We have developed ultrasensitive biosensors for the analysis of neurotransmitters such as glutamate, GABA and lactate. These sensors have micrometer to submicrometer sizes. They are based on biomolecule immobilization on optical fiber probe surfaces. The miniaturized fiber probes are fabricated by either pulling or etching conventional optical fibers. For example, surface immobilized glutamate dehydrogenase (GDH) is being used for glutamate analysis. GDH has been directly immobilized onto an optical fiber probe surface through a new optical fiber sensor fabrication technique using covalent binding mechanisms. None of the direct or indirect physical confinement methods, such as mechanical confinement, gel trapping or membrane immobilization, has been used for the sensor preparation. An optical fiber surface is initially activated by silanization, which adds amine groups (-NH₂) to the surface. We then affix functional groups -CHO to the optical fiber surface by employing a bifunctional cross-linking agent, glutaraldehyde. The amino acids of GDH enzyme molecules (or other biomolecules) readily attach to these free -CHO groups on the fiber surface. The sensor is able to detect its substrate, glutamate, by monitoring the fluorescence of reduced nicotinamide adenine dinucleotide (NADH), a product of the reaction between nicotinamide adenine dinucleotide (NAD⁺) and glutamate. Similar procedures and principle have been used for the development of lactate and GABA sensors. Our biomolecule based biosensors have been applied to the study of single living cell neurophysiological responses.

In order to determine the maximum efficiency obtainable for the Mesa photodiodes used in our integrated bio/chemical microsystems, devices of different geometry have been integrated with optical waveguides and a simple microfluidic system in a test chip. Also, in order to study the effect of increasing the interaction length between the chemicals in the microchannel and the measuring light beam, a second integrated system was designed and fabricated. Two different geometries were studied, one in which the light is transmitted through the liquid in a direction perpendicular to the axis of a microchannel 50 micrometer wide and another in which the light is transmitted along the axis of a microchannel 5 mm long. The latter geometry was used in order to increase the interaction length between the light and the liquid by a factor of 100, thus, increasing the detection sensitivity by approximately 20 dB. The systems were fabricated employing two substrates which were bonded together, a silicon wafer containing the optical circuitry, plus one half of the microchannel circuitry, and a Pyrex wafer containing the other half of the microchannel circuitry. The planar multimode waveguides which formed the optical circuits were low loss ((alpha) less than or equal to 0.5 dB/cm) germanosilicate glass structures while the photodetectors were special, 'end-fire,' coupled Mesa structures having a good sensitivity (maximum R approximately equals 0.5 A/W) and broad spectral response ((lambda) approximately equals 350 - 1000 nm). In order to make more realistic studies of the detection sensitivity, simple chemical analyses such as phosphate determinations were performed using the chips made.

We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A prototype twin microcalorimeter based on the Seebeck effect has been fabricated by IC technology and micromachined postprocessing techniques. A biocompatible liquid rubber membrane supports two identical 0.5 X 2 cm² measurement chambers, situated at the cold and hot junction of a 666-junction aluminum/p+-polysilicon thermopile. The chambers can house up to 10⁶ eukaryotic cells cultured to confluence. The advantage of the device over microcalorimeters on the market, is the integration of the measurement channels on chip, rendering microvolume reaction vessels, ranging from 10 to 600 (mu) l, in the closest possible contact with the thermopile sensor (no springs are needed). Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The small thermal inertia of the microchannels results in the short response time of 70 s, when filled with 50 (mu) l of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 20%. Our results show that it is feasible to apply this large-area, small- volume whole-cell biosensor for drug discovery, where the binding assays that are commonly used to provide high- throughput need to be complemented with a functional assay. Solutions are brought onto the sensor by a simple pipette, making the use of an industrial microtiterplate dispenser feasible on a nx96-array of the microcalorimeter biosensor. Such an array of biosensors has been designed based on a new set of requirements as set forth by people in the field as this project moved on. The results obtained from the prototype large-area sensor were used to obtain an accurate model of the calorimeter, checked for by the simulation software ANSYS. At present, the sensor chip has been designed. Future publication(s) will deal with this part of the work.

The goal of our TU Delft interfaculty research program is to develop intelligent molecular diagnostic systems (IMDS) that can analyze liquid samples that contain a variety of biochemical compounds such as those associated with fermentation processes. One specific project within the IMDS program focuses on photon sensors. In order to analyze the liquid samples we use dedicated microarrays. At this stage, these are basically miniaturized micro titre plates. Typical dimensions of a vial are 200 X 200 X 20 micrometer³. These dimensions may be varied and the shape of the vials can be modified with a result that the volume of the vials varies from 0.5 to 1.6 nl. For all experiments, we have used vials with the shape of a truncated pyramid. These vials are fabricated in silicon by a wet etching process. For testing purposes the vials are filled with rhodamine solutions of various concentrations. To avoid evaporation glycerol-water (1:1, v/v) with a viscosity of 8.3 times the viscosity of water is used as solvent. We aim at wide field-of-view imaging at the expense of absolute sensitivity: the field-of-view increases quadratically with decreasing magnification. Small magnification, however, implies low Numerical Aperture (NA). The ability of a microscope objective to collect photons is proportional to the square of the NA. To image the entire microarray we have used an epi-illumination fluorescence microscope equipped with a low magnification (2.5 X/0.075) objective and a scientific CCD camera to integrate the photons emitted from the fluorescing particles in the solutions in the vials. From these experiments we found that for this setup the detection limit is on the order of micromolar concentrations of fluorescing particles. This translates to 10⁸ molecules per vial.

Metal clusters excited by light exhibit high local field enhancement and nanoscale resonant behavior. Absorptive properties of these metal clusters bound to a surface are the basis of various new and highly promising setups to transduce biorecognitive interactions into an optical signal. Multilayered highly resonant systems had been proposed and recently demonstrated employing a metal mirror, a nanometric polymer distance layer, a biomolecule interaction layer and biorecognitively bound metal nano clusters. The optochips clearly exhibit strong reflection minima induced by the resonant behavior of the metal cluster layer. At least one narrow reflection minimum can be shifted to the red or infra red spectral range and therefore far away from spherical gold colloids (less than 520 nm) and human plasma absorption. The setup enabled us to replace conventional binding assays (like ELISA) overcoming the various technological limits as there are multiple incubation steps, harmful reagents and spatial resolution. A modified setup (the metal island coated swelling polymer over mirror system) employing an optical thin-layer system consisting of a metal mirror, an active analyte-induced swelling polymer, and a metal cluster (island) film as the topmost layer was used to transduce human plasma ion concentrations.

The organization and assembly of molecules in cellular membranes is orchestrated through the recognition and binding of specific chemical signals. A simplified version of the cellular membrane system has been developed using a synthetically prepared membrane receptor incorporated into a biologically derived lipid bilayer. Through an interplay of electrostatic and van der Waals interactions, aggregation or dispersion of molecular components could be executed on command using a specific chemical signal. A pyrene fluorophore was used as an optical probe to monitor the aggregational state of the membrane receptors in the bilayer matrix. The pyrene excimer emission to monomer emission (E/M) intensity ratio gave a relative assessment of the local concentration of receptors in the membrane. Bilayers were prepared with receptors selective for the divalent metal ions of copper, mercury, and lead. Addition of the metal ions produced a rapid dispersion of aggregated receptor components at nano- to micro-molar concentrations. The process was reversible by sequestering the metal ions with EDTA. Receptors for proteins and polyhistidine were also prepared and incorporated into phosphatidylcholine lipid bilayers. In this case, the guest molecules bound to the membrane through multiple points of interaction causing aggregation of initially dispersed receptor molecules. The rapid, selective, and sensitive fluorescence optical response of these lipid assemblies make them attractive in sensor applications for aqueous phase metal ions and polypeptides.

A new method is being developed for quickly screen for the human exposure potential to polycyclic aromatic hydrocarbons (PAHs) and organochlorines (OCs). The development involves two key elements: identifying suitable signals that represent intracellular changes that are specific to PAH and OC exposure, and constructing a device to guide the biological cell growth so that signals from individual cells are consistent and reproducible. We are completing the identification of suitable signals by using synchrotron radiation-based (SR) Fourier-transform infrared (FTIR) spectromicroscopy in the mid-infrared region (4000 - 400 cm^-1). Distinct changes have been observed in the IR spectra after treatment of human cells in culture medium with PAHs and OCs. The potential use of this method for detecting exposure to PAHs and OCs has been tested and compared to a reverse transcription polymerase chain reaction (RT-PCR) assay that quantifies increased expression of the CYP1A1 gene in response to exposure to PAHs or OCs.

Semiconductor microlasers are attractive components for micro- analysis systems because of their ability to emit coherent, intense light from a small aperture. By using a surface- emitting semiconductor geometry, we were able to incorporate fluid flow inside a laser microcavity for the first time. This confers significant advantages for high throughput screening of cells, particulates and fluid analytes in a sensitive microdevice. In this paper we discuss the intracavity microfluidics and present preliminary results with flowing blood and brain cells.

Over the last ten years, LLNL has been developing Microtechnology for instrumentation with applications in the biosciences and environment. In order to build and field high- performance instruments, we have often had to alter our original premises and assumptions, significantly. This meant that we were forced to abandon materials and dimensions that were appealing to us when we began the R&D. Examples include our work on silicon-based electrophoresis systems, etched- fluidics for sample/sheath flow nozzles in flow cytometers, and polymerase-chain-reaction thermal-cycling chambers based on silicon-nitride. This presentation will discuss these and our work on other devices and instruments.

T-Sensors have been demonstrated to allow both absorption- and fluorescence-based detection of analytes directly in complex samples such as whole blood and contaminated environmental samples, without prior separation of blood cells or other soluble and insoluble components. In this paper, we present the implementation of electrochemical detection methods in T- Sensors, as well as their optical validation. Microelectrodes integrated with etched microfluidic flow channels allow traditional electroanalytical techniques to be performed on microliter and smaller volumes and also enable new detection techniques which are based upon the interaction between the microelectrodes and the diffusional mixing that occurs between flow lamina at low Reynolds numbers. Conductivity versus mixing measurements, anodic stripping voltammetry, and isolation of microelectrodes by sheath flow are demonstrated for an experimental device. In a T-Sensor, a sample solution, a receptor solution, and, optionally, a reference solution (a known analyte standard) are introduced in a common channel (T- SensorTM), and laminarly flow next to each other until the exit the structure. Smaller particles such as ions or small proteins diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles show no significant diffusion within the time the two flow streams are in contact. Two interface zones are formed between the fluid layers. The ratio of a property (e.g., fluorescence intensity, stripping voltammetry signal) of the two interface zones is a function of the concentration of the analyte, and is largely free of cross-sensitivities to other sample components and instrument parameters.

We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 10² - 10³ fold discrimination between matched and mismatched sequences.

We have developed a process for the production of microchannel arrays on bonded glass substrates up to 14 X 58 cm, for DNA sequencing. Arrays of 96 and 384 microchannels, each 46 cm long have been built. This technology offers significant advantages over discrete capillaries or conventional slab-gel approaches. High throughput DNA sequencing with over 550 base pairs resolution has been achieved. With custom fabrication apparatus, microchannels are etched in a borosilicate substrate, and then fusion bonded to a top substrate 1.1 mm thick that has access holes formed in it. SEM examination shows a typical microchannel to be 40 X 180 micrometers by 46 cm long; the etch is approximately isotropic, leaving a key undercut, for forming a rounded channel. The surface roughness at the bottom of the 40 micrometer deep channel has been profilometer measured to be as low as 20 nm; the roughness at the top surface was 2 nm. Etch uniformity of about 5% has been obtained using a 22% vol. HF/78% Acetic acid solution. The simple lithography, etching, and bonding of these substrate enables efficient production of these arrays and extremely precise replication from master masks and precision machining with a mandrel.

A slide-together compression package and microfluidic interconnects for microfabricated devices requiring fluidic and electrical connections is presented. The package assembles without tools, is reusable, and requires no epoxy, wirebonds, or solder, making chip replacement fast and easy. The microfluidic interconnects use standard HPLC PEEK tubing, with the tip machined to accept either an o-ring or custom molded ring which serves the dual function of forming the seal and providing mechanical retention strength. One design uses a screw to compress the o-ring, while others are simply plugged into a cartridge retained in the package. The connectors are helium leak-tight, can withstand hundreds of psi, are easy to connect and disconnect, are low dead volume, have a small footprint, and are adaptable to a broad range of microfabricated devices.

Intelligent Molecular Diagnostic Systems (IMDS)- The objective of this multidisciplinary research program is to design and develop an analytical system that is able to measure and interpret concentrations of various analytes which are dispensed on a micro-array. The analytes are detected by means of fluorescence or (chemi)luminescence measurement. Furthermore, the collected data are combined and interpreted using modern reasoning techniques. Micro-injection- Dispensing picoliters (pl) of reagents (enzymes, antibodies, etc.) and liquid samples on a micro-array requires special techniques. At the moment we are working on a technique which will allow for accurately dispensing liquid volumes less than 100 pl on a micro-array. Detection of (beta) -D-glucose- (beta) -D-glucose standards are dispensed on a micro-array, after which a solution of Amplex Red reagent, horse radish peroxidase (HARP), and glucose oxidase in a mixture of ethylene glycol and water is added. Ethylene glycol is added to prevent evaporation. The (beta) -D-glucose reacts with glucose oxidase to D-gluconolactone and H₂O₂. The H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) with a 1:1 stoichiometry to produce highly fluorescent resorufin. The formation of resorufin with time is followed with a Zeiss Axioskop microscope equipped with a KAF Photometrics CCD camera, in order to determine the sensitivity, concentrations, and volumes associated with the dispensed fluids.

Thin-walled, compliant plastic structures for meso-scale fluidic systems were fabricated, tested and used to demonstrate valving, pumping, metering and mixing. These structures permit the isolation of actuators and sensors form the working fluid, thereby reducing chemical compatibility issues. The thin-walled, compliant plastic structures can be used in either a permanent, reusable system or as an inexpensive disposable for single-use assay systems. The implementation of valving, pumping, mixing and metering operations involve only an elastic change in the mechanical shape of various portions of the structure. Advantages provided by the thin-walled plastic structures include reduced dead volume and rapid mixing. Five different methods for fabricating the thin-walled plastic structures discussed including laser welding, molding, vacuum forming, thermal heat staking and photolithographic patterning techniques.

We describe the development and performance of microchips that interface capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The chip contains an open channel where CE is performed. The open channel functions as the CE column and is used to separate the mixture. Once separation occurs, the solvent is evaporated and the chip placed in the ionization source of a Fourier transform mass spectrometer. To perform the MALDI, a buffer will be used in the CE that will also function as matrix once the solvent is evaporated. Preliminary results will be described showing: (1) the design and construction of a new ionization source for an external source FTMS that will handle the microchip, (2) the feasibility of the CE on an open channel and (3) the feasibility of MALDI on an open channel. Two chips made of glass with groves cut on the surface have been fabricated for these experiments. The rates of evaporation of different solvent mixtures indicate that evaporation will not be a problem during the CE analysis. The rates of evaporation are considerably slower than the speed of the separation. To determine the feasibility of CE, a colored dye was placed on a 2 cm long column and high voltages attached to the two ends. Movement of a colored dye on the chip was observed under an electric field correspond to about 500 V/cm. This experiment indicates that CE can be performed on an open channel. The first experiments with MALDI of biomolecules, in this case oligosaccharides have been performed. (beta) -Cyclodextrin, a seven-membered cyclic oligosaccharide, was mixed with 3,5-dihydroxybenzoic acid (matrix) on an open channel. Striking the grove with a 337 nm beam from a N₂ laser produces the mass spectrum of the compound with excellent resolution and high signal-to-noise.

Tissue cells bind to extracellular matrix (ECM), and this attachment to ECM plays an essential role in their growth, function, and even survival. Furthermore, the geometry of ECM is known to play an additional role in regulation for these cells to proliferate and differentiate so that tissues with normal morphologies can be formed or maintained. We attempted to culture fibroblast, osteoblast, and bone marrow derived cells in previously described microchannel arrays and newly created micropit arrays, both coated with ECM protein collagen, to examine usefulness of microfabricated structures for elucidating 'what is geometry?' for cells. Cells were inseminated in the well in front of a microchannel array and their movement and stretching behavior against the microchannel array including the entrance and exit terraces were observed using a microscope-TV camera-time lapse video recorder system for 24 hours. Cells entered into the entrance terrace and showed active motions including extending pseudopodia into the channels and whole cell passage through the channels, and fully stretched in the entrance terrace in 8 hours or so. Those cells, however, voluntarily detached themselves from the area in another 8 hours or so probably because of worsening condition of nutrient supply there. Micropit arrays used in the present study consist of a regular arrangement of circular or square pits of diameter or side length of 25, 50, 100, 200, 400, and 600 micrometer and depth of 10 micrometer with one size per array or chip. The total area of the pits was designed to be equal to the rest surface area. After four days of incubation of bone marrow derived cells, the total number of cells and the number of cells in the pits were counted. The former and the ratio of the latter to the former appeared to become maximal when the pits of diameter or side length of 100 and 50 micrometer were used, respectively.

Fluid flow in capillary microchannels is used in numerous applications in biotechnology (such as protein separation, fast DNA analysis, drug deliveries systems and viral filtration), in solid-state devices, and in catalytic devices. The current work presents the experimental validation for the electrokinetic theory in microchannels. Retardation of polar liquids, including de-ionized water, ethanol and propyl alcohol, is studied in microfabricated channels of several diameters. It was found that polar liquids flow about 6 percent more slowly than predicted by the classical hydrodynamic theory in microchannels, with the hydraulic diameter equal to 90 microns. For small microchannels with a hydraulic diameter of several microns, observed retardation is on the order of 70 percent. Collected experimental data have good correspondence with the electrokinetic model presented. Electrokinetic retardation of polar liquids in microchannels is based on the charge separation principle. Electrical charges are separated at the interface (near the channel wall). When liquid is forced downstream, it causes charge accumulation at one end of the microchannel. The streaming potential produced causes an upstream current that creates upstream counterflow. The resultant fluid flow is less than it would be for non-polar liquids. The higher the zeta-potential at the microchannel wall and the smaller the channel, the larger the resulting retardation. Modifications for the friction factor, as applied to microfluidics, are suggested. Recommendations to improve fluid flow in microchannels are made.