Under this NIH-funded project, we are developing a lab-on-a-card platform to identify enteric bacterial pathogens in patients presenting with acute diarrhea, with special reference to infections that might be encountered in developing countries. Component functions that are integrated on this platform include on-chip immunocapture of live or whole pathogens, multiplexed nucleic acid amplification and on-chip detection, sample processing to support direct use of clinical specimens, and dry reagent storage and handling. All microfluidic functions are contained on the lab card. This new diagnostic test will be able to rapidly identify and differentiate Shigella dysenteriae serotype 1, Shigella toxin-producing Escherichia coli, E. coli 0157, Campylobacter jejuni, and Salmonella and Shigella species. This presentation will report on progress to date on sample and bacteria processing methodologies, identification and validation of capture antibodies and strategy for organism immunocapture, identification and validation of specific polymerase chain reaction (PCR) primer sequences for over 200 clinical isolates of enteric pathogens, and implementation of on-chip nucleic acid extraction for a subset of those pathogens.

The development of a lab-on-chip system which allows the parallel detection of a variety of different parameters of a water sample is presented. Water analysis typically comprises the determination of around 30 physical and chemical parameters. An even larger number can arise when special contaminations of organic molecules are of interest. A demonstration system has been realised to show the feasibility and performance of an integrated device for the determination of physical quantities like electrical conductivity, light absorption and turbidity. Additionally, chemical quantities like the pH-value and the content of inorganic and organic contaminations are also determined. Two chips of credit card size contain the analytical functions and will be fabricated by injection moulding. First prototypes have been manufactured by milling or precision milling for the optical components.

Rapid and automated preparation of PCR (polymerase chain reaction)-ready genomic DNA was demonstrated on a multiplexed CD (compact disk) platform by using hard-to-lyse bacterial spores. Cell disruption is carried out while beadcell suspensions are pushed back and forth in center-tapered lysing chambers by angular oscillation of the disk - keystone effect. During this lysis period, the cell suspensions are securely held within the lysing chambers by heatactivated wax valves. Upon application of a remote heat to the disk in motion, the wax valves release lysate solutions into centrifuge chambers where cell debris are separated by an elevated rotation of the disk. Only debris-free DNA extract is then transferred to collection chambers by capillary-assisted siphon and collected for heating that inactivates PCR inhibitors. Lysing capacity was evaluated using a real-time PCR assay to monitor the efficiency of Bacillus globigii spore lysis. PCR analysis showed that 5 minutes' CD lysis run gave spore lysis efficiency similar to that obtained with a popular commercial DNA extraction kit (i.e., IDI-lysis kit from GeneOhm Sciences Inc.) which is highly efficient for microbial cell and spore lysis. This work will contribute to the development of an integrated CD-based assay for rapid diagnosis of infectious diseases.

A novel water chemical toxin sensor has been successfully developed and evaluated as a working portable laboratory prototype. This sensor relies on a disposable plastic biochip prepared with a 4x4 micro-laboratory (μLab) chambers array of Escherichia coli reporter cells and micro-fluidic channels for liquids translocation. Each bacterial strain has been genetically modified into a bioluminescent reporter that responds to a pre-determined class of chemical agents. When challenged with a water sample containing a toxic chemical, the sensor responds with an increased bioluminescent signal from the biochip that is monitored over time. The signal is received by a motorized photomultiplier-based analyzer and interpreted by signal processing software. We have performed several levels of analysis: (i) the change in the bioluminescent signal from the sensor bacteria serves as a rapid indication for the presence of toxic chemicals in the water sample; (ii) the intensity of the change indicates the toxin concentration level; and (iii) the pattern of the responses for the different members of the bacterial panel on the biochip characterizes the biological origin of the toxin. The analyzer contains housing mechanics, electro-optics for signal acquisition, motorized readout calibration accessories, hydro-pneumatics modules for water sample translocation into biochip micro laboratories, electronics for overall control and communication with the host computer. This prototype has a demonstrated sensitivity for broad classes of water-borne toxic chemicals including naladixic acid (a model genotoxic agent), botulinum and acetylcholine esterase inhibitors. This work has initiated an investigation of a novel handheld field-deployable Water Toxicity Analysis (WTA) device.

We present the fabrication and characterization of single mode waveguides fabricated monolithically in SU-8. SU-8 is a negative resist, which can be structured by UV lithography followed by a baking step to induce cross-linking. As a material platform, SU-8 is well suited for systems used for biochemical analysis, as it possesses very high chemical resistance and good mechanical stability. Here we show that single mode embedded waveguides can be fabricated using SU-8 as core material and the modified SU-8, mr-L 6050XP, as cladding material. The refractive index difference between the two materials of the final waveguides is around 0.004. All waveguides fabricated in this work have a height of 4.5 μm and their widths are 3, 5 or 10 μm. We have characterized the losses of these waveguides with the cut-back method both at 635 nm and 1535 nm. We have furthermore studied changes in the refractive index of the material with changes in the processing of the SU-8 material. Finally, absorption measurements in the visible spectral range and mode profile analysis have also been performed. Because of the low optical absorption at wavelengths above 700 nm in combination with the fast, simple and cost-efficient fabrication process, we show that SU-8 is well suited as structuring material for waveguides for integrated optics.

Our research group is interested in environmental sensing of heavy metals that are involved in pollution of aqueous environments. As a result, we are developing chemical sensors within integrated microfluidic systems for sensitive and selective detection of these pollutants. Our approach is to combine established chemical sensing strategies with microfluidic structures, especially in plastic devices, to achieve a total heavy metal analysis system. In this regard, the combination of three complementary techniques - optical waveguide spectroscopy, electrochemistry and chemical partitioning offers the required selectivity and sensitivity essential for many environmental samples. On-chip optical waveguide spectroscopy promises to yield the necessary high sensitivity but relies on fabrication of optical structures with a material of appropriate refractive index, optical quality, and chemical stability by methods consistent with established fabrication methods. SU-8, the epoxy-based negative photoresist, appears to satisfy these requirements and, thus, has become one of our candidate materials for waveguide fabrication on plastic microchips. Although the SU-8 has been previously used for waveguide fabrication, its optical properties and more specifically the influence of processing conditions on resultant optical properties have not been thoroughly characterized. This work presents an evaluation of SU-8-based multimode waveguides on glass and plastic substrates. Optical constants of waveguides have been characterized by spectroscopic ellipsometric and prism coupling techniques. Additionally, using the latter method, evaluation of propagation losses of various structures with different thicknesses has been made. Ellipsometric and prism coupling measurements gave comparable refractive indices for variously cured SU-8 waveguide materials. Prism coupling analyses proved to be more useful for analysis of the many SU-8 waveguide structures fabricated in the thickness range of 5 to 75 μm.

The objective of this effort was to fabricate a waveguide integrated in a polymer microfluidic chip in order to deliver excitation light to fluorescent probes contained in a fluidic channel. Instead of exciting the volume at a certain point along a fluidic channel, the goal herein was to excite all the probes contained along the length of the fluidic channel. An air-waveguide structure was designed and integrated into a polymer microfluidic chip. Fabrication of the microfluidic chip was carried out by double-sided hot embossing of poly methyl methacrylate (PMMA) in sheet form. The efficacy of the waveguide was evaluated by coupling light from a laser into it and testing the fluorescence intensity from dye contained in the microfluidic channel. The results demonstrate illumination of the entire length of the microfluidic channel with excitation wavelength light from the waveguide. Details of the design, fabrication process and initial experimental results are presented in the course of this paper.

Lab-on-chip systems become increasingly more relevant for biochemical analyses. Here is presented a concept for realizing a small footprint chip by combining fluorescence detection and on-chip spectrometry. The chip is to be fabricated using a single mask process based on the negative photoresist SU-8. The various subcomponents are discussed; in particular a spectrometer is presented and interfaced to a linear CCD. The integrated spectrometer combined with the CCD displays a resolving power of 175 for HeNe laser light. The fluidic system is a simple passive microfluidic network which can withstand a pressure in excess of 22 kPa without leakage as long as the sidewall is 10 μm or thicker.

High-precision micromilling was employed as a cost-efficient method preparation of metal masters useful in fabrication of polymer microfluidic devices through replication techniques. In first application, a brass mold master was used for hot embossing of microchip electrophoresis devices in poly(methyl methacrylate) (PMMA). The sidewalls of the milled microstructures were characterized by a maximum average roughness (R_a) of 110 nm and mean peak height (R_pm) of 320 nm. SEM imaging showed a transfer of the sidewall roughness from the molding tool to the polymer microdevice. The electroosmotic flow (EOF) values for micromilled-based microchannels were comparable to ones in the LiGA-prepared devices (sidewall R_a = 20 nm) with values of ca. 3.7 x 10^-4 cm²V^-1s^-1 (20 mM TBE buffer, pH 8.2), indicating insignificant effects of wall roughness on the bulk EOF. Numerical simulations showed that the additional volumes present in an injection cross due to curvature of the corners produced by micromilling lead to elongated sample plugs. PMMA microchip electrophoresis devices were used for a separation of pUC19 Sau3AI double-stranded DNA. The plate numbers achieved exceeded 1 million m^-1 and were comparable to the plate numbers for the LiGA-based devices of similar geometry. In second application brass master was used as tool for preparation of poly(dimethylsiloxane) PDMS stencils for patterning of DNA microarrays onto a PMMA substrate. Four zip code probes immobilized onto the PMMA surface directed allele-specic ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of crosshybridization or misligation.

Silicon-based microelectrode chips are useful tools for temporal recording of neurotransmitter releasing from neural cells. Both invasive and non-invasive methods are targeted by different group researchers to perform electrical stimulating on neural cells. A microfabricated microelectrodes integrated biochip will be presented in this paper, which describes the dopaminergic cells growing on the chip directly. The dopamine exocytosis can be detected non-invasively from drug incubated dopaminergic cells growing on the chip. The abovementioned silicon-based electrochemical sensor chip has been designed with an electrode array located on the bottom of reaction chamber and each electrode is individually electrical controlled. MN9D, a mouse mesencephalic dopaminergic cell line, has been grown on the surface of the biochip chamber directly. Dopamine exocytosis from the chip-grown MN9D cells was detected using amperometry technology. The amperometric detection limit of dopamine of the biochip microelectrodes was found from 0.06μM to 0.21μM (S/N=3) statistically for the electrode diameters from 10 μm to 90 μm, the level of dopamine exocytosis from MN9D cells was undetectable whithout drug incubation. In contrast, after MN9D cells were incubated with L-dopa, a dopamine precursor, K+ induced dopamine extocytosis was temporally detected. The microelectrodes integrated biochip provides a non-invasive, temporal detection of dopamine exocytosis from dopaminergic cells, and holds the potential for applications in studying the mechanisms of dopamine exocytosis, and drug screening. It also provides a tool for pharmaceutical research and drug screening on dopaminergic cells, extendably to be used for other cell culture and drug effects study.

The first on-chip injection and manipulation of optically encoded, silicon microbeads in a microfluidic platform is reported. Encoded microbeads of different shapes and sizes were fabricated in silicon via standard microfabrication techniques. The optical signature consisted of a series of lithographically defined bar-codes, which can be identified by a laser detection system. In-situ identification of encoded microbeads was possible at microbead velocities ≤ 50 cm per second. The microbeads can also be transported within a channel network in accordance with the encoded optical signature of each bead. The microbead transport is controlled by the laminar flow of a liquid in pressure driven microchannels. Hydrodynamic pulsing facilitated single and multiple injection of microbeads from a reservoir into the laminar fluid stream of a branched microfluidic network. Careful control of the fluid velocity and alteration of the microchannel geometry also enabled manipulation of microbead velocity. The incorporation of five pillars to retain microbeads at a specific location within the microchannel network formed the basis of a reaction chamber for on chip functionalization of microbeads. The principle of hydrodynamic switching was utlized to re-direct the transport of microbeads at a branched microfluidic network. In the final part of this research we verify that this microbead technology is suitable for detection of specific target DNA.

A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

In this research, first a modular polymer-based (PMMA) injection micromixer prototype has been designed, fabricated and tested. This micromixer is easy to be integrated into biochemical microfluidic systems under development for BioMagnetICs DARPA funded project at CAMD. To improve the mixing efficiency, layout of micronozzles of the mixer was optimized according to the simulation results. Also because SU-8, an epoxy-based negative photoresist, has high chemical resistance, an SU-8 injection mixer was designed and fabricated to run some biochemical sample liquids. Internal stress in patterned SU-8 structures has been reduced and multi-layer SU-8 processing has been successfully developed to fabricate SU-8 injection mixer.

We report the control of flow direction of a dilute salt solution in a microchannel by external voltage control of an ac electrokinetic micropump. Reversal of the flow velocity is achieved when a voltage of 2.0 V is applied to the drive electrodes and we show that the reverse velocity increases non-linearly with voltage above this value. Velocities of more than 250 um/s at a distance of 75 μm above the electrode surfaces have been measured, which is much greater than in the forward direction. A possible mechanism based on faradaic charge injection is proposed to explain the onset and propagation of reverse flow. We have also shown how multi-directional flow control can be utilized to promote mixing of fluids in a microchannel, and suggest a biochip application which can benefit from this new pumping technology.

Valves for microfluidic systems have, for various reasons, proven to be difficult to fabricate, cumbersome to operate, and/or unreliable. We have explored the performance of a novel microfluidic valve formed by creating a flow channel past a Peltier junction, and developed methods for fabricating multiple such valves in an integrated device. Using the Peltier junction as a thermoelectric cooler causes the fluid in the valve to freeze, forming a plug that blocks flow through the valve. Reversing the current in the Peltier junction causes the fluid to melt, reopening the valve. This type of valve is fundamentally leak-free, has no moving parts, and is electrically actuated. We have also developed a finiteelement thermal model of the valve, and exercised it to optimize valve design. Current prototype valves have cycle times under 100 ms, and optimized valves are expected to be able to operate in less than 10 ms.

In this paper, we report on design and fabrication of a passive microfluidic mixer capable of mixing at low Reynolds numbers (Re). Passive mixers typically use channel geometry to mix fluids, and many previously reported designs that work only at moderate to high Reynolds numbers and are often difficult to fabricate. Our design uses diamond-shaped obstructions inside the microchannel to break up and laminate the flow, thus enhancing mixing. Both numerical and experimental studies show that the mixer is efficient at mixing fluids at low Reynolds numbers. We benchmarked our mixer design against a conventional T-mixer. Results show that the new design exhibits rapid mixing at Re < 0.1. The new mixer has a planar design which is easy to fabricate and thus will benefit a wide range of lab-on-a-chip applications.

Fluid flow in microfluidic systems can be achieved by electroosmosis (EO) pumping, with its own unique characteristics and advantages. In practice, multi-fluid (one fluid displacing another fluid) flows are frequently encountered. Understanding of multi-fluid EO flow associated with non-uniform liquid properties is of importance to precise flow control. This paper reports an EO-driven, two-fluid displacement flow in a microcapillary. The electrical current monitoring method is adopted for investigating the dynamic flow response. The nonlinear change of the electrical current with time under a constant applied voltage is observed during the displacing processes. The theoretical and experimental results validate the hypothesis that the non-uniform zeta potential and electric field induce local pressure gradients in the two different fluids. This results in the deviation of the velocity flow profile from the ideal plug-like flow profile expected for EO flow. The model predictions agreed well with the experimental data when a low concentration fluid displaces a high concentration fluid, but not vice versa. The time of displacement, and thus the flow velocity, is found to be dependent on the displacing flow direction, which is hitherto not reported. The underlying mechanisms are postulated, but require further investigation.

A DNA extraction system was designed and fabricated using an AOM (aluminum oxide membrane) with 200 nm pores and PDMS microfluidic channels. The membrane was patterned using soft lithography techniques and SU-8 photolithography on the membrane. After making the pattern with SU-8, the AOM was observed using an SEM (scanning electro microscope) to verify the AOM structure was not damaged. From the SEM images, the AOM structure was not different after modification with SU-8. To complete the system, a PDMS mold for the microfluidic channels was made by soft lithography. Using the SU-8 mold, PDMS microchannels were cast using PDMS with a low polymer to curing agent ratio to provide adhesion between the patterned membrane and microfluidic channel. Then, the patterned membrane was sandwiched between PDMS microfluidic channels in a parallel format. The completed system was tested with 10ug of Lambda DNA mixed with the fluorescent dye SYBR Green I. Following DNA extraction, the surface of each well was examined with fluorescence microscopy while embedded in the microfluidic system. Extracted and immobilized DNA on the AOM was observed in almost every separation well. This microsystem, referred to as a membrane-on-a-chip, has potential applications in high-throughput DNA extraction and analysis, with the possibility of being integrated into polymer-based microfluidic systems.

Digital In-line Holographic Microscopy (DIHM) is a technique that provides depth and lateral resolution of the order of the wavelength throughout a volume of several cubic centimeters for visible light. This outstanding characteristic is reached by means of a simple optical setup and numerical reconstruction of the recorded holograms. It makes DIHM the right tool for applications in many microscopic studies. In this paper we study microfluidic phenomena by means of DIHM. To this end we seed a fluid with micron-size trackers (latex microspheres) and follow their displacement within an observation volume. We apply this technique to several situations such as the flow around a big sphere, flow through microchannels, bubbles in a fluid, bacterial motion in a diatom and the swimming behavior of paramecia and algae in water. By taking advantage in DIHM of the plane-to-plane reconstruction through a large depth of field, we generate 3D renderings of the paths followed by the trackers to produce a complete picture of the flow pattern, i.e. streamlines and velocity fields.

This paper reports on the design, fabrication, and analysis of an electrochemical (ECM) microactuator. The driving mechanism of the ECM actuator is based on the reversible electrolysis process of water. The expansion and reduction of gas bubbles generated in a micro electrochemical chamber during a reversible electrolysis process can be used to provide a pressure difference in microlfuidic systems. The ECM actuator has a very simple design consisting of inlet/outlet channels, reservoirs, and electrochemical reaction chamber. The fluidic components of the ECM actuator were fabricated on a glass substrate using UV lithography of SU-8 using both Pt black and Ag/AgCl electrodes. The Pt black and Ag/AgCl coated electrodes were supplied with controlled electropotentials for active control of expansion and shrinkage of gas bubbles using reproducible electrochemical reactions. The theoretical volume change rate of gas bubbles was simulated as a function of time using the ideal gas law and compared with the measured volume change. The results show that the simulation can be used to predict trends of the volume change by the electrochemical reactions and also, the device can serve as a promising microactuator for microfluidic applications.

With the continuing advances in the miniaturization of analytical techniques, and in particular of microchip capillary electrophoresis, the challenge of rapid metal complex speciation can now be taken up. The discrimination between different chemical species of metal ions is important as the environmental toxicity, biological activity and availability of many metals is often species dependent. Extensive electrophoresis studies have focused on speciation of low molecular weight metal complex speciation. However, limited research has focused on the separation of different stoichiometries of species within the same metal-ligand system. This new research demonstrates the first rapid separation of individual species in equilibrium within selected metalligand systems by microchip electrophoresis. The transfer of speciation ability within a single metal-ligand system from capillary electrophoresis to microchip electrophoresis has been demonstrated with considerable success. This emphasises the immense future potential for microchip electrophoresis in the field of rapid chemical speciation.

The biodegradable controlled-release system with large array of micro chambers is a new controlled drug delivery system. The structure of the system is designed using the MEMS technology, combining the drug release condition and the biodegradable characteristic of the polymer. This type of drug delivery system has some unique advantages in controlled long-term drug delivery, such as more drug loading than the matrices release systems, easier control the release rate, and so on. It is necessary to optimum the structure for the long-term drug delivery. This paper founds the simulated modeling which can simulate the erosion and the drug delivery from this controlled-release system. Further more the thickness of the chamber's wall is taken as the design variable. An optimized modeling for the drug delivery from the biodegradable controlled-release system is developed. This model can optimize the structure of the controlled release system for the desired release characteristics. The results indicate that this method can be used to design the drug delivery system based on the biodegradable polymer.

This paper advances a kind of micro spectrometer, which is based upon Fabry-Perot antrum's character of filtering the waves. The basic structure of the micro spectrometer is the array of Fabry-Perot antrum which contains many different length of antrum on the substrate of silicon, consequently we can achieve the detector for several wavelengths simultaneously. The unit of probing is a Fabry-Perot antrum, which is made up of the substrate of silicon--metal film--silicon dioxide layer-- metal film. We carried out the corresponding simulation. In the basic structure of aluminum film(14nm)- silicon dioxide layer - silver film(39nm), the resolution can reach 15nm. When the area of a unit of probing is 0.14mm x 0.14mm only, it can reach the luminous flux of miniature grating spectrum instrument(the minimum volume in the order of cm),but the volume of the part of spectrum detector is only the order of mm. The design size of the micro spectrometer is in a few millimeters. Furthermore it has no movable parts and could detect several wavelengths at the same time. It is possible to fabricate such micro spectrometer through existing process methods of IC technology.

In recent years, highly sensitive and selective as well as cost-effective sensing and detection of bio-molecules (e.g. virus, bacterial, DNA and protein) by MEMS/NEMS (Micro-/Nano Electro-Mechanical-System) structures have attracted extensive attention for its importance in clinical diagnostics, treatment, and various genome projects. Meanwhile, substantial research efforts have been spent on the improvement of sensitivity of BioMEMS structures. Among a variety of methods that have been investigated, surface modification by nanoparticles (NPs) turns out to be an attractive way, which provides a platform for the enhancement of the sensitivity for biosensor devices. However, conventional applications for surface modification were mostly implemented on microelectrodes. This paper is going to present the self-assembly surface binding of nano-gold particle and functional MWCNT on the cantilever sensor, which can easily facilitate biomolecular detection by resonance frequency shift. Its sensitivity can be improved due to the large binding area of probes to the targeting biomolecules. The LPCVD SiN low-stress rectangular cantilever is produced by laser micromachining and alkaline KOH etching, which is a maskless, simple, convenient, fast-prototyping way to produce such cantilever sensor for biomolecular detection. The commercially available Atomic Force Microscopy (AFM) cantilevers are also used to verify the concept.