Construction of a panel of glucose indicator proteins for continuous glucose monitoring

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi-pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0-200 μM) within living cells through FRET imaging microscopy measurement.     

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.     

Continuous monitoring of blood glucose

Continuous blood glucose monitoring aims to: better evaluate glycaemic variations; better detect hypoglycaemia; and, ultimately, automatize insulin delivery (artificial beta cell). The sensors can be fully implantable, with the challenge of constructing durable systems to avoid repeated implantations. In-dwelling needle-like electrodes and microdialysis fibres with a pump that brings the dialysate to the glucose sensor are inserted in the subcutaneous tissue through the skin. The GlucoWatch is an almost non-invasive technique that extracts the extracellular fluid by iontophoresis. In these systems, the glucose oxidase generates the electrical signal, proportional to the glucose concentration. Non-invasive techniques aim at measuring the glucose concentration without breaching the skin, using absorption of light in the infrared spectrum. These techniques have not reached the necessary reliability for use as glycaemic alarms, and even less as artificial beta cells. Currently, glucose sensors are mainly used as glycaemic holters to help in the management of insulin therapy.     

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.     

Biosensors for in vivo glucose measurement: can we cross the experimental stage

The development of in vivo working glucose sensors needs two decades, so far. The availability of long term functional implantable biosensors for continuous glucose measurings is a basic prerequisite for the individualized optimum insulin treatment of diabetics. Enzymatic electrochemical sensors are described which realize a functional stability over more than 2 years in vitro, however their function in vivo is limited due to certain bioincompatibility expressed by inflammation of the surrounding tissue, exudates, and immun reactions. The paper reflects an overview concerning different sensor covering materials used as more or less suitable diffusion membranes. From experimental studies in animals and human volunteers conclusions are drawn for further developmental steps of biosensors for in vivo use and for the applicability of glucose sensors for transient diagnostic purposes and as a basis for glucose controlled therapeutic measures. The results demonstrate that further progress aimed at long term biostability of implanted biosensors needs to solve technological problems and the serial production of sensors with really comparable qualities as a prerequisite for clinical trials.     

Concanavalin A for in vivo glucose sensing: a biotoxicity review

Over the last two decades there as has been surging scientific interest in employing the glucose- and mannose-specific lectin Concanavalin A (ConA) in affinity biosensors for in vivo glucose monitoring in diabetics. Numerous research groups have successfully shown in in vitro and in vivo studies that ConA-based affinity sensors can monitor glucose very accurately and reproducibly over many months, making ConA-based sensors an extremely interesting prospect for long-term implantation in humans. Despite this progress, there remains concern over the safety of ConA, which has widely been reported as a toxin in the literature. In this article, we review in vitro and in vivo studies related to ConA toxicity in order to assess the health risks posed by ConA in the context of an implantable biosensor. Based on the wealth of information available and on data from our own studies, we can conclude that the site of implantation (subcutaneous skin tissue) and the small amount of ConA (<10 microg/microl) being used in implantable glucose-sensitive detector devices like those proposed by various research groups would pose little or no health risk to its bearer even in the event of unexpected sensor rupture.     

In vitro testing of a simply constructed, highly stable glucose sensor suitable for implantation in diabetic patients

We have constructed and tested in vitro a potentially implantable, needle-type amperometric enzyme electrode which is suitable for continuous monitoring of glucose concentrations in diabetic patients. The major requirements of stability during operation and ease of manufacture have been met with a sensor design which involves a simple dip-coating procedure for applying to a platinum base electrode an inner membrane of glucose oxidase immobilised in polyhydroxyethyl methacrylate (pHEMA), and an outer membrane composed of a pHEMA/polyurethane mixture. Sensors were operated at 700 mV for detection of hydrogen peroxide. Calibration curves for the sensor were linear to at least 20 mM glucose and were unaffected by a reduction in PO2 from 20 to 5 kPa. During continuous operation in 5 mM buffered glucose solutions in vitro, sensors suffered no significant loss of response over periods of up to 60 h. Such electrodes are, therefore, useful for development as in vivo glucose sensors.     

The role of H2O2 outer diffusion on the performance of implantable glucose sensors

The performance of an implantable glucose sensor is strongly dependent on the ability of their outer membrane to govern the diffusion of the various participating species. In this contribution, using a series of layer-by-layer (LBL) assembled outer membranes, the role of outwards of H(2)O(2) diffusion through the outer membrane of glucose sensors has been correlated to sensor sensitivity. Glucose sensors with highly permeable humic acids/ferric cations (HAs/Fe(3+)) outer membranes displayed a combination of lower sensitivities and better linearities when compared with sensors coated with lesser permeable outer membranes (namely HAs/poly(diallyldimethylammonium chloride) (PDDA) and poly(styrene sulfonate) (PSS)/PDDA). On the basis of a comprehensive evaluation of the oxygen dependence of these sensors in conjunction with the permeability of H(2)O(2) through these membranes, it was concluded that the outer diffusion of H(2)O(2) is crucial to attain optimized sensor performance. This finding has important implications to the design of various bio-sensing elements employing perm-selective membranes.     

A long-term flexible minimally-invasive implantable glucose biosensor based on an epoxy-enhanced polyurethane membrane

This paper describes the preparation method as well as the in vitro and in vivo evaluation of a novel flexible glucose biosensor designed for long-term subcutaneous implantation. An epoxy-enhanced polyurethane membrane, which includes ca. 30–40% epoxy resin adhesive and 50–70% polyurethane, has been developed and used for the first time as the outer protective membrane of the sensor. This new membrane was developed to increase the in vivo durability and lifetime of implantable biosensors. This epoxy-polyurethane membrane was shown to be porous and is of excellent durability. A sensor with such a membrane shows excellent long-term stability and can last for 4–8 months in solutions at room temperature. To verify the in vivo performance of the sensor, nine sensors were implanted in three rats and tested regularly. Eight sensors kept functioning well in the rats for 10–56 days. The ninth sensor was damaged during implantation. All original sensitivity data as well as four response curves obtained at days 7, 17, 52 and 56, respectively are presented.     

An acoustic glucose sensor

In vivo glucose monitoring is required for tighter glycaemic control. This report describes a new approach to construct a miniature implantable device based on a magnetic acoustic resonance sensor (MARS). A ≈ 600-800 nm thick glucose-responsive poly(acrylamide-co-3-acrylamidophenylboronic acid) (poly(acrylamide-co-3-APB)) film was polymerised on the quartz disc (12 mm in diameter and 0.25 mm thick) of the MARS. The swelling/shrinking of the polymer film induced by the glucose binding to the phenylboronate caused changes in the resonance amplitude of the quartz disc in the MARS. A linear relationship between the response of the MARS and the glucose concentration in the range ≈ 0-15 mM was observed, with the optimum response of the MARS sensor being obtained when the polymer films contained ≈ 20 mol% 3-APB. The MARS glucose sensor also functioned under flow conditions (9 μl/min) with a response almost identical to the sensor under static or non-flow conditions. The results suggest that the MARS could offer a promising strategy for developing a small subcutaneously implanted continuous glucose monitor.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

The development of an implantable electrochemical glucose sensor: response to glucose in bovine serum ultrafiltrate.

A glucose sensor based on organic oxidation reactions at a platinum electrode is being developed as the key component for an implantable artificial beta cell for diabetic patients. Sensitivity of a membrane-covered platinum electrode to changing glucose concentration in bovine serum ultrafiltrate has been demonstrated, with current response as high as 60 microamperes per 100 mg/dl change in glucose concentration. Reproducibility of measurements probably will depend on satisfactory mechanical assembly of the membrane-electrode combination. An approach to selectivity based on multiple current measurements is suggested.     

Direct glucose determination in blood using a reagentless optical biosensor

This paper demonstrates that glucose determination in blood can be done directly (without sample pretreatment) using a reagentless reversible biosensor based on the intrinsic spectroscopic properties of peroxidase (HRP). The biosensor, prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix, works in continuous mode, presents a linear response range from 1.5 × 10⁻⁶ up to 5.5 × 10⁻⁵ M and can be used for at least 750 measurements; in the best conditions (0.1 M pH 6 phosphate buffer, HRP and GOx amounts in the polymersation mixture for the sensor film preparation 0.0165 and 0.0010 g, respectively) the minimum samples rate is 30 h⁻¹. For glucose determination, blood is simply diluted in water (until haemolysis is completed) and fed into the sensor without a cleaning step between samples; the blood absorption is corrected in a simple way by working at a proper reference wavelength. The biosensor signals have been mathematically modeled in order to facilitate the design of sensors based on the same idea for other biochemical compounds.     

Biosensor for continuos glucose monitoring

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.     

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.     

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.     

The function of a hydrogen peroxide-detecting electroenzymatic glucose electrode is markedly impaired in human sub-cutaneous tissue and plasma

Electroenzymatic glucose sensors implanted into sub-cutaneous (s.c.) tissue of human subjects and experimental animals exhibit lower sensitivities to glucose than in buffer solutions before implantation. The mechanism of the decrease of sensitivity is not known. Sensors used in this study were fabricated from platinum wires (diameter 0.125 mm) with covalently bound glucose oxidase at the tip of the wire. After coating the tip with polyurethane, wires were placed into 27 gauge steel needles. Sensors were operated potentiostatically at 700 mV against Ag/AgCl pseudo-reference electrodes. These sensors were implanted s.c. in 6 diabetic patients for 7 h. In 4 patients, sensors were responsive to successive increases of plasma glucose levels. Mean sensitivity to glucose in s.c. tissue was 29% of in vitro sensitivity. In 2 patients there was a sudden decrease of sensor currents, unrelated to glucose, shortly after implantation. Sensors were inhibited in human plasma to a similar extent. When sensors were exposed to native plasma and to plasma ultrafiltrate (mol. wt. <10 kDa) for 10 h, identical decreases of signals were found. Exposure to dialysed plasma (mol. wt. >12 kDa) caused much less decrease of sensor signals. Losses of sensor sensitivities to glucose in s.c. tissue and in plasma were totally reversible upon re-exposure of sensors to buffer solutions. We conclude that sensor inactivation in plasma and possibly in s.c. tissue is caused by low molecular weight substances not retained by the polyurethane membrane.     

A new amperometric glucose microsensor: in vitro and short-term in vivo evaluation

For biosensor fabrication, it is important to optimize materials and methods in order to create predictable function in vitro and in vivo. For this reason, we designed a new glucose sensor ('revised protocol') that utilized an outer permselective membrane made of amphiphobic polyurethane which allows glucose passage through hydrophilic segments. An inner polyethersulfone membrane, stabilized with a trimethoxysilane, provided specificity. Before application of the inner membrane, it was necessary to etch the platinum electrode with a radio frequency oxygen plasma. The revised protocol sensors (n=185) were compared with sensors fabricated with an earlier ('original') protocol (n=204) which used an outer polyurethane without hydrophilic segments and a complex inner membrane of cellulose acetate and Nafion. The function of revised protocol sensors was more predictable in vitro as evidenced by a much lower variation of glucose sensitivity than the original protocol sensors. Revised and original protocol sensors were nearly linear up to a glucose concentration of 20 mM. In vitro interference from 0.1 mM acetaminophen was minimal in both groups of sensors and would be expected to represent about 2% of the total sensor response at normal glucose levels for revised protocol sensors. Prolonged testing of the revised protocol sensors for 11 days during immersion in buffer revealed stable sensitivities (day 1: 6.12+/-1.34 nA/mM; day 3: 6.33+/-1.40; day 8: 7.13+/-1.39; and day 11: 7.56+/-1.47; sensitivity for day 1 vs. each other day: not significant) and no critical loss of glucose oxidase activity. The response of the revised protocol sensors (n=7) to intraperitoneal glucose was tested in rats approximately one day after subcutaneous implantation and the sensors tracked glucose closely with a slight lag of 3-6 min.     

On the possibility of real-time monitoring of glucose in cell culture by microdialysis using a fluorescent glucose binding protein sensor

Although glucose sensors with millimolar sensitivity are still the norm, there is now a developing interest in glucose sensors with micromolar sensitivity for applications in minimally invasive sampling techniques such as fast microdialysis and extraction of interstitial fluid by iontophoresis and laser poration. In this regard, the glucose binding protein (GBP) with a binding constant for glucose in the micromolar range is of particular relevance. GBP is one of the soluble binding proteins found in the periplasmic space of Gram-negative bacteria. Because of its hinge-like tertiary structure, glucose binding induces a large conformational change, which can be used for glucose sensing by attaching a polarity sensitive fluorescent probe to a site on the protein that is allosterically responsive to glucose binding. Correspondingly, the resulting optical biosensor has micromolar sensitivity to glucose. Because binding is reversible, the biosensor is reusable and can be stored at 4 degrees C for 6 months without losing its sensitivity. In this paper, we show the feasibility of using the GBP biosensor to monitor glucose in microdialysis. The effect of perfusion rate, bulk glucose concentration and temperature on microdialysis efficiency was determined. Additionally, the glucose concentrations in mammalian cell culture were monitored to demonstrate the applicability of this sensor in complex and dynamic processes over a period of time. As the sensor is sensitive to micromolar glucose, high dialysis efficiency is not required when the bulk glucose concentration is within the millimolar physiological range. Thus, a perfusion rate of 10 microL/min or faster can be used, resulting in delay times of 1 min or less.     

Thermal stabilization of glucose oxidase and glucoamylase by physical entrapment.

Physical entrapment was used as an approach to achieve thermal stabilization of enzymes. The t 1/2 values for the thermoinactivation of glucose oxidase and glucoamylase were increased several-fold by their entrapment in polyacrylamide gels. In polyacrylate gels the individual enzymes behaved differently, probably owing to microenvironmental effects arising by the polyelectrolyte nature of the carrier.