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.     

Improved electrochemical microsensor for the real-time simultaneous analysis of endogenous nitric oxide and carbon monoxide generation

An amperometric dual NO/CO microsensor was developed on the basis of a working electrode incorporating dual Pt microdisks (each diameter, 76 μm) and a Ag/AgCl reference electrode covered with a gas permeable membrane. One of the Pt disks was sequentially electrodeposited with Pt and Sn; the other Pt disk was deposited with Pt-Fe(III) oxide nanocomposites. The first showed activity for the oxidation of both NO and CO; the second showed activity only for NO oxidation. In the copresence of NO and CO, the currents measured at each electrode, respectively, represented the concentrations of CO and NO. The sensor showed high stability during the monitoring of organ tissue for at least 2.5 h and high selectivity to NO over CO at the Pt-Fe(III) oxide working electrode. Real-time coupled dynamic changes of NO and CO generated by a living C57 mouse kidney were monitored simultaneously and quantitatively in response to a NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester), for the first time. CO was found to increase and NO decreased upon addition of the inhibitor, suggesting a possible reciprocal interaction between these endogenous gases.     

Stationary-state oxidized platinum microsensor for selective and on-line monitoring of nitric oxide in biological preparations

Despite the multifaceted biomedical significance of NO, little progress has been achieved so far in the quantitative understanding of the signal transduction mechanisms where NO is involved. To help progress in this area, we propose a simple electrochemical NO sensor here, consisting of a glass sealed platinum microdisk electrode coated with cellulose acetate to reduce both surface fouling by proteins and response to potential interferences. A differential amperometry protocol is optimized to improve selectivity and provide a stationary oxidation state of the platinum surface, which prevents loss in sensitivity during long-term use. We found the oxidation of NO by O2 second order in [NO] with a rate constant of (8.0 +/- 0.4) x 10(6) M(-2) s(-1), in good agreement with literature data obtained by other than electrochemical methods. The release rates of NO detected in cultures of activated macrophages were on the order of 20 pmol/ (10(6)cells s) and correlated well with the nitrite content determined by the spectrophotometric Griess assay.     

Electrochemical system for the simultaneous monitoring of algal motility and phototaxis

A compact electrochemical system equipped with dual electrodes was developed for the simultaneous monitoring of motility and phototaxis of flagellates. Photoinduced behavior of unicellular flagellate alga Chlamydomonas reinhardtii in the presence of diltiazem, azide, or ethanol was recorded as changes in the redox currents for a coexisiting redox marker. The system responded to the chemicals in significantly different ways; it allows qualitative and semiquantitative evaluation of influences of chemical species on the algal cells. Therefore, the present system is potentially applicable to not only aquatic risk assessment but also screening of newly found and synthesized compounds in terms of hazard probability and severity. Additionally, it unveiled some new and interesting behaviors of the algae.     

On two pairs of simultaneous dual integral equations

Summary This paper considers a system of coupled pairs of dual integral equations with constant coefficients involving Bessel functions of orders zero and unity. A solution is obtained in terms of the coefficients by reducing the system to a single integral equation of the Wiener-Hopf type with both the sum and difference kernels present.A simple transformation of the system causes the coefficient of the sum kernel to vanish. The transformation leaves the Wiener-Hopf equation unaltered except for the coefficients which become complex. An equation of this type was solved by Spence in 1967. Although Spence's solution does not cover complex coefficients it can be modified to do so.The result is quoted in this paper and is used to solve the system of coupled pairs of dual integral equations of the present paper.The adhesive contact problem recently solved by Gladwell is one in which the solution technique of the present paper has proved useful.On leave of absence from the Department of Mathematics, University of Southampton.     

Modified dual lifetime referencing method for simultaneous optical determination and sensing of two analytes

Simultaneous fluorometric sensing of two analytes becomes possible using a modified dual lifetime referencing (m-DLR) method. In this scheme, two luminescent indicators are needed that have overlapping absorption and emission spectra but largely different decay times. They are excited by a single light source, and both emissions are measured simultaneously. In the frequency domain m-DLR method, the phase of the short-lived fluorescence of a first indicator is referenced against that of the long-lived luminescence of the second indicator. The analytical information is obtained by measurement of the phase shifts at two modulation frequencies. The method is demonstrated to work for the case of dually sensing oxygen and carbon dioxide. It benefits from simple instrumentation and optical setup. The approach is perceived to be of wide applicability. Examples include (1) analysis of two luminescent analytes, (2) analytical determinations that make use of two probes, and (3) sensing of two species such as carbon dioxide and oxygen (as demonstrated here), or oxygen and chlorophyll, provided the luminophores meet the condition of having largely different decay times and overlapping absorption and emission spectra.     

Can continuous glucose monitoring be used for the treatment of diabetes.

In the case of the glucose sensor, clinicians and chemists must cooperate in interdisciplinary research to carefully define the analytical problem. Although not specifically discussed in this article, another group that must participate in this effort is engineers. Their expertise is needed to design the monitoring and control unit that contains the alarm and pump systems. The glucose sensor must operate reliably in an in vivo environment, provide the clinical information needed, and be easy to operate and manufacture.     

双视角三维测量系统同时标定方法

针对现有标定方法在相机无公共视场情况下的局限性,本文提出使用双平面标定板对双相机进行同时标定的方法.通过推导两个相机与两个标定板间的坐标变换,将待标定相机与参考相机的相对位姿关系的求解转换为较为成熟的手眼标定方程求解.通过实验验证:该方法可实现双相机的同时标定,且方法的绝对误差不超过0.089 mm,较为可靠;在双视角...     

On-line simultaneous monitoring of polarization mode dispersion and chromatic dispersion

We have developed new expressions for power fading and average power fading owing to polarization mode dispersion (PMD) and chromatic dispersion in terms of the angle of precession of the output state of polarization about the PMD vector. Based on these expressions, a simple and novel pilot-tone-based technique for simultaneous monitoring of chromatic dispersion and PMD is proposed. Experimental results demonstrate the effectiveness of the proposed technique; these results agree well with the theoretical analysis.     

Optimal alarm-signal algorithms for gas analyzers in simultaneous concentration monitoring for several substances

A decision rule with multiple alternatives has been formulated for generating an alarm signal in simultaneous concentration monitoring for several substances in several gas analyzers, together with the conditions for limiting the false-alarm probability. Examaples are given, including ones where the uninformative parameters are not known a priori. A statistic is proposed for generating the false alarm signal for simultaneous concentration measurement.Translated from Izmeritel'naya Tekhnika, No. 12, pp. 47–50, December, 1993.     

Combined system for the simultaneous optical and electrochemical monitoring of intra- and extracellular NO produced by glioblastoma cells

A combined, optospectroscopic and electrochemical assay system for the simultaneous monitoring of intra- and extracellular production of biologically important species has been developed and assessed. The present model system evaluates intra- and extracellular nitric oxide produced by stimulated glioblastoma multiform cell line (A172). The production of endogenous NO was induced by phorbol-12-myristate-13-acetate and inhibited by N(omega)-nitro-l-arginine methyl ester. Intracellular production of NO was monitored via fluorescence image analysis using a 4,5-diaminofluorescein probe, while extracellular NO release was monitored via a chemically modified electrode, which was incorporated into an optically transparent cell chip. The results indicated that there was no mutual interference between the optical and electrochemical measurement systems. The response time of the combined optical/electrochemical system was found to be in the range of a few tens of seconds.     

Integrated circuit microsensor for selectively detecting nitrogen dioxide and diisopropyl methylphosphonate

A novel gas-sensitive microsensor, known as the interdiginatated gate electrode field effect transistor (IGEFET), was realized by selectively decositing a chemically-active, electron-beam evaporated copper phthalocyanine (CuPc) thin film onto an integrated circuit (IC). When isothermally operated at 150 °C, the microsensor can selectively and reversibly detect parts-per-billion concentration levels of nitrogen dioxide (NO₂) and diisopropyl methylphosphonate (DIMP). The selectivity feature of the microsensor was established by operating it with a 5 V peak amplitude, 2 μs duration, 1000 Hz repetition frequency pulse, and then analyzing its time- and frequency-domain responses. The envelopes associated with the normalized-difference Fourier transform magnitude spectra, and their derivatives, reveal features which unambiguously distinguish the NO₂ and DIMP challenge gas responses. Furthermore, the area beneath each response envelope may correspondingly be interpreted as the microsensor's sensitivity for a specific challenge gas concentration. Scanning electron microscopy (SEM) was used to characterize the morphology of the CuPc thin film. Additionally, infrared (IR) spectroscopy was employed to verify the - and β-phases of the sublimed CuPc thin films and to study the NO₂- and DIMP-CuPc interactions.     

High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts

Adult zebrafish is a well-known small animal model for studying heart regeneration. Although the regeneration of scars made by resecting the ventricular apex has been visualized with histological methods, there is no adequate imaging tool for tracking the functional recovery of the damaged heart. For this reason, high-frequency Doppler echocardiography using dual mode pulsed wave Doppler, which provides both tissue Doppler (TD) and Doppler flow in a same cardiac cycle, is developed with a 30 MHz high-frequency array ultrasound imaging system. Phantom studies show that the Doppler flow mode of the dual mode is capable of measuring the flow velocity from 0.1 to 15 cm s⁻¹ with high accuracy (p-value = 0.974 > 0.05). In the in vivo study of zebrafish, both TD and Doppler flow signals were simultaneously obtained from the zebrafish heart for the first time, and the synchronized valve motions with the blood flow signals were identified. In the longitudinal study on the zebrafish heart regeneration, the parameters for diagnosing the diastolic dysfunction, for example, E/Em < 10, E/A < 0.14 for wild-type zebrafish, were measured, and the type of diastolic dysfunction caused by the amputation was found to be similar to the restrictive filling. The diastolic function was fully recovered within four weeks post-amputation.     

Nitric oxide microsensor for high spatial resolution measurements in biofilms and sediments

Nitric oxide (NO) is a ubiquitous biomolecule that is known as a signaling compound in eukaryotes and prokaryotes. In addition, NO is involved in all conversions of the biogeochemical nitrogen cycle: denitrification, nitrification, and the anaerobic oxidation of ammonium (Anammox). Until now, NO has not been measured with high spatial resolution within microbial communities, such as biofilms, sediments, aggregates, or microbial mats, because the available sensors are not robust enough and their spatial resolution is insufficient. Here we describe the fabrication and application of a novel Clark-type NO microsensor with an internal reference electrode and a guard anode. The NO microsensor has a spatial resolution of 60-80 microm, a sensitivity of 2 pA microM-1, and a detection limit of approximately 30 nM. Hydrogen sulfide (H2S) was found to be a major interfering compound for the electrochemical detection of NO. The application of the novel NO microsensor to nitrifying biofilms and marine sediments revealed dynamic NO concentration profiles with peaks in the oxic parts of the samples. The local concentrations suggested that NO may be an important bioactive compound in natural environments. The consumption and production of NO occurs in separate regions of stratified microbial communities and indicates that it is linked to distinct biogeochemical cycles.     

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.     

Highly sensitive carbon nanotube-based sensing for lactate and glucose monitoring in cell culture

Monitoring of metabolic compounds in cell cultures can provide real-time information of cell line status. This is particularly important in those lines not fully known, as the case of embryonic and mesenchymal cells. On the other hand, such approach can pave the way to fully automated systems for growing cell cultures, when integrated in Petri dishes. To date, the main efforts emphasize the monitoring of few process variables, like pH, pO(2), electronic impedance, and temperature in bioreactors. Among different presented strategies to develop biosensors, carbon nanotubes exhibit great properties, particularly suitable for high-sensitive detection. In this work, nanostructured electrodes by using multiwalled carbon nanotubes are presented for the detection of lactate and glucose. Some results from simulations are illustrated in order to foresee the behavior of carbon nanotubes depending on their orientation, when they are randomly dispersed onto the electrode surface. A comparison between nonnanostructured and nanostructured electrodes is considered, showing that direct electron-transfer between the protein and the electrode is not possible without nanostructuration. Such developed biosensors are characterized in terms of sensitivity and detection limit, and are compared to previously published results. Lactate production is monitored in a cell culture by using the developed biosensor, and glucose detection is also performed to validate lactate behavior.     

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.     

Microfabricated electrophoresis chips for simultaneous bioassays of glucose, uric acid, ascorbic acid, and acetaminophen

A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.     

Microseparation chips for performing multienzymatic dehydrogenase/oxidase assays: simultaneous electrochemical measurement of ethanol and glucose

This report describes a new "lab-on-a-chip" protocol integrating on-line precolumn biocatalytic reactions of multiple (oxidase and dehydrogenase) enzymes and substrates with effective capillary electrophoresis microseparations and amperometric detection. The operation of the new oxidase/dehydrogenase reaction/separation microchip is illustrated for the simultaneous measurement of glucose and ethanol in connection to the corresponding glucose oxidase and alcohol dehydrogenase reactions, respectively. The enzymatic reactions generate hydrogen peroxide and NADH species that are separated (on the basis of their different charges) and detected amperometrically at the end-column thick-film detector. A driving voltage of 2000 V results in peroxide and NADH migration times of 74 and 230 s, respectively. Operating the gold-coated carbon detector at +1.0 V allows simultaneous anodic detection of both reaction products. Factors influencing the reaction, separation, and detection processes are examined and optimized. The applicability of the new multienzyme assay to wine samples is illustrated.