Oxygen optrode for use in a fiber-optic glucose biosensor

An optical fiber oxygen sensor, based on the dynamic quenching of the luminescence of tris(1,10-phenanthroline)-ruthenium(II) cation by molecular oxygen, is presented. The complex is adsorbed onto silica gel, incorporated in a silicone matrix possessing a high oxygen permeability, and placed at the tip of the optical fiber. Oxygen has been monitored continuously in the 0-750 Torr range, with the detection limit being as low as 0.7 Torr. The device has been applied to the development of a fast responding and highly sensitive fiber-optic glucose biosensor based on this highly sensitive oxygen transducer. The sensor relates oxygen consumption (as a result of enzymatic oxidation) to glucose concentration. The enzyme is immobilized on the surface of the oxygen optrode; carbon black is used as an optical isolation in order to prevent ambient light and sample fluorescence to interfere. Measurements have been performed in a flow-through cell in air-equilibrated glucose standard solutions of pH 7.0. The effects of enzyme immobilization procedures (including enzyme immobilization on carbon black) as to response times (around 6 min), analytical ranges (0.06-1 mM glucose), reproducibility in sensor construction, and long-term stability have been studied as well.     

A glucose sensor made of an enzymatic clay-modified electrode and methyl viologen mediator

A novel glucose sensor has been contrived by immobilizing glucose oxidase between two nontronite clay coatings on glassy carbon electrode with methyl viologen as mediator. The sandwich configuration proved to be very effective in the determination of glucose. The response of the glucose sensor was determined by measuring cyclic voltammetric peak current values under aerobic solution conditions. The effects of the amount of enzyme immobilized, the operating pH, and the common interferences on the response of the glucose sensor were studied. The detection limit was 5 μM, with a linear range extending to about 6 mM, giving a dynamic range of over 3 orders of magnitude for 0.1 mM methyl viologen. When stored in pH 7 phosphate buffer at 4 °C, the sensor shows almost no change in performance after operating for at least 2 months. A mechanism for the operation of the glucose sensor is also proposed.     

Needle-type dual microsensor for the simultaneous monitoring of glucose and insulin

A miniature needle-type sensor suitable for the simultaneous amperometric monitoring of glucose and insulin is described. The integrated microsensor consists of dual (biologically and chemically) modified carbon-paste working electrodes inserted into a 14-guage needle. The glucose probe is based on the biocatalytic action of glucose oxidase, and the insulin one relies on the electrocatalytic activity of ruthenium oxide. The analytical performance of the dual sensor is assessed under flow injection conditions. The needle dual detector exhibits a very rapid response to dynamic changes in the concentrations of glucose and insulin. No apparent cross reactivity is observed in mixtures containing millimolar glucose levels and nanomolar insulin concentrations. The response is highly linear (to at least 1000 nM insulin and 14 mM glucose) and reproducible (RSD = 2.6-4.1%). The combination microsensor holds great promise for real-time measurements of the insulin/glucose ratio and for improved management of diabetes.     

Affinity-based turbidity sensor for glucose monitoring by optical coherence tomography: toward the development of an implantable sensor

We investigated the feasibility of constructing an implantable optical-based sensor for seminoninvasive continuous monitoring of analytes. In this novel sensor, analyte concentration-dependent changes induced in the degree of optical turbidity of the sensing element can be accurately monitored by optical coherence tomography (OCT), an interferometric technique. To demonstrate proof-of-concept, we engineered a sensor for monitoring glucose concentration that enabled us to quantitatively monitor the glucose-specific changes induced in bulk scattering (turbidity) of the sensor. The sensor consists of a glucose-permeable membrane housing that contains a suspension of macroporous hydrogel particles and concanavalin A (ConA), a glucose-specific lectin, that are designed to alter the optical scattering of the sensor as a function of glucose concentration. The mechanism of modulation of bulk turbidity in the sensor is based on glucose-specific affinity binding of ConA to pendant glucose residues of macroporous hydrogel particles. The affinity-based modulation of the scattering coefficient was significantly enhanced by optimizing particle size, particle size distribution, and ConA concentration. Successful operation of the sensor was demonstrated under in vitro condition where excellent reversibility and stability (160 days) of prototype sensors with good overall response over the physiological glucose concentration range (2.5-20 mM) and good accuracy (standard deviation 5%) were observed. Furthermore, to assess the feasibility of using the novel sensor as one that can be implanted under skin, the sensor was covered by a 0.4 mm thick tissue phantom where it was demonstrable that the response of the sensor to 10 mM glucose change could still be measured in the presence of a layer of tissue shielding the sensor aiming to simulate in vivo condition. In summary, we have demonstrated that it is feasible to develop an affinity-based turbidity sensor that can exhibit a highly specific optical response as a function of changes in local glucose concentration and such response can be accurately monitored by OCT suggesting that the novel sensor can potentially be engineered to be used as an implantable sensor for in vivo monitoring of analytes.     

Amperometric glucose biosensor based on assisted ion transfer through gel-supported microinterfaces

A novel amperometric glucose sensor was developed based on the facilitated proton transfer across microinterfaces between two immiscible electrolyte solutions. The combination of a 1,3:2,4-dibenzylidene sorbitol/2-nitrophenyl octyl ether gel membrane and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine as the ionophore allows the transfer of protons from water to the gellified organic phase; the gel membrane is supported on arrays of microholes drilled on a polyester film. The protons are generated as the result of the dissociation of gluconic acid produced during the enzymatic degradation of glucose by glucose oxidase. The characteristics of the glucose sensor were investigated using several experimental conditions, namely, the concentration of ligand and enzyme. The electrochemical response is typical of an enzymatic electrode and displays a linear behavior in the range 0.2-3 mM glucose. The effect of the experimental parameters of the voltammetric technique was also optimized with the aim of improving sensor sensitivity.     

In situ assembled mass-transport controlling micromembranes and their application in implanted amperometric glucose sensors

Micromembranes were assembled by sequentially chemisorbing polyanions and polycations on miniature (5 x 10(-4) cm2) enzyme electrodes. The sequential chemisorption process allowed the simultaneous tailoring of their sensitivity, dynamic range, drift, and selectivity. When assembled on tips of 250-microm-diameter gold wires coated with redox polymer-"wired" glucose oxidase, they allowed tailoring of the glucose electrodes for > 2 nA/mM sensitivity; 0-30 mM dynamic range; drift of < or =5% per 24 h at 37 degrees C at 15 mM glucose concentration; and < or =5% current increment by the combination of 0.1 mM ascorbate, 0.2 mM acetaminophen, and 0.5 mM urate. The membranes also retained transition metal ions that bound to and damaged the redox polymer "wiring" the enzyme. The electrodes were tested in the jugular veins and in the intrascapular subcutaneous region of anaesthetized and heparinized nondiabetic Sprague-Dawley rats, in which rapid changes of glycemia were forced by intravenous injections of glucose and insulin. After one-point in vivo calibration of the electrodes, all of the 152 data points were clinically accurate when it was assumed that after insulin injection the glycemia in the subcutaneous fluid lags by 9 min behind that of blood withdrawn from the insulin-injected vein.     

A fluorescence affinity hollow fiber sensor for continuous transdermal glucose monitoring

A novel concept of a fluorescence affinity hollow fiber sensor for transdermal glucose monitoring is demonstrated. The glucose-sensing principle is based on the competitive reversible binding of a mobile fluorophore-labeled Concanavalin A (Con A) to immobile pendant glucose moites inside of intensely colored Sephadex beads. The highly porous beads (molecular weight cutoff of 200 kDa) were colored with two red dyes, Safranin O and Pararosanilin, selected to block the excitation and spectrum of the fluorophore Alexa488. The sensor consists of the dyed beads and Alexa488-Con A confined inside a sealed, small segment of a hollow fiber dialysis membrane (diameter 0.5 mm, length 0.5 cm, molecular weight cutoff 10 kDa). In the absence of glucose, the majority of Alexa488-Con A resides inside the colored beads bound to fixed glucose. Thus, excitation light at 490 nm impinging on the sensor is strongly absorbed by the dyes, resulting in a drastically reduced fluorescence emission at 520 nm from the Alexa488-Con A residing within the beads. However, when the hollow fiber sensor is exposed to glucose, glucose diffuses through the membrane into the sensor chamber and competitively displaces Alexa 488-Con A molecules from the glucose residues of the Sephadex beads. Thus, Alexa 488-Con A appears in the void space outside of the beads and is fully exposed to the excitation light, and a strong increase in fluorescence emission at 520 nm is measured. At a medium to high loading degree of Sephadex with Alexa488-Con A (10 mg mL(-1) bead suspension), the absolute fluorescence increase due to 20 mM glucose was very large. It exceeded the response of other sensor devices based on FRET by a factor of 50 (Meadows and Schultz Anal. Chim. Acta 1993, 280, 21-30; Russell et al. Anal. Chem. 1999, 71, 3126-3132). The new sensor featured a glucose detection range extending from 0.15 to 100 mM, exhibiting the strongest dynamic signal change from 0.2 to 30 mM. It showed a reasonably fast response time (4-5 min). The combination of all the beneficial sensor features makes this sensor extremely attractive for future in vivo implantation studies for glucose monitoring in subdermal tissue.     

ENFET glucose biosensor produced with mesoporous silica microspheres

The biosensors based on ENFETs for direct glucose oxidase (GOD) concentration analysis have been fabricated. Mesoporous silica (MS) microspheres of different pore sizes were used as the substrates for immobilizing enzyme to construct ENFETs. The MS microspheres show significantly improved enzyme immobilization capacity. The glucose sensitive ENFET based on the modification of the gate surface of ion-sensitive field-effect transistors (ISFETs) with MS microspheres and glucose oxidase (GOD) shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. MS microspheres of different pore sizes were investigated and the sensor with remodeled MS microspheres which experienced the pore expanding treatment show better performance than untreated MS microspheres. The fabricated sensors have a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.10 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability and reproducibility.     

Design and in vitro studies of a needle-type glucose sensor for subcutaneous monitoring.

A new miniaturized glucose oxidase based needle-type glucose microsensor has been developed for subcutaneous glucose monitoring. The sensor is equivalent in shape and size to a 26-guage needle (0.45-mm o.d.) and can be implanted with ease without any incision. The novel configuration greatly facilitates the deposition of enzyme and polymer films so that sensors with characteristics suitable for in vivo use (upper limit of linear range greater than 15 mM, response time less than 5 min, and sensitivity yielding a 5:1 signal-to-background ratio at normal basal glucose levels) can be prepared in high yield (greater than 60%). The sensor response is largely independent of oxygen tension in the normal physiological range. It also exhibits good selectivity against common interferences except for the exogenous drug acetaminophen.     

A novel non-enzymatic glucose sensor based on nickel (II) oxide electrospun nanofibers

A new enzymeless glucose sensor has been fabricated via electrospinning technology and subsequent calcination. The morphology and structure of the as-prepared nanofibers have been characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The electrocatalytic oxidation of glucose in alkaline medium at nickel oxide modified glassy carbon electrodes has been investigated. The modified electrodes offer excellent electrocatalytic activity toward the glucose oxidation at low positive potential (0.3 V). Glucose has been determined chronoamperometrically at the surface of NiO nanofibers modified electrode in 0.5 mM NaOH. Under the optimized condition, the calibration curve is linear in the concentration range of 2 x 10(-3) mM - 1 mM, and 1 mM - 9.5 mM. The detection limit (signal-to-noise 3) and response time are 3.394 x 10(-6) M and 2 s, respectively. The NiO electrospun nanofibers is easy to prepare and feasible in economy. The modified electrode is steady and can be used repeatedly, so it is reasonable to expect its broad use in non-enzymatic glucose sensor.     

Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic beta-cells

Glucose gradients generated by an artificial source and beta-cells were measured using an enzyme-based glucose microsensor, 8-microm tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 +/- 0.4 pmol.cm(-2).s(-1) (mean +/- SEM, n = 7) with a sensor sensitivity of 7.0 +/- 0.4 pA/ mM (mean +/- SEM, n = 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from beta-cell clusters, measured in the presence of 5 mM glucose, was 61.7 +/- 9.5 fmol.nL(-1).s(-1) (mean +/- SEM, n = 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 microM) transiently increased, subsequently decreasing to below basal by 93 +/- 16 and 56 +/- 6%, respectively (mean +/- SEM, n = 5). Consumption was decreased after the application of 10 microM rotenone by 74 +/- 5% (mean +/- SEM, n = 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment.     

Paper bioassay based on ceria nanoparticles as colorimetric probes

We report the first use of redox nanoparticles of cerium oxide as colorimetric probes in bioanalysis. The method is based on changes in the physicochemical properties of ceria nanoparticles, used here as chromogenic indicators, in response to the analyte. We show that these particles can be fully integrated in a paper-based bioassay. To construct the sensor, ceria nanoparticles and glucose oxidase were coimmobilized onto filter paper using a silanization procedure. In the presence of glucose, the enzymatically generated hydrogen peroxide induces a visual color change of the ceria nanoparticles immobilized onto the bioactive sensing paper, from white-yellowish to dark orange, in a concentration-dependent manner. A detection limit of 0.5 mM glucose with a linear range up to 100 mM and a reproducibility of 4.3% for n = 11 ceria paper strips were obtained. The assay is fully reversible and can be reused for at least 10 consecutive measurement cycles, without significant loss of activity. Another unique feature is that it does not require external reagents, as all the sensing components are fixed onto the paper platform. The bioassay can be stored for at least 79 days at room temperature while maintaining the same analytical performance. An example of analytical application was demonstrated for the detection of glucose in human serum. The results demonstrate the potential of this type of nanoparticles as novel components in the development of robust colorimetric bioassays.     

A disposable amperometric sensor screen printed on a nitrocellulose strip: a glucose biosensor employing lead oxide as an interference-removing agent

A new type of disposable amperometric sensor is devised by screen printing thick-film electrodes directly on a porous nitrocellulose (NC) strip. The chromatographic NC strip is then utilized to introduce various sample pretreatment layers. As a preliminary application, a glucose biosensor based on hydrogen peroxide detection is constructed by immobilizing glucose oxidase (GOx) on the NC electrode strip and by formulating a strong oxidation layer (i.e., PbO2) at the sample loading area, placed below the GOx reaction band. The screen-printed PbO2 paste serves as a sample pretreatment layer that removes interference by its strong oxidizing ability. Samples applied are carried chromatographically, via the PbO2 paste, to the GOx layer, and glucose is catalyzed to liberate hydrogen peroxide, which is then detected at the electrode surface. The proposed NC/PbO2 strip sensor is shown to be virtually insusceptible to interfering species such as acetaminophen and ascorbic and uric acids and to exhibit good performance, in terms of the sensor-to-sensor reproducibility (standard deviation, +/-0.026 - +/-0.086 microA), the sensitivity (slope, -0.183 microA/mM), and the linearity (correlation coefficient, 0.994 in the range of 0-10 mM).     

Simulations of the frequency response of implantable glucose sensors

The response of enzyme electrode glucose sensors implanted in tissues to physiologic blood glucose oscillations is simulated. Models describe both oxygen-based and peroxide-based glucose sensors in spatially homogeneous medium simulating some mass transfer properties of tissue. Pass-through ratios and delays are reported as a function of frequency for the oxygen-based sensor, and the effects on continuous blood glucose monitoring are illustrated using data from the literature. Certain peroxide-based sensor designs may produce common signals for different glucose concentrations, a characteristic not found in oxygen-based sensors. The dynamic response depends on the frequency of glucose oscillation and is sensitive to sensor type, enzyme activity, and diffusional resistance.     

Monomolecular enzyme films stabilized by amphiphilic polyelectrolytes for biosensor devices

This paper describes the use of new amphiphilic polyelectrolytes for protein immobilization. Monomolecular films of glucose oxidase (GO) and monoamine oxidase (MAO) stabilized by amphiphilic polyelectrolytes (polyethyleneimine and poly-4-vinylpyridine derivatives modified by lauryl chains) were formed on a water surface in a Langmuir-Blodgett trough. The compressed films containing the enzymes were transferred onto the surface of a polypropylene membrane of a Clark electrode according to the Langmuir-Schaefer method. The analytical responses of the resulting biosensors were linear over the range 1–10 mM of glucose and 8–100 μM of tyramine. Furthermore, direct functional coupling of GO and ferrocenecarboxylic acid in multilayer films stabilized by amphiphilic polyelectrolytes was demonstrated. The amperometric response of such a sensor was linear over the range 1–20 mM of glucose. The dependence of the kinetic parameters of the enzymes on the amphiphilic polyelectrolyte structures is discussed.     

Electrochemically mediated electrodeposition/electropolymerization to yield a glucose microbiosensor with improved characteristics.

A procedure is described that provides for electrochemically mediated deposition of enzyme and a polymer layer permselective for endogenous electroactive species. Electrodeposition was first employed for the direct immobilization of glucose oxidase to produce a uniform, thin, and compact film on a Pt electrode. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)trimethoxysilane was constructed by electrochemically assisted cross-linking. This hybrid film outside the enzyme layer contributed to the improved stability and permselectivity. The resulting glucose sensor was characterized by a short response time (<4 s), high sensitivity (1200 nA/mM x cm2), low interference from endogenous electroactive species, and working lifetime of more than 50 days.     

Real-time glucose sensing by surface-enhanced Raman spectroscopy in bovine plasma facilitated by a mixed decanethiol/mercaptohexanol partition layer

A new, mixed decanethiol (DT)/mercaptohexanol (MH) partition layer with dramatically improved properties has been developed for glucose sensing by surface-enhanced Raman spectroscopy. This work represents significant progress toward our long-term goal of a minimally invasive, continuous, reusable glucose sensor. The DT/MH-functionalized surface has greater temporal stability, demonstrates rapid, reversible partitioning and departitioning, and is simpler to control compared to the tri(ethylene glycol) monolayer used previously. The data herein show that this DT/MH-functionalized surface is stable for at least 10 days in bovine plasma. Reversibility is demonstrated by exposing the sensor alternately to 0 and 100 mM aqueous glucose solutions (pH approximately 7). The difference spectra show that complete partitioning and departitioning occur. Furthermore, physiological levels of glucose in two complex media were quantified using multivariate analysis. In the first system, the sensor is exposed to a solution consisting of water with 1 mM lactate and 2.5 mM urea. The root-mean-squared error of prediction (RMSEP) is 92.17 mg/dL (5.12 mM) with 87% of the validation points falling within the A and B range of the Clarke error grid. In the second, more complex system, glucose is measured in the presence of bovine plasma. The RMSEP is 83.16 mg/dL (4.62 mM) with 85% of the validation points falling within the A and B range of the Clarke error grid. Finally, to evaluate the real-time response of the sensor, the 1/e time constant for glucose partitioning and departitioning in the bovine plasma environment was calculated. The time constant is 28 s for partitioning and 25 s for departitioning, indicating the rapid interaction between the SAM and glucose that is essential for continuous sensing.     

ENFET glucose biosensor produced with dendrimer encapsulated Pt nanoparticles

The biosensors based on ENFETs for direct glucose concentration analysis have been fabricated by introducing dendrimer encapsulated Pt nanoparticles and glucose oxidase (GOx) via a layer-by-layer self-assembly method. The free amine groups located on each poly(amidoamine) dendrimer molecule were exploited to immobilize enzyme through covalent attachment. Depending on metal nanoparticles within dendrimers and biocompatibility of dendrimers, the fabricated glucose sensitive ENFET shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. The fabricated sensor has a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.15 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability, which response could be used for detecting of glucose samples at intervals for at least 1 month when it stored in dry state at 4 °C.     

Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film

o-Phenylenediamine has been used for glucose oxidase (GOx) immobilization on Pt electrodes by electrochemical polymerization at +0.65 V vs SCE. By this approach the enzyme is entrapped in a strongly adherent, highly reproducible thin membrane, whose thickness is around 10 nm. This one-step procedure produces a glucose sensor with a response time less than 1 s, an active enzyme loading higher than 3 units/cm2 of electrode surface, a high sensitivity, and a sufficiently wide linear range. The glucose response shows an apparent Michaelis-Menten constant, K'm = 14.2 mM, and a limiting current density, jmax of 181 microA/cm2. The product kD of partition and diffusion coefficients of glucose in the polymer film is on the order of 10(-13) cm2/s. Due to permselectivity characteristics of the membrane, the access of ascorbate, a common interfering species, to the electrode surface is blocked. To our knowledge, this represents the first report of a membrane capable, at the same time, of immobilizing GOx and rejecting ascorbate. The interesting electrode behavior can be rationalized by using an existing model predicting the amperometric response of an immobilized GOx system.     

Design and characterization of immobilized enzymes in microfluidic systems.

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.