line of research is to obtain procedures that permit producing behavior based controllers that work on real robots operating in real environments as independently of the platform as possible. In this particular paper we will concentrate on different aspects regarding the inclusion of virtual sensors as a way to make improved use of the capabilities of the different platforms and on the reuse of behavior modules. This reuse will be contemplated within the same behavioral architecture and from the point of view of transferring behavior modules from one platform to a different one.     

Photochemical oxygen evolution by Chlorella fusca in glucose

Summary Oxygen is evolved from illuminated Chlorella fusca, in absence of CO₂, on addition of glucose, fructose, mannose, and, to a small extent, galactose. The amount of oxygen, as measured by manometry, is equivalent to the amount of carbon from glucose incorporated into lipids, as determined radiochemically. This amount is much more than that incorporated in light in presence of CO₂, or in the dark. The rate of evolution of oxygen with glucose is about twice that with acetate of similar molarity, after an initial burst of respiration in case of acetate.     

Synthesis and performance of novel hydrogels coatings for implantable glucose sensors

Novel hydrogel polymers were prepared, characterized, coated on implantable glucose sensors, and tested in vitro and in vivo. The effects of 2,3-dihydroxypropyl methacrylate (DHPMA) on the swelling, morphology, glass transition (T(g)), and water structure were studied. The results show that the degree of swelling increases with increasing DHPMA content. Scanning electron microscopy (SEM) studies identified uniform, porous structures in samples containing 60-90 mol % DHPMA. Glass-transition temperatures did not change significantly with DHPMA content, but the ratio of freezing to nonfreezing water tended to increase with DHPMA content. Sensors coated with different hydrogels were prepared and in vitro evaluations were performed. The 80% DHPMA hydrogels exhibited optimum sensitivity, response, and stability when coated directly onto the sensor or top of a polyurethane (PU) layer. The histology results show that 80% DHPMA samples exhibit reduced fibrosis and reduced inflammation, resulting in a longer functional life.