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.     

Rechargeable glucose electrodes for long-term implantation

A unique enzyme electrode was designed using glucose oxidase immobilized on fine graphite powder. The graphite-enzyme is in a fluid state which enables recharging of the system when the enzyme activity decreases, therefore allowing the system to have a long lifetime in a diabetic patient. The sensor was tested using glucose concentration in the range of 20–300 mg d⁻¹. The electrode construction includes a hydrophobic membrane and a platinum electrode for detection of hydrogen peroxide. The signal output is large with minimal noise when tested in buffer. The sensor has been performing at ambient temperature for 4 months when stored overnight at 4° C.     

The assessment of potentially interfering metabolites and dietary components in blood using an osmotic glucose sensor based on the concanavalin A-dextran affinity assay

Continuous surveillance of blood glucose is a prerogative of maintaining a tight glycaemic control in people suffering from diabetes mellitus. Implantable sensor technology offers the potential of conducting direct long term continuous glucose measurements, but current size restrictions and operational challenges have limited their applications. The osmotic sensor utilises diffusion to create a hydrostatic pressure that is independent of sensor operation and power consumption. This permits ultra-low power architectures to be realized with a minimal start-up time in a package suitable for miniaturization. In contrast, osmotic sensors suffer from the inability of their membranes to discriminate between different constituents in blood or the interstitial fluid that are of comparable size to glucose. By implementing an affinity assay based on the competitive bonding between concanavalin A and dextran, the selectivity of the membrane can be transferred to the glucose specific recognition of the affinity assay. The osmotic effect from the physiological levels of several key metabolites and nutritional components has been addressed identifying in particular ethanol, lactate and amino acids as potential interfering constituents. Both ascorbic acid and mannose would have a normal physiological concentration that is too low to be detected. The studies shows that an osmotic glucose sensor equipped with the con A-dextran affinity assay, is able to filter out potential interfering constituents present in blood, plasma and the interstitial fluid yet retaining a pressure that is proportional to glucose only.     

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

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

A solid-phase optoelectronic sensor for serum albumin

A simple solid-phase optoelectronic sensor for serum albumin is described. Bromocresol green covalently attached to a cellophane membrane is sandwiched between a red light emitting diode and a silicon photodiode with integral amplifier. Adsorption of serum albumin to the membrane at pH 3.8 causes a characteristic yellow to blue-green color change in the membrane and is thus monitored as a fall in the output voltage of the detector system. The response is reproducible and linear over the range 5–35 mg/ml albumin concentration. This report describes an investigation of this inexpensive and potentially reagentless albumin sensor.     

Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.     

A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor

A miniature Clark-type oxygen electrode was fabricated by anisotropically etching silicon. A two-gold-electrode configuration was used and a double-layered gas-permeable membrane was formed directly on the electrolyte, poly(vinyl-4-ethylpyridinium bromide) in the sensitive area. These materials improved the electrode's stability in long-term storage and sterilization tolerance to a practical level. The 90% response time averaged 80 s and residual current 10%, with a good linear calibration curve. The oxygen electrode was also used to make an integrated sensor for the simultaneous determination of glucose and oxygen. The glucose sensor's response time was 50-110 s, with good linearity in glucose concentrations between 56 microM and 1.1 mM at 37 degrees C, pH 7.0.     

A flow-through amperometric sensor based on dialysis tubing and free enzyme reactors.

A generic flow-through amperometric microenzyme sensor is described, which is based on semi-permeable dialysis tubing carrying the sample to be analyzed. This tubing (300 microm OD) is led through a small cavity, containing the working and reference electrode. By filling this cavity with a few microl of an appropriate enzyme solution, an amperometric enzyme sensor results. As the dialysis tubing is impermeable for large molecular species such as enzymes, this approach does not require any immobilization chemistry, and as a consequence the enzyme is present in its natural free form. Based on this principle, amperometric sensors for lactate, glucose, and glutamate were formed by filling cavities, precision machined in Perspex, with buffered solutions containing respectively, lactate-, glucose-, and glutamate-oxidase. All sensors showed a large linear range (0-35 mM for glucose, 0-3 mM for lactate, and 0-5 mM for glutamate) covering the complete physiological range. The lower detection limit was in the order of 15-50 microM. Applicability in flow injection analysis systems is demonstrated.     

Glucose sensor with improved haemocompatibilty

A new biocompatible copolymer has been synthesised and used in an electrochemical enzyme-based glucose sensor. The copolymer incorporates three segments including a monomer with an electrically neutral phosphorylcholine head group that is able to reject protein adsorption and two segments that increase the affinity to polyurethane substrate. Peel and solution circulation tests showed that this material has high attachment to polyurethane. With the new copolymer as the outermost layer and the polyurethane as the diffusion-limiting membrane, the sensor showed extended linearity up to 50 mM glucose and stable output in bovine serum for 70 h. During in vivo tests, the sensor exhibited a steady current signal and a rapid transient response when the glucose concentration was raised. These results imply that the haemocompatibility of the glucose sensor coated with the new copolymer has been improved, which is crucial for a sensor used for clinical real-time monitoring. The material may also be suitable for application to other implantable devices.     

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.     

An immersible manometric sensor for measurement of humidity and enzyme mediated changes in dissolved gas

An immersible manometric sensor was made by covering the gaseous cavity of a pressure transducer with a 1 microm controlled pore membrane. Transfer of gas across the membrane allowed the pressure transducer to record changes in humidity or dissolved gas when immersed in solution. By immersing the sensor in distilled water, atmospheric humidity could be estimated by the deficit of atmospheric vapor pressure from saturation. In another application of the sensor, CO(2) was monitored continuously. This was not possible in previous closed-reactor type manometric sensors, and may allow the new technology to be used in applications requiring continuous monitoring of a process or stream. By coupling the sensor with enzymes liberating or consuming dissolved gas, different chemicals could be estimated. Urea was estimated by first hydrolyzing it with urease and then measuring the resulting CO(2) gas in solution. Glucose was measured through its enzymatic oxidation by glucose oxidase. The sensitivity to urea over the range 0-2.5 mM was about 1.02 kPa/mM, and the standard error was 0.086 mM. Due to the lower solubility of oxygen, the sensitivity to glucose in a range from 0 to 10 microM was over 100 kPa/mM, with a standard error of only 0.76 microM. This sensitivity was not possible in closed-reactor type manometric sensors due to constraints of dimensioning the head space gas volume for reproducibility and effective mass transfer. The 90% rise times for the sensor ranged from about 1-60 min for the different applications. The dynamic characteristics of the device may be improved by using a membrane with greater porosity, higher rigidity and lower thickness, and by reducing the dimensions of the cavity volume in the sensor through integrated microfabrication of the membrane onto the transducer.     

The use of co-immobilization of Trichosporon cutaneum and Bacillus licheniformis for a BOD sensor

The microorganisms Trichosporon cutaneum and Bacillus licheniformis were used to develop a microbial biochemical oxygen demand (BOD) sensor. It was found that T. cutaneum gave a greater response to glucose, whereas B. licheniformis gave a better response to glutamic acid. Hence, co-immobilized T. cutaneum and B. licheniformis were used to construct a glucose and glutamic acid sensor with improved sensitivity and dynamic range. A membrane loading of T. cutaneum at 1.1x10(8 )cells ml(-1) cm(-2) and B. licheniformis at 2.2x10(8) cells ml(-1) cm(-2) gave the optimum result: a linear range up to 40 mg BOD l(-1) with a sensitivity of 5.84 nA mg(-1) BOD l. The optimized BOD sensor showed operation stability for 58 intermittent batch measurements, with a standard deviation of 0.0362 and a variance of 0.131 nA. The response time of the co-immobilized microbial BOD sensor was within 5-10 min by steady-state measurement and the detection limit was 0.5 mg BOD l(-1). The BOD sensor was insensitive to pH in the range of pH 6.8-7.2.     

Sensitive immunosensor for tumor necrosis factor α based on dual signal amplification of ferrocene modified self-assembled peptide nanowire and glucose oxidase functionalized gold nanorod

Sensitive electrochemical immunosensor for the detection of protein biomarker tumor necrosis factor α (TNF-α) was reported that uses ferrocene carboxylic acid (Fc) functionalized self-assembled peptide nanowire (Fc-PNW) as sensor platform and glucose oxidase (GOx) modified gold nanorod (GNR) as label. Greatly enhanced sensitivity is achieved based on a dual signal amplification strategy: first, the synthesized Fc-PNW used as the sensor platform increased the loading of primary anti-TNF-α antibody (Ab(1)) onto electrode surface due to its large surface area. At the same time, the Fc moiety on the nanowire is used as a mediator for GOx to catalyze the glucose reaction. Second, multiple GOx and secondary anti-TNF-α antibody (Ab(2)) molecules are bounded onto each GNR to increase the sensitivity of the immunosensor. After the preparation of the immunosensor based on the traditional sandwich protocol, the response of the immunosensor towards glucose was used as a signal to differentiate various concentrations of TNF-α. The resulting immunosensor has high sensitivity, wide linear range (0.005-10ng/mL) and good selectivity. This immunosensor preparation strategy is a promising platform for clinical screening of protein biomarkers.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Adaptation of a manometric biosensor to measure glucose and lactose

A manometric sensor previously developed to measure urea was modified to measure glucose and lactose through enzymatic oxidation. Change in pressure in an enclosed cavity was correlated to the depletion of oxygen resulting from the enzymatic oxidation of glucose or lactose. The response of the sensor was linear and could be made adjustable over a large range by adjusting the amount of sample loaded into the fixed volume reactor. Because of the slow mutarotation of glucose, the oxidation of glucose was not allowed to proceed to completion. Therefore, the precision of the sensor (approximately 0.2 mM in a range from 0 to 5 mM) was limited by variations in the oxidation rate of glucose by glucose oxidase. Because the assay for lactose measured glucose subsequent to the hydrolysis of lactose by beta-galactosidase, the same degree of precision was observed in lactose. Milk lactose, typically at concentrations of about 150 mM, was estimated using the lactose assay after first diluting the samples. For many fluids such as milk, the use of manometric sensors for oxidizable substrates may be preferable to optical and electrochemical methods because they are robust and suffer a low degree of optical and chemical interferences. Glucose and lactose are representative of many important oxidizable substrates, which may be determined in this manner, many of which do not suffer from limitations caused by mutarotation. In theory, detection limits less than 1 microM may be achieved using these methods.     

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.     

Characterization of a low-cost PIN photodiode for dosimetry in diagnostic radiology

A commercial silicon PIN-photodiode was tested and characterized as ionizing radiation detector for radiological applications. A current-to-voltage amplification stage was designed and realized in order to acquire the photodiode signal in current mode. The system was tested with clinical beams routinely used for radiography and mammography. A Monte Carlo simulation of the detector was performed with the MCNPX code in order to model and fully understand, in particular, the impact of the sensor casing on the low energy response of the device. A reproducible output linearity was found over the dose range 0.03–4.5 mGy of great clinical relevance. The system sensitivity was found to be stable at 0.2 V s Gy⁻¹ for effective X-ray energies between 17 and 40 keV. The batch-to-batch reproducibility of the diodes was also experimentally investigated for two different batches of 14 diodes each. An inter-comparison with dosimeters routinely used in medical physics (i.e. Barracuda MPD RTI) showed a linear correlation between PIN-photodiode readout and absorbed dose measured with Barracuda, in the range of doses received by mammography and radiology patients.     

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.     

Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, beta-galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or beta-galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60-180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2-16% sucrose, 2.8-8.3% lactose, or 0.1-1% starch. The relative errors were +/-4% for sucrose and +/-3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.