As biosensors become more sophisticated and commercially available, the general appreciation for their capabilities also increases. We now focus on multi-analyte sensors and address the problems inherent in discriminating multiple simultaneous signals without loss in assay speed, specificity or sensitivity. Furthermore, the goals of portability, simplicity and low cost have not diminished in importance. NRL is developing a multi-analyte sensor designed to be portable, inexpensive, and easy to use. To achieve these goals, we use a room temperature CCD, a diode laser, and a disposable waveguide. While our goals of using automated fluidics and automated image processing are not yet completely realized, we have fabricated a prototype biosensor which fits into a tackle box with a associated portable computer. Simple microscope slides are used as waveguides and precoated with arrays of immobilized antibodies. Fluorescence immunoassays are performed on these waveguides using as many as 6 samples at a time and assaying for up to 5 different analytes in each samples Sensitivities in the 1-10 ng/ml range have been achieved in 10-minute assays. Initial studies in clinical fluids indicate that assays can be run on samples such as whole blood, plasma, urine, saliva and nasal secretions.

This paper describes a design for an inexpensive phase fluorimeter based on blue or green light emitting diodes for use with chemical and biological sensors with fluorescence and phosphorescence lifetime-based transduction. The phase fluorimeter is based on a personal computer, two frequency synthesizers and off-the-shelf optical and electronic components and has an estimated lifetime resolution better than 10 ps. The phase fluorimeter is especially well-suited for implementation with arrays of chemically sensitive elements. The data acquisition system allows rapid monitoring of light emission from fluors or phosphors immobilized in the chemically sensitive array elements, each of which can be designed to be responsive to a particular chemical analyte. This paper describes chemically sensitive elements based on ultrathin films of porous polymers and on self-assembled monolayers. The paper focuses on methods for detection of low levels of luminescence emission from micropatterned array elements that comprise sensor arrays. Of particular importance is the detection of low levels of fluorescence and phosphorescence from SAMs of alkanethiolates on thin gold and silver films. Methods for enhancing the luminescence yield from these SAMs include optimization of the dielectric environment of the luminescent dyes and surface plasmon resonance enhanced excitation.

We propose a novel method for individual immobilization. Biomaterials were first immobilized enlarges the size of biomaterials to the size of sensor element. Then the support is arranged on the sensor element by self-assembling. The element was microfabricated to have a microstructure for self-assembling. When both size of biomaterial and the element are the same, self-assemble is expected to give one- to-one correspondence, and then individual response. Mixed glass beads immobilized each of various enzyme were put near the pits fabricated one chip and the chip was slanted to roll the beads into the pits. When the beads immobilized only with peroxidase were arranged, the addition of luminol and hydrogen peroxide gave chemiluminescence at almost every site. Next, the beads immobilized only with glucose oxidase as dummy enzyme were mixed with HRP-beads and arranged to the sites. Addition of substrate gave limited number of luminescence-giving sites, though every site had an enzyme- immobilized bead. These results show that two kinds of enzymes were separately arranged in the site in one-to-one correspondence.

Silicon biomedical microdevices generally require surface modifications to improve their biocompatibility. ONe of the challenges in the field is the development of molecular coatings for devices with nanofeatures.In this paper we report the results of our investigation of synthetic poly(ethylene glycol) (PEG) and monomethoxypoly(ethylene glycol) (MPEG) coatings incorporating functional groups that in turn immobilize self-assembled monolayers. The properties of the modified surfaces were characterized by ellipsometry and scanning electron microscopy. Protein adsorption as well as platelet adhesion to the treated surfaces were studied to evaluate the non-fouling property of the PEG-enriched surface layers. Both PEG- and MPEG-modified surfaces showed significant suppression in plasma protein adsorption and platelet adhesion.

Uniform, conformal, and ultrathin coatings are needed on the surface of biomedical microdevices such as microfabricated silicon filters, in order to regulate hydrophilicity and minimize unspecific protein adsorption. Currently, the predominant coating methods involve the assembly of normally a silane 'monolayer' onto silicon surfaces in organic solution. A typical prototype molecule is alkyltrichloro- silane. However, this type of molecule is very sensitive to moisture. Even trace amount of water in the organic solution or its environment lead to polymerization.This causes the formation of multilayers with variable thickness, and submicron aggregates or islands on the silicon surface. In this communication we present a vapor-phase coating method of forming a uniform and nanometer thick silanes on silicon surface at ambient pressure using nitrogen as a carrier gas. The modified surface is extremely smooth, with no detectable aggregates. The coatings and subsequent treatments are characterized with ellipsometry, scanning electron microscopy, contact angles, SFG spectroscopy, and zeta potential in water. The method of deposition is particularly advantageous whenever it is necessary to coat irregular shapes or channels in microdevices, where liquids may have difficult access due to capillary forces.

Surface plasmon resonance (SPR) is a phenomenon wherein the reflectance versus angle-of-incidence profile for a thin gold film illuminated with p-polarized light has a distinct minimum at a particular angle. This minimum of reflectance is due to an absorption of the light energy by the surface electron plasma of the metal occurring when the surface- parallel components of the light and plasmon propagation vectors match up. The value of this particular angle of incidence changes in proportion to the degree of adsorption of analytes to the metal film. This allows SPR to be used as a simple, noninvasive, optical tool for measuring the binding of chemical analytes. With a predetermined pattern of chemically specific receptors bound to the gold film, it is possible to detect a variety of species and concentrations of analytes, provided that one has a sensor platform capable of resolving the different reactions in each element of the receptor array. We have developed such a platform which is capable of optically monitoring an array of analyte receptors immobilized on gold coated microscope slides in real time. Moreover, the optical resolution of sensor platform allows the receptors to be micro-patterned.

Silicon-based biocapsules have been microfabricated with uniform and well-controlled pore dimensions in the tens of nanometer range to provide effective immunoisolation of cell xenografts. Surface and bulk micromachining were integrated in the fabrication process, resulting in a diffusion membrane with mechanical and chemical stability, surrounded by an anisotropically-etched silicon wafer, which serves as the encapsulation cavity. The membrane allows the diffusion of essential nutrients while blocking the passage of immune molecules, which may destroy cellular transplants. Preliminary short term studies on both primary pancreatic islets and insulinoma cell lines encapsulated within microfabricated biocapsules were conducted to determine the toxicity and biocompatibility of biocapsules, the viability and functionality of encapsulated cells, as well as the overall immunoprotective capabilities of the biocapsule. Results seem to indicate that microfabricated biocapsules are non-toxic and do not elicit any adverse inflammatory reactions when implanted. Furthermore, encapsulated insulinoma cells remained viable and functional within microfabricated environments in vivo. These results show the feasibility and potential application of microfabricated biocapsules for several pathologies.

A new type of spectroelectrochemical sensor that embodies tow modes of instrumental selectivity in addition to selective partitioning through an applied film barrier is described. The sensor consists of a planar optical substrate/electrode coated with a chemically-selective film. SEnsing is based on the change in the attenuation of light passing through the guided wave substrate which accompanies a chemical reaction of an analyte induced by electromodulation. Threefold selectivity for a chosen analyte relative to other environmental components is obtained by the choice of coating material, the electrolysis potential, and the wavelength for optical monitoring. The sensor concept is demonstrated with an indium tin oxide coated glass guided wave device that has been over-coated with a sol-gel derive charge-selective thin film. One such selective coating used was a charge-selective sol-gel processed Nafion-SiO₂ composite film. Prototype analytes have been used to demonstrate that the change in the transmittance of the waveguide resulting from electrochemical oxidation/reduction can be used to quantify an analyte.

Two new series of chemically-selective optical materials have been made and tested on chemical sensor. One new series of materials was based on polymer blending in a host of glutaraldehyde cross-linked poly(vinyl alcohol). Chemically- selective dopants in this host demonstrate property- selective separations of chemicals from mixtures. We have optimized the composition, optical properties and the coating procedures for several specific blends for optical sensing. These blends have clear UV and visible spectral regions for direct spectroscopic sensing and they are excellent absorber of many inorganic and organic charged species from aqueous environments. A second new series consists of polyelectrolyte containing silica composites prepared by sol-gel processing. The thickness of spin-coated films of these materials on glass can be varied from 0.1 micrometers to 4 micrometers . These materials are ion exchangeable and less brittle than the parent silica substrate due to the incorporation of the organic polyelectrolyte. These new composites retain the nano-scale porosity and optical transparency into the UV of the parent silica sol-gel processed glasses making them attractive host matrices for the immobilization of a variety of chemical reagents. Results obtained with film-clad sensors from both new series of materials are presented.

A fiber optic based deep UV-absorption sensor system is characterized, using fibers for light delivery and a liquid core waveguide (LCW) for analyzing liquids. UN-improved fibers with 500 micrometers core diameter are capable of transmitting light intensities below 230 nm with spectral radiant powers above 500 nW/nm at 214 nm. Their short-term behavior and lifetime in respect to UV-stability have been investigated, using a broadband deuterium lamp. To raise the sensitivity of the total system, the absorption path length has been increased significantly using the lightguiding properties of the LCW consisting of a cylindrical glass tube with a Teflon AF 2400 inner coating of about 50 micrometers thickness. Due to lower refractive index of Teflon in comparison to water, the LCW concept offers significant advantages, especially for long optical pathlengths. However, the basic attenuation of the liquid in dependence on the wavelength as to be taken into account. Results on the use of such a system monitoring concentrations of acetylsalicylic acid, acetone and toluene in water are reported and discussed.

Micromachining technologies were applied to fabricate metal- electrode-instrumented microchannels with cross-sectional dimensions similar in size to blood cells. The instruments enable electric impedance measurement of femptoliter quantities of materials and solutions. The completed micro- electric impedance devices were characterized with varying concentrations of phosphate buffered saline solutions, DI water, and air in the recording zone. With the microdevices on the stage of an inverted light microscope, individual living cells were positioned tightly between metal electrodes using mechanical suction. Impedance spectra form 100 Hz to 2 MHz measured in isolated toadfish red blood cells (RBCs) and human neutrophils were distinct and demonstrated the ability to permeate the cell membrane at high frequency. The cell/shunt path cut-off frequency were approximately 400 kHz at -3dB indicating that non- invasive electric impedance characterization of the cytoplasm may be feasible for his configuration. In addition, the area specific membrane capacitance was estimated for both cell types by fitting the data to a simple RC circuit model.

Reports of enhanced photostability of organic florescent dyes when entrapped in a sol-gel matrix have led us to examine the behavior of the near-IR dye IR-125 coated onto the end of single mode optical fibers. Various fiber tip geometries were fabricated and a specially prepared rounded tip was found to be optimal for long-term adhesion of thick sol-gel coatings to the fiber. Fluorescence captured back into the fibers was measured over one month and different initial concentrations of dye were also examined. The porosity of sol-gel thin films has been explored in the past to fabricate pH, oxygen, and other enzyme-based sensors. We have tested the porosity of our coatings by showing a non- reversible pH sensitivity, however current data is insufficient to rule out the possibility of a structural change in the coating also effecting these results. Using the rounded tip geometry, eighty percent of our samples lasted over two months without cracking while still maintaining fluorescence activity. Exposed to air, the coated fibers were each able to be examined an average of 200 times with 20 microsecond(s) ec interrogation pulses of 1.5 mW from a 785 nm semiconductor laser diode before photobleaching reduced the resulting signal to background.

We will discuss two recent directions of our work: (1) The influence of submicron length scales on polymer dynamics, (2) Ultra-rapid mixing via sub-micron hydrodynamic focusing. (1) Polymer dynamics at sub-micron length scales. We have explored the changes in the dynamics of long polymers as the thickness of the quasi-2 dimensional space is varied from 0.09 microns to 10 microns. We will show how the thickness of this space, scaled with the persistence length of the polymer, changes the dynamics of the polymer. The consequences of this qualitative change in polymer dynamics is quite important, since it controls the elongation of the polymer at a given force field and hence the ability of he array to fractionate the polymer. (2) Mixing at the sub- micron length scale cannot be tubulent but only diffusive in nature. We will show how it is possible using hydrodynamics to produce liquid jets of width under 20 nanometers which can mix fluids in under 1 microsecond times.

A new technique for fabricating 2D artificial gels for DNA electrophoresis is presented. The technique differs from previous approaches in that the entire device is fabricated as a monolithic unit using exclusively planar processing techniques borrowed from semiconductor electronics fabrication. The height of the fluid gap between the dielectric floor and ceiling is determined by the thickness of a sacrificial layer which is removed by a wet chemical etch. This allows precise control and excellent uniformity of the gap over an entire silicon wafer. Gap control better than 5 nm has been demonstrated for floor-to-ceiling height for the fluid gap. The lateral resolution which can be attained is limited only by available lithographic techniques. In this work, 1 micrometers diameter pillars are defined with i-line photolithography. Fluid interconnects are established with a liquid meniscus to the hedge of the device.

Particle image velocimetry (PIV), a technique commonly used at the macroscopic level to measure velocity vectors of particle-seeded flows, is adapted to measure both instantaneous and ensemble-averaged flow fields in microfluidic MEMS devices, where micro-scale spatial resolution is critical. Adapting PIV to the microscopic level presents a number of challenges, including: (1) visualizing tracer particles that are smaller than the wavelength of light, (2) minimizing errors due to the Brownian motion of the tracer particles, and (3) recording particle images with short exposure times, so that their motion does not cause particle streaking in the image field. The PIV technique is used to measure a low Reynolds number Hele-Shaw flow around a roughly 30 micrometers elliptical obstruction and a low Reynolds number flow through a 20 X 200 micrometers capillary tube. Velocity vector fields are presented with a spatial resolution of 6.9 X 6.9 X 1.5 micrometers . In principle, super-resolution particle tracking velocimetry can be used to extend the spatial resolution of the velocity measurements down to approximately 1.5 X 1.5 X 1.5 micrometers .

Microfluidic devices fabricated in silicon are quickly finding use in many areas of technology. Exploration of new applications of this technology has shown both advantages and disadvantages to extreme miniaturization of chemical assays. While accuracy, efficiency and smaller sample volumes are among the advantages, interactions between the walls of the micro-channels and the fluid or particles it contains are among the disadvantages. Our group is applying this technology to chemical and biological warfare (CBW) agent purification and detection. We present preliminary result towards achieving a long-term antifouling surface in our detection system. A microfluidic device was anisotropically etched in a (100) silicon wafer and attached to a Pyrex glass slip to create an enclosed channel. Poly(ethylene glycol) (PEG) silane was covalently bonded to the hydroxyls of an oxide layer on the silicon device and the Pyrex cover slip. Fluorescently labeled ovalbumin, a CBW simulant, was in contact with an unmodified and PEG-modified channel. The extent of adsorption was determined using fluorescence microscopy.

We are investigating fabrication techniques that can be used to form arbitrarily shaped fluid capillaries at dimensions below 1 micrometers . We are also considering processes and materials for forming optical waveguides in the same devices with the same fabrication processes. The intent is to develop fabrication methods that can be used to make optical/fluid-flow systems for greater miniaturization, integration and parallelism of optical excitation and detection systems, for the sampling of small volumes. We have demonstrated fabrication processes that enable the creation of functional fluid channels and waveguide in a single step. The independent operation of capillary channels and waveguides has been demonstrated and a system is designed for future testing of in-plane optical excitation of fluorescence. Capillaries with widths below 1 micrometers dimensions have been fabricated using photolithography and reactive ion etching in glass and silicon substrates. We have driven dye labeled DNA molecules electrophoretically through the micrometer size channels and observed individual molecules fluorescence. Surface energy on the high relative surface area channels is significant in the filling of the channels with aqueous solutions and treatment of the liquid contacting surfaces has influence on the system behavior. Light is coupled into the waveguide through gratings fabricate by electron-beam lithography.

The ability to separate 30-100 nm particles - nanofiltration - is critical for many biomedical applications. Where this filtration needs to be absolute, such as for viral elimination in the blood fractionation process, the large variations in pore size found with conventional polymeric filters can lead to the unwanted presence of viruses in the filtrate. To overcome this problem, we have developed a filter with micromachined channels sandwiched between two bonded silicon wafers. These channels are formed through the selective deposition and then removal of a thermally-grown oxide, the thickness of which can be controlled to +/- 4 percent for 30 nm pores. In this paper, we will present both the gas and liquid characterization, and the filtration studies done on 44 and 100 nm beads.

We have realized a valveless micropipetting device with an integrated sensor which can aspirate and dispense liquid volumes without any valves, hence without any reflow or dead volume. With an external pneumatic actuation, we have demonstrated aspirating and dispensing from 190nl of 6 (mu) l of water. Measurements showed a standard deviation of down to 1 percent. An integrated capacitive sensor will allow monitoring of the pressure throughout the pipetting process and detect malfunctions, e.g. clotting of the pipetting tip. It is our intention to use this demonstrated precise aspiration mechanism in combination with a micromachined reaction chamber and a miniaturized optical analysis system.

In this paper,several candidate bonding materials are reviewed for use in biomedical microdevices. These include poly propylmethacrylate (PPMA), poly methylmethacrylate (PMMA), a copolymer of poly methacrylate and two types of silicone gels. They were evaluated based on their cytotoxicity and bond strength, as well as several other qualitative assessments. The cytotoxicity was determined through a cell growth assay protocol in which cells were grown on the various substrate and their growth was compared to cells grown on control substrate. The adhesive strength was assessed by using a pressurized plate test in which the adhesive interface was pressurized to failure. All of the substrate were found to be non-cytotoxic in an inert manner except for the industrial silicone adhesive gel. The adhesive strengths of the various materials are compared to each other and to previously published adhesive strengths. All of the materials were found to have a sufficient bonding strength for biomedical applications, but several other factors were determined that limit the use of each material.

The microfabrication of DNA sample preparation, electrophoretic separation and detection devises is making possible a new generation of high-speed, high-throughput DNA analysis systems. HIgh quality DNA fragment sizing and short tandem repeat analyses have been preformed on microfabricated capillary electrophoresis devices that are 10-100 times faster than conventional electrophoretic separations. Microfabricated capillary array electrophoreses (CAE) plates increase the throughput of these devices by permitting the analysis of multiple samples in parallel. The practical utility of these devices has been improved by developing elastomer sample well and electrode arrays with standard microtiter dish spacings. We initially developed a CAE microplate that can analyze 48 samples using an array of 12 capillaries. More recently, we have developed an improved microplate with an array of 48 capillaries capable of analyzing up to 96 DNA samples in < 8 minutes. These highly parallel, miniaturized DNA analysis systems are a significant step toward fast and inexpensive genetic analysis.

We have microfabricated devices to size and sort microscopic biological objects, ranging from cells to single molecules of DNA. Sizing is accomplished by fluorescent excitation and detection. The devices are fabricated in a silicone elastomer using a replica method. Single molecules of DNA have been sized to 10 percent accuracy, and manipulation of E. Coli cells has been demonstrated.

Possibility of cell sorting by cellular deformability was examined using yeast cells and previously described microchannel arrays. Cells harvested at every hour during incubation were washed and suspended in sorbitol solution at a concentration of O.D. 0.3. An aliquot of each suspension was caused to flow through the microchannel concentration of O.D. 0.3. An aliquot of each suspension was caused to flow through the microchannel arrays by applying 20 cmH₂O suction. Cells at two hours of incubation could not enter into the microchannels, while cells at 4-5 hours of incubation could enter into the microchannels despite their larger size due to budding than the preceding ones and some few cells were observed to pass through 8 micrometers width microchannels. The number of cells that could enter into the microchannels decreased at 7-8 hours and re-increased at 9- 10 hours, but the synchronism in this second cycle appeared to decrease. Protoplasts prepared by treatment with zymolyase from cells at 4-5 hours of incubation showed no appreciable resistance to the microchannel passage.

In this paper we report investigations of semiconductor laser microcavities for use in detecting changes of human blood cells during lysing. By studying the spectral before and during mixing of blood fluids with di-ionized water, we are able to qualify the cell shape and concentration of hemoglobin in real time during the dynamical process of lysing. We find that the spectra can detect subtle changes that are orders of magnitude smaller than can be observed by standard optical microscopy. Such sensitivity in observing cell structural changes has implications for measuring cell fragility, monitoring apoptitic events in real time, development of photosensitizers for photodynamic therapy, and in-vitro micromanipulation techniques.

At this early phase in the development of microfabricated fluidic systems, only a few components or functions have been microfabricated. Some sort of interface to the remaining 'off chip' components is required. For example, a variety of analysis techniques have been demonstrated in microfabricated channels, and cells, but sample preparation is to date still mostly performed off chip, involving pipetting, tubing and titer plate interfacing. The transition from micro to macro components has been to date rather crude, consisting mostly of tubing glued into or over holes etched into silicon or glass substrates. This paper presents new, micromachinable, joining and interconnecting structures that enable the modular, plug-in assembly of fluidic components to one another, to tubing, and into a fluid channel breadboard. Micro-to-miniature interfacing elements for making connections between microchannels and standard tubing, and both horizontal and vertical channel- to-channel interconnects will be demonstrated. Excellent seals are created using photopatternable silicone O-rings that are held in compression by the connecting structure. This technology allows one to assemble a fluidic microsystem with both custom and off the shelf, micro or miniature components. The connections are all reversible, making the system design reconfigurable and components easily exchanged.

For the last two decades, the majority of research and development at LLNL in microtechnology has focused on photonics devices and bulk micromachining, including micro- electro-mechanical systems (MEMS) and associated areas. For the last ten years, we have used these capabilities to address our analytical instrumentation needs. Just as the integrated circuits in the 1960's enabled the fabrication of portable electronics, MEMS and miniature photonics have enabled the fabrication of analytical instruments that are either higher performance, smaller, more portable, or are combinations of these. Examples of these are our portable thermal cyclers for DNA analysis, our hand-held gas chromatography, our flow-stream-waveguide-based flow cytometer, and our etched-microchannel electrophoresis systems. This presentation will describe these and some related developments.