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.     

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.     

基于组织液超滤提取的MEMS血糖检测传感器

基于超滤原理提取组织液、并对其进行后续葡萄糖检测,是实现长期血糖持续监测的一种有效途径．本文提出一种可用于组织液超滤提取及葡萄糖持续检测的传感器微系统．该系统主要由微流控底座和葡萄糖传感器芯片组成．其中微流控底座由PDMS微通道、SU一8单向阀等微加工器件组成,在压力作用下可完成组织液提取及将检测过的组织液排出的功能．采用体硅加工方法制作葡萄糖传感器芯片微型腔体及腔体底部的微孔膜,研制出具有扩散控制功能的三电极检测芯片,并在其上通过琼脂糖包埋方法固定葡萄糖氧化酶、基于电化学原理实现葡萄糖浓度的检测．实验结果表明,该系统可以实现液体的灵活提取,并且葡萄糖检测响应时间小于5s,在0．4V工作电压下线性测量范围达0．2～20mmol／L,灵敏度为9．76nA／（mmol·L-1）,相关系数为0．9954．多次测量5mmoL／L样本,差异系数3．48％．可见该传感系统具有较好的稳定性,并且体积小、易于集成,有望用于组织液灵活提取及其葡萄糖持续监测．     

Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes

This paper reports the first demonstration of a fluorescence resonance energy transfer based glucose sensor, wherein a competitive binding (CB) assay is encapsulated into polyelectrolyte microcapsules. The work supports the concept that microcapsules are superior to hydrogel systems or other matrixes for competitive-binding-based system, as they provide free movement of the sensing elements within the capsule interior while constant total sensing assay concentration is maintained. The transduction approach employed in these preliminary experiments is also a novel CB system based on a model apoenzyme, apo-glucose oxidase (AG), which is highly specific to beta-d-glucose, as the model target-binding protein. The glucose sensitivity of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothiocyanate-AG encapsulated in microcapsules showed 5 times greater specificity for beta-D-glucose over other sugars, with sensitivity (change in intensity ratio) in the range of 2-6%/mM. It was observed that the sensitivity and range of the response can be tailored by controlling the assay concentration using different FITC-dextran molecular weight and total capsule concentration. The findings support the concepts of using microcapsules to encapsulate CB assays for reversible and stable sensors and the use of apoenzymes as specific molecular recognition elements in CB assays. Further, characterization results for microcapsule glucose sensors demonstrate their suitability for monitoring physiological glucose levels.     

Microscale enzymatic optical biosensors using mass transport limiting nanofilms. 2. Response modulation by varying analyte transport properties

Microscale implantable fluorescent sensors that can be transdermally interrogated using light are being pursued as a minimally invasive biochemical monitoring technology for in vivo applications. Previously, we reported the development of an enzymatic-based sensing platform characterized using glucose as a model biochemical analyte for minimally invasive diabetic monitoring. In this work, surface-adsorbed polyelectrolyte nanofilms were employed to modulate the relative fluxes of glucose and oxygen into the sensor, allowing response characteristics, namely, analytical range and sensitivity, to be tuned. Modulation of substrate transport properties were obtained by varying surface-adsorbed nanofilm thicknesses, ionic strength of assembly conditions, and outermost constituents. In general, increasing film thickness through additional cycles of adsorption resulted in consistently decreased glucose flux, correspondingly decreasing sensitivity and increasing range. While the two components of the nanofilms remained the same [poly(allylamine hydrochloride), PAH; poly(sodium 4-styrenesulfonate)}, the assembly conditions and terminal layer were found to strongly influence sensor behavior. Specifically, without added salt in assembly conditions, glucose diffusion was significantly decreased when films were capped with PAH, resulting in reduced sensitivity and extended range of response. With added salt, however, sensor response was the same for films of the same thickness but different terminal materials. These findings demonstrate that sensor response may be customized to cover the hypo- (0-80 mg/dL), normo- (80-120 mg/dL), and hyperglycemic levels (>120 mg/dL) from a single batch of particles through appropriate selection of coating structure and assembly conditions. Furthermore, the results indicate nanofilms of only 12-nm thickness could significantly affect response behavior, confirming predicted behavior by models of sensor reaction-diffusion kinetics. These findings demonstrate the ability to engineer sensor response properties using a simple, cost-effective means and lay the groundwork for developing additional highly sensitive biochemical monitors.     

Surface-acoustic-wave (SAW) flow sensor

The use of a surface-acoustic-wave (SAW) device to measure the rate of gas flow is described. A SAW oscillator heated to a suitable temperature above ambient is placed in the path of a flowing gas. Convective cooling caused by the gas flow results in a change in the oscillator frequency. A 73-MHz oscillator fabricated on 128 degrees rotated Y-cut lithium niobate substrate and heated to 55 degrees C above ambient shows a frequency variation greater than 142 kHz for flow-rate variation from 0 to 1000 cm(3)/min. The output of the sensor can be calibrated to provide a measurement of volume flow rate, pressure differential across channel ports, or mass flow rate. High sensitivity, wide dynamic range, and direct digital output are among the attractive features of this sensor. Theoretical expressions for the sensitivity and response time of the sensor are derived. It is shown that by using ultrasonic Lamb waves, propagating in thin membranes, a flow sensor with faster response than a SAW sensor can be realized.     

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.     

Genetic engineering of an allosterically based glucose indicator protein for continuous glucose monitoring by fluorescence resonance energy transfer

Real-time monitoring of blood glucose could vastly reduce a number of the long-term complications associated with diabetes. In this article, we present a novel approach that relies on a glucose-binding protein engineered such that a 20% reduction in fluorescence due to the fluorescence resonance energy transfer occurs as a result of glucose binding. This change in fluorescence provides a signal for the optical detection of glucose. The novel glucose indicator protein (GIP) was created by fusing two fluorescent reporter proteins (green fluorescent proteins) to each end of an Escherichia coli glucose-binding protein in such a manner that the spatial separation between the fluorescent moieties changes when glucose binds, thus generating a distinct optical signal that can be used for glucose detection. By placing the GIP within a dialysis hollow fiber sensor, a microsensor has been developed for continuous monitoring of glucose. The sensor had a response time to sudden glucose changes within 100 s and was reversible. The sensor was shown to have an optional range on the order of 10 microM of glucose.     

A wireless, remote query glucose biosensor based on a pH-sensitive polymer

This paper describes a wireless, remote query glucose biosensor using a ribbonlike, mass-sensitive magnetoelastic sensor as the transducer. The glucose biosensor is fabricated by first coating the magnetoelastic sensor with a pH-sensitive polymer and upon it a layer of glucose oxidase (GOx). The pH-responsive polymer swells or shrinks, thereby changing mass, respectively, in response to increasing or decreasing pH values. The GOx-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the polymer mass. In response to a time-varying magnetic field, a magnetoelastic sensor mechanically vibrates at a characteristic resonance frequency, the value of which inversely depends on sensor mass loading. As the magnetoelastic films are magnetostrictive, the vibrations launch magnetic flux that can be remotely detected using a pickup coil. Hence, changes in the resonance frequency of a passive magnetoelastic transducer are detected on a remote query basis, without the use of physical connections to the sensors.The sensitivity of the glucose biosensors decreases with increasing ionic strength; at physiological salt concentrations, 0.6 mmol/L of glucose can be measured. At glucose concentrations of 1-10 mmol/L, the biosensor response is reversible and linear, with the detection limit of 0.6 mmol/L corresponding to an error in resonance frequency determination of 20 Hz. Since no physical connections between the sensor and the monitoring instrument are required, this sensor can potentially be applied to in vivo and in situ measurement of glucose concentrations.     

Extended-range glucose sensor employing engineered glucose dehydrogenases

An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy. This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range. We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold). We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range. The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error. The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system. Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp. The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes.     

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.     

Microscale enzymatic optical biosensors using mass transport limiting nanofilms. 1. Fabrication and characterization using glucose as a model analyte

"Smart tattoo" sensors-fluorescent microspheres that can be implanted intradermally and interrogated noninvasively using light-are being developed as potential tools for in vivo biochemical monitoring. In this work, a platform for enzymatic tattoo-type sensors is described and prototype devices evaluated using glucose as a model analyte. Sensor particles were prepared by immobilizing Pt(II) octaethylporphine (PtOEP), a phosphorescent dye readily quenched by molecular oxygen, into hybrid silicate microspheres, followed by loading and subsequent covalent immobilization of glucose oxidase. Rhodamine B-doped multilayer nanofilms were subsequently assembled on the surfaces of the particles to provide a reference signal and provide critical control of glucose transport into the particle. The enzymatic oxidation of glucose within the sensor results in the glucose concentration-dependent depletion of local oxygen levels, enabling indirect monitoring of glucose by measuring relative changes in PtOEP emission. A custom testing apparatus was used to monitor the dynamic sensor response to varying bulk oxygen and glucose levels, respectively. For the prototypes tested, dynamic test results indicate that the sensors respond rapidly (t(95) = 84 s) and reversibly to changes in bulk glucose levels, while demonstrating high baseline stability. The sensitivity (change in intensity ratio) of these devices was determined to be 4.16 +/- 0.57%/mg dL(-1). The analytical range for the prototypes was determined to be 2-120 mg/dl, though this can be extended to cover the physiologically relevant range by tailoring the nanofilm coatings. These findings confirm the potential for enzymatic microscale optical and pave the way for extension of this initial demonstration with glucose to target other biochemical species relevant to metabolic monitoring.     

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.     

Microtubule sensors and sensor array based on polyaniline synthesized in the presence of poly(styrene sulfonate)

Microtubule sensors for glucose, urea, and triglyceride were fabricated based on poly(styrene sulfonate)-polyaniline (PSS-PANI) composites synthesized within the pores of track-etched polycarbonate membranes. The synthesis of a sufficiently thick and conducting PSS-PANI film at pH 5 provided the advantage of immobilizing enzymes during polymerization. This resulted in the improvement of sensor response for urea and triglyceride by a factor of approximately 10(2) with a significant increase in the linear region of response compared to polyaniline-based sensors, where the enzymes were immobilized by physical adsorption after the polymerization. The sensors based on urea and triglyceride were found to have a higher linear range of response, better sensitivity, improved multiple use capability, and faster response time compared to the potentiometric and amperometric sensors based on polyaniline. A microtubule sensor array for glucose, urea, and triglyceride based on PSS-PANI was fabricated by immobilization of three different sets of enzymes on three closely spaced devices and its response was found to be free from cross-interference when a sample containing a mixture of the above analytes was analyzed in a single measurement.     

Characterization of glucose microsensors for intracellular measurements.

Ultrasmall glucose sensors have been constructed by using platinum-deposited carbon ring microelectrodes with glucose oxidase. Response times as low as 270 ms have been obtained with these sensors. Moreover, there is a linear relationship between sensor tip diameter and response times. The use of these sensors has been demonstrated in the detection of glucose in single-cell cytoplasm of the large dopamine cell of the pond snail Planorbis corneus. Current responses obtained at these sensors implanted into a cell increase following injection of 2 pL of glucose solution (3 M) into the cell. Results obtained from these experiments show that these sensors are suitable for glucose monitoring in ultrasmall environments. In addition, characterizations of these sensors have been investigated under different O2 concentrations. At atmospheric oxygen concentrations, glucose levels in the submillimolar range can be measured without oxygen interference; however, oxygen interference can be substantial at low oxygen concentrations.     

Theoretical and experimental studies of a novel cone-jet sensor

Modeling of a novel cone-jet sensor using two-dimensional (2-D) finite element analysis was investigated for dimensional measurement. Theoretical and experimental studies demonstrated that a cone-jet sensor supplied with air can be used to accurately measure displacement, and its work range of 1.5 to 4.2 mm is some ten times greater than a simple back-pressure sensor. It is anticipated that this type of sensor will find wide applications in manufacturing industry due to its wider working range, high precision, and other features     

The frequency-dependent directivity of a planar fabry-perot polymer film ultrasound sensor

A model of the frequency-dependent directivity of a planar, optically-addressed, Fabry-Perot (FP), polymer film ultrasound sensor is described and validated against experimental directivity measurements made over a frequency range of 1 to 15 MHz and angles from normal incidence to 80 degrees. The model may be used, for example, as a predictive tool to improve sensor design, or to provide a noise-free response function that could be deconvolved from sound-field measurements in order to improve accuracy in high-frequency metrology and imaging applications. The specific question of whether effective element sizes as small as the optical-diffraction limit can be achieved was investigated. For a polymer film sensor with a FP cavity of thickness d, the minimum effective element radius was found to be about 0.9 d, and that an illumination spot radius of less than d/4 is required to achieve it.     

Modeling the response function of dual-enzyme microbiosensors

A general theoretical model for competitive dual-enzyme microbiosensors based on self-assembled monolayers (SAM) is presented. The model is derived for amperometric dual-enzyme ATP sensors and provides excellent agreement with experimental ATP measurements at 25 microm diameter microelectrodes. In this model, the statistical probability of a glucose molecule in competition between two enzymes, glucose oxidase (GOD)/hexokinase (HEX), at the ATP sensor surface is combined with the enzymatic reaction rate. Thereby, a simple model predicting the sensor signal for varying surface concentrations of GOD and HEX, glucose concentration, and ATP concentration is obtained. Excellent agreement of the predicted current signal with experimentally obtained sensor signals was achieved at ATP concentrations between 10 and 300 microM in a buffer containing glucose at physiologically relevant levels. Consequently, the development time for new dual-enzyme biosensors can be reduced, and an analytical model for the sensor response function is provided facilitating the calibration of enzymatic biosensors.     

PVC-Based 2,2,2-Cryptand Sensor for Zinc Ions

A PVC-based membrane of 2,2,2-cryptand exhibits a very good response for Zn(2+) in a wide concentration range (from 2.06 ppm to 6.54 × 10(3) ppm) with a slope of 22.0 mV/decade of Zn(2+) concentration. The response time of the sensor is <10 s, and the membrane can be used for more than 3 months without any observed divergence in potentials. The proposed sensor exhibits very good selectivity for Zn(2+) over other cations and can be used in a wide pH range (2.8-7.0). It has also been possible to use this assembly as an indicator electrode in potentiometric titrations involving zinc ions.     

Sol-Gel-Based Integrated Optical Microring Resonator Humidity Sensor

A novel humidity sensor based on sol-gel clad polymer microring resonator is presented. This sensor is based on the principle of change in refractive index of sol-gel on exposure to moisture. This change causes a shift in the resonant wavelength of the microring resonator. The performance of this sensor is analyzed and different ways of tailoring the sensor response are suggested. This sensor has a sensitivity of 16 pm/% relative humidity (RH) and a dynamic range up to 72% RH. The response time of this sensor can be tailored to be less than 200 ms, which could be utilized for monitoring human breathing condition in medical applications.