Search

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.

The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.

Significant advances have been made in the development of micro-scale technologies for biomedical and drug discovery applications. The first generation of microfluidics-based analytical devices have been designed and are already functional. Microfluidic devices offer unique advantages in sample handling, reagent mixing, separation, and detection. We introduce and review microfluidic concepts, microconstruction techniques, and methods such as flow-injection analysis, electrokinesis, and cell manipulation. Advances in micro-device technology for proteomics, sample preconditioning, immunoassays, electrospray ionization mass spectrometry, and polymerase chain reaction are also reviewed.

Microfluidic structures for the generation of laminar fluid diffusion interfaces (LFDIs) for sample preparation and analysis are discussed. Experimental data and the results of fluid modeling are shown. LFDIs are generated when two or more streams flow in parallel in a single microfluidic structure without any mixing of the fluids other than by diffusion of particles across the diffusion interface. It has been shown that such structures can be used for diffusion-based separation and detection applications. The method has been applied to DNA desalting, the extraction of small proteins from whole blood samples, and the detection of various constituents in whole blood, among other examples. In this paper the design and manufacture of self-contained microfluidic cartridges for the extraction of small molecules from a mixture of small and large molecules by diffusion is demonstrated. The cards are operated without any external instrumentation, and use hydrostatic pressure as the driving force. The performance of the cartridges is illustrated by separating fluorescein from a mixture of fluorescein and dextran of molecular weight 2 x 10(6). In a single pass, 98.6% of dextran was retained in the product whereas 43.1% of fluorescein was removed. The method is adjustable for different separation requirements, and computational fluid dynamics (CFD) models are shown that demonstrate the tuning of various microfluidic parameters to optimize separation performance. Other applications of LFDIs for establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients are shown qualitatively. Microfluidic chips have been designed for high-throughput screening applications that enable the uniform and controlled exposure of cells to lysing agents, thus enabling the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure.

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires1 microl of reagents and sample, and is applicable to a wide range of analytes.

An integrated microfluidic design, modeling, and rapid prototyping process is presented. It is based on laser cutting and lamination of individual thin layers of plastic. The process allows the rapid and low-cost manufacturing of both simple and complex 3-dimensional microfluidic flow structures that are routinely designed, fabricated, and tested within the space of 24 hours. It has yielded microfluidic elements and systems, such as mixers, separators, and detectors, as well as complete microfluidic integrated circuits that have been used with complex biological samples such as whole blood. Both “active”, machine-controlled microfluidic disposables as well as “passive”, self-contained cards that do not require any external instrument are presented. Such devices include a disposable hematology analyzer chip, as well as blood separation and analysis tools.

Microfluidic flow conditions allow the design of highly effective, yet simple devices for on-chip sample preparation and cleanup. Over the past few years, Micronics has developed a number of novel microfluidic structures that are compatible with complex samples such as whole blood or contaminated environmental fluids. The H-Filter is a technology based on the parallel laminar flow of two or more miscible streams in contact with each other. Such streams do not mix, but chemicals contained in these streams can diffuse from one stream into the other, with smaller molecules diffusing faster than larger ones. This principle can be used, for example, to remove salt from a solution containing DNA, or to extract smaller molecules from whole blood. The T-Sensor is based on the same laminar flow diffusion principle, but combines sample preparation with self-calibration and detection. These devices can be used not only in stand-alone research and point-of-use testing applications, but they can also be integrated, as...

The T-sensor is a recently developed microfluidic chemical measurement device that exploits the low Reynolds number flow conditions in microfabricated channels. The interdiffusion and resulting chemical interaction of components from two or more input fluid streams can be monitored optically, allowing measurement of analyte concentrations on a continuous basis. In a simple form of T-sensor, the concentration of a target analyte is determined by measuring fluorescence intensity in a region where the analyte and a fluorescent indicator have interdiffused. An analytical model has been developed that predicts device behavior from the diffusion coefficients of the analyte, indicator, and analyte--indicator complex and from the kinetics of the complex formation. Diffusion coefficients depend on the local viscosity which, in turn, depends on local concentrations of all analytes. These relationships, as well as reaction equilibria, are often unknown. A rapid method for determining these unknown parameters by interpreting T-sensor experiments through the model is presented.

In channels with dimensions much less than 1 mm, fluids with viscosities similar to or higher than that of water and flowing at low velocities exhibit laminar behavior. This allows the adjacent flow of fluids and particles in a channel without mixing other than by diffusion. We demonstrate here the use of a 3-input microfluidic device known as a T-Sensor for the analysis of blood. A sample solution (e.g. whole blood), a receptor solution (e.g. an indicator solution), and a reference solution (a known analyte standard) are introduced into a common channel (T-Sensor), and flow side by side until they leave the structure. Smaller particles such as ions or small proteins diffuse rapidly across the fluid boundaries, whereas larger molecules diffuse more slowly. Large particles (e.g. blood cells) show no significant diffusion within the time the flow streams are in contact. Two interface zones are formed between the fluid layers. The ratio of a property (e.g. fluorescence intensity) of the outer portions 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. This device allows, for example, one-time or continuous monitoring of the concentration of analytes in microliters of whole blood without the use of membranes or prior removal of blood cells. The principle is illustrated by the determination of pH and human albumin in whole blood and serum. Results are also presented for zero-gravity experiments performed with a T-Sensor on board a NASA experimental plane. Due to its microfluidic flow characteristics, a T-Sensor functions independently of orientation and strength of the gravitational field. This was demonstrated by exposing a T-Sensor to variations in gravity from 0 to 1.8 g in a NASA KC135A plane flying repetitive parabolic flight curves.

Many infectious diseases, as well as some cancers, that affect global health are most accurately diagnosed through nucleic acid amplification and detection. There is a great need to simplify nucleic acid-based assay systems for use in global health in low-resource settings as well as in settings that do not have convenient access to laboratory staff and equipment such as doctors’ offices and home care settings. In developing countries, unreliable electric power, inadequate supply chains, and lack of maintenance for complex diagnostic instruments are all common infrastructure shortfalls. Many elements of instrument-free, disposable, nucleic acid amplification assays have been demonstrated in recent years. However, the problem of instrument-free,1 low-cost, temperature-controlled chemical heating remains unsolved. In this paper we present the current status and results of work towards developing disposable, low-cost, temperature-controlled heaters designed to support isothermal nucleic acid amplification assays that are integrated with a two-dimensional paper network. Our approach utilizes the heat generated through exothermic chemical reactions and controls the heat through use of engineered phase change materials to enable sustained temperatures required for nucleic acid amplification. By selecting appropriate exothermic and phase change materials, temperatures can be controlled over a wide range, suitable for various isothermal amplification methods, and maintained for over an hour at an accuracy of +/- 1°C.

Monitoring for drug-induced liver injury (DILI) via serial transaminase measurements in patients on potentially hepatotoxic medications (e.g., for HIV and tuberculosis) is routine in resource-rich nations, but often unavailable in resource-limited settings. Towards enabling universal access to affordable point-of-care (POC) screening for DILI, we have performed the first field evaluation of a paper-based, microfluidic fingerstick test for rapid, semi-quantitative, visual measurement of blood alanine aminotransferase (ALT). Our objectives were to assess operational feasibility, inter-operator variability, lot variability, device failure rate, and accuracy, to inform device modification for further field testing. The paper-based ALT test was performed at POC on fingerstick samples from 600 outpatients receiving HIV treatment in Vietnam. Results, read independently by two clinic nurses, were compared with gold-standard automated (Roche Cobas) results from venipuncture samples obtained in parallel. Two device lots were used sequentially. We demonstrated high inter-operator agreement, with 96.3% (95% C.I., 94.3–97.7%) agreement in placing visual results into clinically-defined “bins” (5x upper limit of normal), >90% agreement in validity determination, and intraclass correlation coefficient of 0.89 (95% C.I., 0.87–0.91). Lot variability was observed in % invalids due to hemolysis (21.1% for Lot 1, 1.6% for Lot 2) and correlated with lots of incorporated plasma separation membranes. Invalid rates, National Institutes of Health (U.S.) (PATH Center to Advance Point of Care Diagnostics for Global Health NIBIB U54-EB007949), National Institute of Neurological Disorders and Stroke (U.S.) (Grant 5F30NS06468)

In many health care settings, it is uneconomical, impractical, or unaffordable to maintain and access a fully equipped diagnostics laboratory. Examples include home health care, developing-country health care, and emergency situations in which first responders are dealing with pandemics or biowarfare agent release. In those settings, fully disposable diagnostic devices that require no instrument support, reagent, or significant training are well suited. Although the only such technology to have found widespread adoption so far is the immunochromatographic rapid assay strip test, microfluidics holds promise to expand the range of assay technologies that can be performed in formats similar to that of a strip test. In this paper, we review progress toward development of disposable, low-cost, easy-to-use microfluidics-based diagnostics that require no instrument at all. We also present examples of microfluidic functional elements--including mixers, separators, and detectors--as well as complete microfluidic devices that function entirely without any moving parts and external power sources.

This chapter provides an introduction to concepts of microfluidics and microfluidic design. First, terms are defined and basic fluid dynamic relationships are introduced. Physical aspects and design consequences of working with fluid flow at the microscale are described. Then, the determination of the preferred scale for various applications of microfluidic devices is discussed. Finally, various kinds of fluid flow typical of microdevices are described in more detail, including gravity- and pressure-driven flow, flow through packed media, electrokinetic flow, capillary-driven flow, and absorption-driven flow. Keywords: microfluidics; microfluidic flow; fluid dynamics; microdevices

This chapter provides insight into practical aspects of microfluidic device design, such as fluid movement, device construction, and feature integration. Several methods of moving fluids into and within microfluidic devices are described, including batch-flow and continuous-flow micropumps, electrokinetic pumps, and external positive displacement pumps. Solutions for the problem of interfacing external fluid sources with a microfluidic device are given. Then, various methods for the construction of microfluidic devices are discussed, such as photolithography, soft lithography, and laminate-based designs. Finally, as a case study, the integration of a microfluidic device for the detection of pathogens is described. Keywords: microfluidics; bioMEMS; lab on a chip; micropump; photolithography; soft lithography; laser ablation; machining; plastic-film laminates; flow sensor

With macroscopic chemical analysis devices, it is usually possible during the development phase to mount flow sensors, temperature probes, and optical detectors at various positions along the instrument pathway to experimentally determine the optimum operational parameters for the device. This approach usually fails for microdevices as standard sensors and probes are typically of the same scale as the microdevice. These relatively large sensors interfere so much with the experiments that any results generated do not represent the actual performance of the system. Fortunately, modeling of microscale processes provides a uniquely useful tool to develop microanalytical devices and optimize their operational parameters, since the chemical and physical processes in the microscale generally follow deterministic physical laws that can be accurately represented in mathematical models. We will discuss some of the methods used to model and design microanalytical assays based on these principles, as well as several ongoing development projects that MicroPlumbers is currently involved in (including a clinical microsensor, a microvolume blood collection device, and a novel clinical assay), and show how modeling and rational design is used during the development processes and yield devices with optimized performance.

Laminar Fluid Diffusion Interfaces are generated when tow or more streams flow in parallel in a microfluidic structure. This technology can be used for diffusion-based separation and detection applications, for example: DNA desalting, the extraction of small proteins from whole-blood samples, and the detection of various constituents in while blood. Additional applications are the establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients, enabling the uniform and controlled exposure of cells to lysing agents, allowing the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure. We have developed integrated systems using machine-controlled disposable cartridges and passive self-contained disposable cards including particle separators, flow cytometers, valves, detection channels, mixers, and diluters that are used in a hematology analyzer, stand-alone blood plasma separators, and a variety of chemical and biological assays. Microfluidic arrays compatible with common well-plate formats have been designed for high-throughout toxicology screening applications. All these devices were manufactured using Micronics' unique rapid-prototyping process yielding low-cost plastic disposable microfluidic chips.

Central to Micronics’ microfluidics technology platforms is the generation and use of Laminar Fluid Diffusion Interfaces (LFDIs). One of the embodiments of LFDIs is in diffusion-based detection technology, which is based on the parallel flow of individual streams in a single microfluidic channel. Micronics’ T-Sensor® based Chemical Analysis System (CAS) harnesses diffusion to identify unknown concentrations of chemical species, allowing researchers to perform multiple assays on a single version of a disposable microfluidic cartridge. Implementations of both a prototype portable device and a benchtop system are demonstrated, with CV’s ranging from 2.6 to 10%.

A novel microcytometry system that monitors leukocyte populations to assess human pathogen exposure is being jointly developed by Micronics and Honeywell. The system contains both an instrument and a disposable card that contains complex microfluidic circuits for blood sample acquisition, reagent storage, erythrocyte lysis, cytometry, and waste storage. This talk discusses the subsystems that provide these functions and shows experimental results qualitatively describing hydrodynamic focusing and leukocyte populations.

Micronics has developed a wide variety of microfluidic devices and integrated systems for clinical diagnostics and life sciences applications. They fall into two general classes: machine-controlled disposable cartridges, and passive self-contained disposable cards. They include particle separators, flow cytometers, valves, detection channels, mixers, and diluters. Current applications for these devices include a hematology analyzer, stand-alone blood plasma separators, and a variety of chemical and biological assays. In this paper, we will focus on microfluidic structures for chemical and cellular analysis. Experimental data as well as the results of fluid modeling will be shown.

Microfluidic disposables are presented that are compatible with standard well plate readers and robotic filling systems. The disposables perform extractions and sample cleanup procedures using the diffusion-based separation principle.

Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this technique are a reduction in reagents, higher sensitivity, minimal preparation of complex samples such as blood, real-time calibration, and extremely rapid analysis.© (2000) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The manipulation of transport transverse to the flow direction has permitted development of microfluidic devices that allow continuous processing of samples. One of these is a rapid competition immunoassay that relies on apparent changes in the diffusivity of antigen due to binding of the antigen to relatively slowly diffusing antibody. It has also been possible to utilize isoelectric focusing to concentrate and separate different types of analytes into separate flowing streams.

A number of microfluidic devices have been developed that do not require any external power source or means for fluid movement. They include particle separators, valves, detection channels, mixers, and diluters. Current applications for these devices include standalone blood plasma separators, and a variety of qualitative and semi-quantitative assays. Experimental data as well as the results of fluid modeling are shown.

An immunoassay format is presented that takes advantage of the microfluidic properties of the H-FilterTM for measuring sample analyte concentration. The method relies on the diffusion of analyte particles into a region containing beads coated with specific antibody. Competitive binding of labeled analyte and sample analyte with a limited number of binding sites allows measurement of the concentration of sample analyte based on the fraction of labeled analyte bound. The fraction of labeled analyte bound can be determined with a microcytometer by measuring the bead fluorescence intensity on the microcytometer portion of an integrated microfluidic chip. It is not necessary to separate the beads from the mixture because the bead intensity can be determined above the background of unbound labeled antigens. Other advantages include the ability to eliminate large interfering particles from samples, continuous sample monitoring, and the ability to concentrate the beads. Microfluidic immunoassay formats also consume smaller volumes of costly reagents and sample.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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 a demonstration of an optical microfluidic system for rapid and simultaneous measurement of several fluid analytes in a microscale volume that is based on fluorescent indicators immobilized on polymer microbeads. The fluorescence properties of such 'reporter beads' can be a function of the concentration of an analyte, and can be monitored in a silicon microfabricated flow channel. The sizes of microbeads can be determined by the scattering signal, so this system can be used to analyze several analytes at the same time by using beads of different sizes that are sensitive to different analytes. The fluorescent indicator Carboxy-SNAFL 1 was immobilized on amino-functionalized polystyrene beads of 5 micrometer diameter. The intensities of two fluorescent peaks (at 560 nm and at 620 nm) were measured and their ratio was dependent on the pH value of the analyte. A 488 nm air-cooled argon laser was used to excite the fluorescent beads flowing through a microfabricated V-groove channel. Two miniature photomultiplier modules and interference filters were used to simultaneously measure both fluorescence intensities.

Over the last 4 years our group has been involved in developing a series of devices for chemical separation and analysis. These devices share a theme of utilizing the low Reynolds number properties of liquids flowing at slow speeds in small channels. These devices allow some types of function that are not possible in larger devices because of the possibility of bringing flows together without convective mixing. We have taken two somewhat different approaches to coping with samples that contain particles that are incompatible with one or more analytical methods to be used.

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.

Based on the recently introduced T-Sensor method, we demonstrate the fluorescence-determination of various analytes directly in whole blood and in serum. The method relies on microfluidic flow in silicon structures, diffusion-based separation, and analyte determination using fluorescent and absorption indicator dyes. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without mixing other than by diffusion. A sample solution (e.g., blood), and a receptor solution containing the indicator dye are introduced in a common channel, and flow laminarly next to each other until they exit the structure. Small ions such as H+, and Na+ diffuse rapidly across the channel, whereas larger molecules diffuse more slowly. Larger particles such as blood cells and polymer beads show no significant diffusion within the time the two flow streams are in contact. The fluorescence emission of indicator dyes is a function of the concentration of the analyte molecules and the dye concentration in the interaction zone between the two streams. This device allows continuous monitoring of the concentration of analytes in whole blood without the use of membranes or prior removal of blood cells. This principle is illustrated by the determination of human albumin, total calcium, and pH in whole blood and serum.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A benchtop optical flow cytometer utilizing microfabricated silicon V-groove flow channels (25 micron diameter) has been constructed and tested using diluted whole blood. A 1.2 mw diode laser probe beam focused onto the flow stream is used to generate scattered light from passing blood cells. Photodiode detectors are used to collect both small and large angle signals which are counted and analyzed for pulse peak intensities. Count rates as high as 1000/second have been obtained using pressure heads of about 0.5 psi. Optical modeling has also been carried out in order to determine the light scattering signature of blood cells passing down various channel flow lines. Results suggest that a significant amount of experimental signal variability may be due to variations in the positions of cells passing through the sampling region, but that this degree of signal variation should not prohibit the discrimination of different cell populations. Experimental and computational results are presented and discussed, as well as the possibility of developing a miniature portable flow cytometer based on microfabricated flow channels.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Most fluids show laminar behavior in miniature flow structures with channel cross-sections below 0.5 mm. Different layers of miscible fluids and particles can flow next to each other in a microchannel without any mixing other than by diffusion. Small particles diffuse faster than larger ones, which allows separation of particles by size. It is possible to design fluidic microchips in which separations, chemical reactions, and calibration-free analytical measurements can be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples.

Monitoring of biologically active agents such as bacteria, viruses, proteins and small molecules in environmental samples poses complex analytical problems. The particulate nature of the analytes and potential interferents is of particular concern for microfluidic systems in which the channels may not be much larger than the particles themselves. For this reason, sample preconditioning upstream of a chemical analytical device will usually be required. However, the small dimensions of microfluidic devices also allow unique methods of sample purification, concentration, and detection. In our laboratory we have developed a series of microfluidic chemical analytical devices for such purposes. These devices rely on the low Reynolds number flow conditions. In such conditions field flow fractionation based on sedimentation, diffusion and electrophoresis perpendicular to the flow direction can be profitably harnessed to precondition samples. The H-filter is one such device in which a simple 4-port device that allows two fluids to be brought into adjacent flow, and then separated downstream into two (or more) flow streams after exchange of material under the influence of one or more fields. It can be fabricated using anodically bonded silicon and Pyrex channels, or using polymeric devices formed using `soft lithography' techniques. We have tested the ability of this device to be used for purification of bacteria and their spores from complex samples containing silica and other interferent particles. We will present results of our tests of this device, as well as initial attempts to integrate the H-filter into a sample preconditioning system that includes on-chip pumps.