Optical biosensor based on hollow integrated waveguides

The first absorbance biosensor based on pure silicon hollow integrated waveguides is presented in this work. With the use of horseradish peroxidase (HRP) as a model recognition element, an enzymatic sensor for the measurement of hydrogen peroxide was fabricated, numerically simulated, and experimentally characterized. Waveguides with widths ranging from 50 to 80 microm, having a depth of 50 microm and lengths up to 5 mm were easily fabricated by just one photolithographic step. These were further modified by covalent immobilization of HRP using silanization chemistry. Simulation studies of the proposed approach showed a sensor linear behavior up to 300 microM H2O2 and a sensitivity of 2.7 x 10(-3) AU/microM. Experimental results were in good agreement with the simulated ones. A linear behavior between 10 and 300 microM H2O2, a sensitivity of 3 x 10(-3) AU/microM, and a signal-to-noise ratio around 20 dB were attained. Also, kinetic studies of the activity of the immobilized enzyme on the silicon waveguide surface gave an apparent Michaelis-Menten constant of 0.44 mM. The simple technology proposed in this work enables the fabrication of cost-effective, easy-to-use, miniaturized biosensor generic platforms, these being envisioned as excellent candidates for the development of lab-on-a-chip systems.     

Enzyme immobilization in porous silicon: quantitative analysis of the kinetic parameters for glutathione-S-transferases

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.     

Microelectromechanical system pressure sensor integrated onto optical fiber by anodic bonding

Optical microelectromechanical system pressure sensors based on the principle of Fabry-Perot interferometry have been developed and fabricated using the technique of silicon-to-silicon anodic bonding. The pressure sensor is then integrated onto an optical fiber by a novel technique of anodic bonding without use of any adhesives. In this anodic bonding technique we use ultrathin silicon of thickness 10 microm to bond the optical fiber to the sensor head. The ultrathin silicon plays the role of a stress-reducing layer, which helps the bonding of an optical fiber to silicon having conventional wafer thickness. The pressure-sensing membrane is formed by 8 microm thick ultrathin silicon acting as a membrane, thus eliminating the need for bulk silicon etching. The pressure sensor integrated onto an optical fiber is tested for static response, and experimental results indicate degradation in the fringe visibility of the Fabry-Perot interferometer. This effect was mainly due to divergent light rays from the fiber degrading the fringe visibility. This effect is demonstrated in brief by an analytical model.     

Ion diffusion in channels containing random arrays of microspheres: an electrochemical time-of-flight method

Rates of chloride ion diffusion in narrow (ca. 3 microm thick), rectangular (ca. 0.1 x 1.0 mm(2)) channels partially filled with polystyrene microspheres are investigated by a potentiometric electrochemical time-of-flight (P-ETOF) method. Lithographically fabricated on glass slides, P-ETOF devices consist of a centrally positioned 10 microm wide, ca. 1 mm long generator microelectrode and two sensor microelectrodes of the same dimensions symmetrically positioned on both side of the generator at a distance of 50 microm. The electrodes are silver-plated and partially oxidized in a chloride electrolyte to form Ag/AgCl deposits. Constant current reduction of AgCl on the generator electrode is used to produce chloride ions at a constant rate. Ag/AgCl deposited on the sensor microelectrodes allows time-dependent potentiometric monitoring of the increasing concentration of chloride ions diffusing across the interelectrode gap. The device is enclosed with a parallel glass plate to form a narrow channel with the polystyrene microbeads serving as spacers. The packing density of the microspheres expressed in terms of the fractional void volume (rho) varied from ca. 0.6 to 1.0. Using rho, we modified a diffusion equation describing the change of chloride ion concentration at the sensor microelectrode to include the effect of the microspheres restricting the void volume. We rely on digital simulations as well as on direct P-ETOF experiments to show that the proposed equation does accurately account for the effect of rho on the diffusion processes. We thus demonstrate that P-ETOF can be used to measure the number of identical microspheres in the active region of a narrow channel device. In the latter context, a future application of P-ETOF as a signal transduction mechanism in biosensors is outlined.     

A chip system for size separation of macromolecules and particles by hydrodynamic chromatography

For the first time, a miniaturized hydrodynamic chromatography chip system has been developed and tested on separation of fluorescent nanospheres and macromolecules. The device can be applied to size characterization of synthetic polymers, biopolymers, and particles, as an attractive alternative to the classical separation methods such as size exclusion chromatography or field-flow fractionation. The main advantages are fast analysis, high separation efficiency, negligible solvent consumption, and easy temperature control. The prototype chip contains a rectangular flat separation channel with dimensions of 1 microm deep and 1000 microm wide, integrated with a 300-pL injector on a silicon substrate. The silicon microtechnology provides precisely defined geometry, high rigidity, and compatibility with organic solvents or high temperature. All flows are pressure driven, and a specific injection system is employed to avoid excessive sample loading times, demonstrating an alternative way of lab-on-a-chip design. Separations obtained in 3 min show the high performance of the device and are also the first demonstration of flat channel hydrodynamic chromatography in practice.     

(Ba(x)La(1-x)(2))In(2)O(5+x) (0.4 < or = x < or = 0.6) electrolyte-supported mixed-potential CO sensors

The dense (Ba(x)La(1-x)(2))In(2)O(5+x) electrolytes with different compositions (x = 0.4, 0.5, 0.6) were synthesized by Pechini method. The obtained sintered (Ba(x)La(1-x)(2))In(2)O(5+x) electrolytes showed a high relative density of approximately 98%, and the major phase of three electrolyte compositions was indexed as a cubic phase. The CO sensing properties of as-fabricated planar-type (Ba(x)La(1-x)(2))In(2)O(5+x)-based sensors coupled with ITO and Pt as the sensing electrode and reference electrode, respectively, were investigated. The effects of factors such as gas flow rate, chemical compositions, and density of the electrolytes on the sensing performance were investigated. The sensors showed good sensitivity to different concentrations of CO from approximately 100 to approximately 500 ppm and excellent selectivity over low concentrations of methane (<500 ppm). Linear relationships between emf of the sensors and CO gas concentrations from approximately 100 to approximately 400 ppm were observed. However, the sensors indicated more sluggish response compared with the sensors coupled with a corresponding porous electrolyte. The probable reason has been discussed. The long-term stability of the sensor for the detection of CO was also investigated, which indicated a reasonably stable sensor signal after an initial decline during the incubation period.     

Integrated electrochemical sensor array for on-line monitoring of yeast fermentations

This paper describes the design, modeling, and experimental characterization of an electrochemical sensor array for on-line monitoring of fermentor conditions in both miniaturized cell assays and in industrial scale fermentations. The viable biomass concentration is determined from impedance spectroscopy. As a miniaturized electrode configuration with high cell constant is applied, the spectral conductivity variation is monitored instead of the permittivity variation. The dissolved oxygen concentration is monitored amperometrically using an ultramicroelectrode array, which is shown to have negligible flow dependence. pH is monitored using an ion-sensitive field effect transistor (ISFET), and a platinum thermistor is included for temperature measurements. All sensors were shown to be sufficiently accurate within the range relevant to yeast fermentations. The sensor array is shown to be very stable and durable and withstands steam-sterilization.     

Wafer-scale fabrication of polymer-based microdevices via injection molding and photolithographic micropatterning protocols

Because of their broad applications in biomedical analysis, integrated, polymer-based microdevices incorporating micropatterned metallic and insulating layers are significant in contemporary research. In this study, micropatterns for temperature sensing and microelectrode sets for electroanalysis have been implemented on an injection-molded thin polymer membrane by employing conventional semiconductor processing techniques (i.e., standard photolithographic methods). Cyclic olefin copolymer (COC) is chosen as the polymer substrate because of its high chemical and thermal stability. A COC 5-in. wafer (1-mm thickness) is manufactured using an injection molding method, in which polymer membranes (approximately 130 microm thick and 3 mm x 6 mm in area) are implemented simultaneously in order to reduce local thermal mass around micropatterned heaters and temperature sensors. The highly polished surface (approximately 4 nm within 40 microm x 40 microm area) of the fabricated COC wafer as well as its good resistance to typical process chemicals makes it possible to use the standard photolithographic and etching protocols on the COC wafer. Gold micropatterns with a minimum 5-microm line width are fabricated for making microheaters, temperature sensors, and microelectrodes. An insulating layer of aluminum oxide (Al2O3) is prepared at a COC-endurable low temperature (approximately 120 degrees C) by using atomic layer deposition and micropatterning for the electrode contacts. The fabricated microdevice for heating and temperature sensing shows improved performance of thermal isolation, and microelectrodes display good electrochemical performances for electrochemical sensors. Thus, this novel 5-in. wafer-level microfabrication method is a simple and cost-effective protocol to prepare polymer substrate and demonstrates good potential for application to highly integrated and miniaturized biomedical devices.     

Fluorinated xerogel-derived microelectrodes for amperometric nitric oxide sensing

An amperometric fluorinated xerogel-derived nitric oxide (NO) microelectrode is described. A range of fluorine-modified xerogel polymers were synthesized via the cohydrolysis and condensation of alkylalkoxy- and fluoroalkoxysilanes. Such polymers were evaluated as NO sensor membranes to identify the optimum composition for maximizing NO permeability while providing sufficient selectivity for NO in the presence of common interfering species. By taking advantage of both the versatility of sol-gel chemistry and the "poly(tetrafluoroethylene)-like" high NO permselective properties of the xerogels, the performance of the fluorinated xerogel-derived sensors was excellent, surpassing all miniaturized NO sensors reported to date. In contrast to previous electrochemical NO sensor designs, xerogel-based NO microsensors were fabricated using a simple, reliable dip-coating procedure. An optimal permselective membrane was achieved by synthesizing xerogels of methyltrimethoxysilane (MTMOS) and 20% (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilane (17FTMS, balance MTMOS) under acid-catalyzed conditions. The resulting NO microelectrode had a conical tip of approximately 20 microm in diameter and approximately 55 microm in length and exhibited sensitivities of 7.91 pA x nM (-1) from 0.2 to 3.0 nM (R (2) = 0.9947) and 7.60 nA x microM (-1) from 0.5 to 4.0 microM ( R (2) = 0.9999), detection limit of 83 pM (S/ N = 3), response time ( t 95%) of <3 s, and selectivity (log K NO, j (amp)) of -5.74, <-6, <-6, <-6, <-6, -5.84, and -1.33 for j = nitrite, ascorbic acid, uric acid, acetaminophen, dopamine, ammonia/ammonium, and carbon monoxide. In addition, the sensor proved functional up to 20 d, maintaining >or=90% of the sensor's initial sensitivity without serious deterioration in selectivity.     

Triple-sensor multiplexed frequency-modulated continuous-wave interferometric fiber-optic displacement sensor

A triple-sensor multiplexed fiber-optic displacement sensor, which can measure the displacements of three different objects or the three-dimensional displacement of a single object, is introduced. The sensor is based on the principles of optical frequency-modulated continuous-wave interference and frequency-division multiplexing. The beat signals from the individual sensors are assigned in the frequency domain and separated with different electrical bandpass filters. The displacements of objects can be determined simultaneously by detecting the phase shifts of the corresponding signals. The cross talk between the individual sensors is evaluated, and an accuracy of 0.08 microm in a dynamic range of 1000 microm is achieved.     

Equilibrium and nonequilibrium electrode processes on porous silicon

The electrode potential of porous silicon (por-Si) in aqueous electrolytes of variable acidity has been measured for the first time. It is shown that por-Si electrode can be a promising pH sensor. The results of the electrode potential measurements show that water condensed in capillaries of capacitive humidity sensors with a thick layer of mesoporous silicon is probably subject to electrolysis.     

Flow injection based renewable electrochemical sensor system

A novel class of electrochemical sensors is proposed utilizing electrically conducting beads to form a disposable electrode as well as nonconducting beads to form renewable layers of immobilized enzymes. The concept, aimed to prevent fouling, is tested on an amperometric sensor coupled to nonconducting beads with different immobilized oxidases: galactose, lactate, alcohol, or glucose oxidase, the latter two being used to determine alcohol and gluocse, respectively, in samples of beer and wine. Glucose oxidase was also immobilized on conducting glassy carbon particles to explore the performance of a biosensor where both enzyme and electrode can be automatically renewed in less than 1 min. The results confirm that the concept of a flow injection renewable electrochemical sensor (FI-RES) is practical. It provides a novel approach to biosensing, to comparing enzyme activity, to studying enzyme immobilization on different supports, and to voltammetry in general.     

Ultrafast microfluidic mixer and freeze-quenching device

The freeze-quenching technique is extremely useful for trapping meta-stable intermediates populated during fast chemical or biochemical reactions. The application of this technique, however, is limited by the long mixing time of conventional solution mixers and the slow freezing time of cryogenic fluids. To overcome these problems, we have designed and tested a novel microfluidic silicon mixer equipped with a new freeze-quenching device, with which reactions can be followed down to 50 micros. In the microfluidic silicon mixer, seven 10-microm-diameter vertical pillars are arranged perpendicular to the flow direction and in a staggered fashion in the 450-pL mixing chamber to enhance turbulent mixing. The mixed-solution jet, with a cross section of 10 microm x 100 microm, exits from the microfluidic silicon mixer with a linear flow velocity of 20 m/s. It instantaneously freezes on one of two rotating copper wheels maintained at 77 K and is subsequently ground into an ultrafine powder. The ultrafine frozen powder exhibits excellent spectral quality and high packing factor and can be readily transferred between spectroscopic observation cells. The microfluidic mixer was tested by the reaction between azide and myoglobin at pH 5.0. It was found that complete mixing was achieved within the mixing dead time of the mixer (20 micros), and the first observable point for this coupled device was determined to be 50 micros, which is approximately 2 orders of magnitude faster than commercially available instruments.     

Production of biosensors with exchangeable enzyme-containing threads

We introduce a simple method for the construction of biosensors, based on coiling an enzyme-containing, thread-shaped material around a cylindrical signal transducer in the form of winding stairs with a variable length of step and so forming a variable biocatalytic membrane on the sensor surface, which can be easily modified for particular purposes. In the model system, we immobilized glucose oxidase (GO) on a nylon thread, formatted from a sheaf of numerous minor filaments and used as a biorecognition element integrated with a Clark-type oxygen sensor. The immobilized enzyme was evenly distributed throughout the thread, and the activity of the enzyme could be measured in units of length. Appropriate pieces of the enzyme-containing thread with a certain amount of GO could be cut for a definite biosensor or bioreactor. The enzyme amount and substrate diffusion parameters, which together control the sensor's working range and sensitivity, could be changed simultaneously with the change of the length of the thread. Besides glucose oxidase, experiments with other enzymes have confirmed the applicability of the proposed technological solution. Thus, the thread-type matrixes enable one to construct sensors with a required range of work, sensitivity, and selectivity, which can be easily customized within seconds.     

Si nanocolumns as novel nanostructured supports for enzyme immobilization

Silicon nanocolumns have been used as novel supports for the high-density immobilization of enzymes. Silicon nanocolumns with diameters of ca. 50-100 nm and a height of 1 micron were constructed using glancing angle deposition. The surfaces were successively treated with 3-aminopropyltriethoxysilane (APTES) and then with an amine reactive polymer, poly(ethylene-alt-maleic anhydride), to attach soybean peroxidase (SBP) to the support. Optimal coverage of APTES, polymer, and SBP was obtained for incorporation of enzyme onto the sidewalls of the nanocolumns. SBP immobilized on the silicon nanocolumns demonstrated an enhancement in biocatalytic activity of 160% over that of the enzyme immobilized on flat silicon wafers with the same projected area. The enzymatic activity decreased with progressive washes for both supports. This decrease in the activity of enzyme was found to be primarily due to the intrinsic deactivation of immobilized enzyme on the silicon surface. Designing nanocolumns with optimal dimensions, spacing, and surface chemistry may lead to the development of high-density arrays of proteins for applications in biotechnology.     

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.     

Nanophase-separated amphiphilic conetworks as versatile matrixes for optical chemical and biochemical sensors

As a novel class of sensor matrixes, nanophase-separated amphiphilic polymeric conetworks (APCNs) open a new dimension for optical chemical and biochemical sensing. These conetworks consist of a hydrophilic phase-we used poly(2-hydroxyethyl acrylate), poly(2-(dimethylamino)ethyl acrylate), or polycationic poly(2-(trimethylammonium)ethyl acrylate)-and of a hydrophobic phase-poly(dimethylsiloxane). Sensors can be prepared by simple impregnation of the matrix. Due to nanophase separation, there is a spatial separation between areas in which the indicator reagents are well immobilized and areas that advantageously take care of the diffusive transport of the analyte, whereby these functionalities of the contrary phases can be exchanged. Thanks to the huge interface between the contrary phases, the accessibility of the indicator reagents is good, which makes it possible to design sensors with high sensitivity. To demonstrate the advantages of APCNs as matrixes, different prototypes of sensors were prepared, e.g., one to determine gaseous chlorine based on its reaction with immobilized o-tolidine and another to determine vaporous acids based on immobilized bromophenol blue dianions. As a breakthrough in biochemical sensing, we are also able to present an easily producible, optically transparent biochemical sensor to determine peroxides in nonpolar organic media-based on coimmobilized horseradish peroxidase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate).     

Toward a miniature wireless integrated multisensor microsystem for industrial and biomedical applications

This paper presents our work toward the integration of a multisensor microsystem with wireless communication, using system-on-chip (SoC) methodology. Four different forms of microelectronic sensors have been fabricated on two separate 5/spl times/5 mm/sup 2/ silicon chips measuring pH, conductivity, dissolved oxygen concentration, and temperature. The sensors are integrated with a sensor fusion chip comprising analog circuitry for sensor operation and signal amplification prior to digital decoding and transmission. The microsystem prototype will be packaged in a miniature capsule, which measures 16 mm /spl times/55 mm including batteries and dissipates 6.3 mW for a minimal life cycle of 12 h.     

Electrochemical multianalyte immunoassays using an array-based sensor

A novel amperometric biosensor for performing simultaneous electrochemical multianalyte immunoassays is described. The sensor consisted of eight iridium oxide sensing electrodes (0.78 mm(2) each), an iridium counter electrode, and a Ag/AgCl reference electrode patterned on a glass substrate. Four different capture antibodies were immobilized on the sensing electrodes via adsorption. Quantification of proteins was achieved using an ELISA in which the electrochemical oxidation of enzyme-generated hydroquinone was measured. The spatial separation of the electrodes enabled simultaneous electrochemical immunoassays for multiple proteins to be conducted in a single assay without amperometric cross-talk between the electrodes. The simultaneous detection of goat IgG, mouse IgG, human IgG, and chicken IgY was demonstrated. The detection limit was 3 ng/mL for all analytes. The sensor had excellent precision (1.9-8.2% interassay CV) and was comparable in performance to commercial single-analyte ELISAs. We anticipate that chip-based sensors, as described herein, will be suitable for the mass production of economical, miniaturized, multianalyte assay devices.     

Development of advanced-type multi-functional electronic personal dosemeter

An advanced-type small, light, multi-functional electronic personal dosemeter has been developed using silicon semiconductor radiation detectors for dose management of workers at nuclear power plants and accelerator facilities. This dosemeter is 62 x 82 x 27 mm(3) in size and approximately 130 g in weight, which is capable of measuring personal gamma ray and neutron dose equivalents, Hp(10), simultaneously. The neutron dose equivalent can be obtained using two types of silicon semiconductors: a slow-neutron sensor (<1 MeV) and a fast-neutron sensor (>1 MeV). The slow neutron sensor is a 10 x 10 mm(2) p-type silicon on which a natural boron layer is deposited around an aluminium electrode. The fast neutron sensor is also a 10 x 10 mm(2) p-type silicon crystal on which an amorphous silicon hydride is deposited. The neutron energy response corresponding to the fluence-to-dose-equivalent conversion coefficient given by ICRP Publication 74 has been evaluated using a monoenergetic neutron source from 250 keV to 15 MeV at the Fast Neutron Laboratory of Tohoku University. As the result, the Hp(10) response to neutrons in the energy range of 250 keV and 4.4 MeV within +/-50% difference has been obtained.