In vivo evaluation of an electroenzymatic glucose sensor implanted in subcutaneous tissue

Cleanroom processing techniques have been used to mass-produce flexible, electroenzymatic glucose sensors designed for implantation in subcutaneous tissue. In vitro characterization studies have shown the sensor's performance to be acceptable. Initial in vivo studies were conducted with the sensor implanted in the subcutaneous tissue of rabbits. Sensors implanted in the subcutaneous tissue of normal human subjects showed an excellent correlation between glucose concentrations measured by the sensor and capillary finger sticks measured with a commercial analyzer.     

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.     

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.     

Variants of the tissue-sensor array window chamber

Sensors are being developed that can be implanted in tissues for continuous monitoring of oxygen, glucose, and other metabolites. However, there have been difficulties in inferring metabolite concentrations in blood from the signals of tissue sensors due to the properties of tissues at the implant site and local physiological phenomena that can affect sensor responses. A multisensor array has been previously developed for implantation in a hamster skinfold window chamber preparation to study these effects. The preparation allows recording of concentration-dependent signals from multiple sensors while nondestructively visualizing the adjacent tissue and microvascular function. Variants of the tissue-sensor array window chamber described here have respective advantages over the original chamber design, including improved tissue visualization and reduced surgical intervention, and allow exposure of the sensor to different tissues. Results indicate that mass transfer within tissues is heterogeneous, and sensor signals are affected by variable perfusion of local microvasculature in addition to vascular metabolite concentration. These observations suggest new strategies for sensor design and operation. Window chamber variants are important tools for validation of implanted sensors.     

Lifespan of subcutaneous glucose sensors and their performances during dynamic glycaemia changes in rats

Performances of a glucose sensor have been investigated during dynamic variations of plasma glucose levels. Subcutaneous glucose concentrations measured by the sensors were calculated by a one-point calibration, performed in basal conditions. A first group of sensors were chronically implanted in the subcutaneous tissue of normal rats. The animals were submitted to glucagon and insulin injection, in order to induce rapid modifications of their glycaemia. This test was repeated at different days after implantation in order to investigate the lifespan and the performance of the sensors. All the sensors were working 1 or 2 days after implantation, and 70% adequately responded to glycaemia variations at day 3 or 4. The quality of the sensors' performance remained constant as a function of the time. With a second group of sensors, we demonstrated that an efficient sterilization procedure did not alter the sensors' characteristics. At the day of implantation, the sterilized sensors' performance, during dynamic variations of plasma glucose levels, was closely similar to that of the non-sterilized sensors. The animals bearing the sterilized devices were rendered diabetic by steptozotocin (STZ) injection. Once the rats had developed a severe hyperglycaemia (1–3 days after STZ), they were injected with intravenous insulin. The subcutaneously implanted glucose sensors correctly followed the decline in plasma glucose levels. We therefore conclude that our sensor could represent a useful tool for short-term continuous blood monitoring.     

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.     

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.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

Calibration in dogs of a subcutaneous miniaturized glucose sensor using a glucose meter for blood glucose determination.

The feasibility of calibrating a glucose sensor by using a wearable glucose meter for blood glucose determination and moderate variations of blood glucose concentration was assessed. Six miniaturized glucose sensors were implanted in the subcutaneous tissue of conscious dogs, and the parameters used for the in vivo calibration of the sensor (sensitivity coefficient and extrapolated current in the absence of glucose) were determined from values of blood glucose and sensor response obtained during glucose infusion. (1) Venous plasma glucose level and venous total blood glucose level were measured simultaneously on the same sample, using a Beckman analyser and a Glucometer II, respectively. The regression between plasma glucose (x) and whole blood glucose (y) was y = 1.12x-0.08 mM (n = 114 values, r = 0.96, p = 0.0001). The error grid analysis indicated that the use of a Glucometer II for blood glucose determination was appropriate in dogs. (2) The in vivo sensitivity coefficients were 0.57 +/- 0.11 nA mM-1 when determined from plasma glucose, and 0.51 +/- 0.07 nA mM-1 when determined from whole blood glucose (t = 1.53, p = 0.18, n.s.). The background currents were 0.88 +/- 0.57 nA when determined from plasma glucose, and 0.63 +/- 0.77 nA when determined from whole blood glucose (t = 0.82, p = 0.45, n.s.). (3) The regression equation of the estimation of the subcutaneous glucose level obtained from the two methods was y = 1.04x + 0.56 mM (n = 171 values, r = 0.98, p = 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Needle-type lactate biosensor

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.     

Distribution of [3H]dexamethasone in rat subcutaneous tissue after delivery from osmotic pumps

Inflammation surrounding implantable glucose sensors may be controlled through local release of dexamethasone at the site of implantation. In the present study, we evaluated the distribution of dexamethasone in rat subcutaneous tissue during the first 2.5 days after local release. Osmotic pumps containing [3H]dexamethasone were implanted into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H]dexamethasone within the subcutaneous tissue at 6, 24, and 60 h after implantation. Measured concentration profiles, near the catheter tip through which the agent was released, were compared to mathematical models of drug diffusion and elimination. The results demonstrate that the majority of the [3H]dexamethasone delivered into the subcutaneous tissue was found within a 3 mm region surrounding the catheter tip. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient for dexamethasone in subcutaneous tissue was found to be D = 4.11 +/- 1.77 x 10(-10) m2/s, and the elimination rate constant was found to be k = 3.65 +/- 2.24 x 10(-5) s(-1). The diffusion coefficient and elimination rate constants for dexamethasone in subcutaneous tissue have not been previously reported. The use of a mathematical model may be useful in predicting the effectiveness of local delivery of dexamethasone around implantable glucose sensors.     

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.     

Screen-printed enzyme sensors for l-lysine determination

Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer.     

First step toward near-infrared continuous glucose monitoring: in?vivo evaluation of antibody coupled biomaterials

Continuous glucose monitoring (CGM) is crucial in diabetic care. Long-term CGM systems however require an accurate sensor as well as a suitable measuring environment. Since large intravenous sensors are not feasible, measuring inside the interstitial fluid is considered the best alternative. This option, unfortunately, has the drawback of a lag time with blood glucose values. A good strategy to circumvent this is to enhance tissue integration and enrich the peri-implant vasculature. Implants of different optically transparent biomaterials (poly(methyl-methacrylate) [PMMA] and poly(dimethylsiloxane) [PDMS]) – enabling glucose monitoring in the near-infrared (NIR) spectrum – were surface-treated and subsequently implanted in goats at various implantation sites for up to 3 months. The overall in vivo biocompatibility, tissue integration, and vascularization at close proximity of the surfaces of these materials were assessed. Histological screening showed similar tissue reactions independent of the implantation site. No significant inflammation reaction was observed. Tissue integration and vascularization correlated, to some extent, with the biomaterial composition. A modification strategy, in which a vascular endothelial-cadherin antibody was coupled to the biomaterials surface through a dopamine layer, showed significantly enhanced vascularization 3 months after subcutaneous implantation. Our results suggest that the developed strategy enables the creation of tissue interactive NIR transparent packaging materials, opening the possibility of continuous glucose monitoring.     

Near‐infrared bulk optical properties of goat wound tissue and human serum: consequences for an implantable optical glucose sensor

Near‐infrared (NIR) spectroscopy offers a promising technological platform for continuous glucose monitoring in the human body. Moreover, these measurements could be performed in vivo with an implantable single‐chip based optical sensor. However, a thin tissue layer may grow in the optical path of the sensor. As most biological tissues are highly scattering, they only allow a small fraction of the collimated light to pass, significantly reducing the light throughput. To quantify the effect of a thin tissue layer in the optical path, the bulk optical properties of serum and tissue samples grown on implanted dummy sensors were characterized using double integrating sphere and unscattered transmittance measurements. The estimated bulk optical properties were then used to calculate the light attenuation through a thin tissue layer. The combination band of glucose was found to be the better option, relative to the first overtone band, as the absorptivity of glucose molecules is higher, while the reduction in unscattered transmittance due to tissue growth is less. Additionally, as the wound tissue was found to be highly scattering, the unscattered transmittance of the tissue layer is expected to be very low. Therefore, a sensor configuration which measures the diffuse transmittance and/or reflectance instead was recommended.

( a ) Dummy sensor; ( b ) explanted dummy sensor in tissue lump; ( c ) removal of dummy sensor from tissue lump; and ( d ) 900 µm slices of tissue lump.     

Thermoelectric enzyme sensor for measuring blood glucose

A new calorimetric sensor has been developed which employs a thin-film thermopile in association with an immobilized enzyme. The thermopile detects the minute temperature rise that occurs when a specific chemical substrate is catalyzed by the enzyme. A prototype sensor is described which generates an equivalent proportional voltage response to glucose concentrations present in either buffer solution or blood. These sensors have remained useful for up to 18 days when operated intermittently for measuring glucose in buffer solutions, or for up to 4 days when operated continuously. When implanted inside cardiovascular shunts on anesthetized dogs, the sensors responded appropriately to changes in the blood glucose concentration.     

Urea sensor with single poly(propylene) membrane

Urease was immobilized on the plasma-aminated surface of a hyfrophobic poly(propylene) (PP) membrane. This membrane, with urease matrix on one side while maintaining its original hydrophobic property on the other, was used to construct the urea sensor. The new urea sensors had response sensitivities ranged from 19 mV/decade to 30 mV/decade depending on the conditions of the plasma reaction. The enzyme electrode using single membrane gave a shorter response time as compared to the corresponding conventional electrode employing two seperate PP membranes. The sensitivity of the enzyme electrode increased with increasing buffer pH and reached a maximal level (40 mV/decade) at pH 7.6. The response sensitivity of the electrode was not affected by the change of buffer strength. Deamination of the plasma-modified hydrophobic PP membrane did not occur in aqueous environment judging from the stability of the urea electrode up to 12 days of operation. (c) 1992 John Wiley & Sons. Inc.     

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.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

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.