Whole Blood Assays Using Microfluidics-Based T-Sensor Technology
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.
•"Labs on a Chip"
•The T-Sensor System
•Applications
Micronics' T-Sensor technology is based on microfluidics—the very special behavior of fluids flowing in channels the size of a human hair. Fluids in this environment show very different properties than in the macro world of our everyday experience. This new field of technology was enabled by advances in microfabrication—the etching of silicon to create very small features. Micronics' study of microfluidic structures resulted in engineering advances that the company believes will provide significant benefits and competitive advantages for In Vitro Diagnostic and other applications.
Silicon microfabrication enabled the development of the integrated circuit, which made possible the PC revolution. Micronics is applying polymer microfabrication techniques to enable the development of miniaturized fluidic circuits, sometimes referred to as Labs-on-a-chip, as building blocks for a revolution in medical diagnostics and chemical analysis.
In these tiny microchips, a multitude of chemical and physical processes for both chemical analysis and synthesis can occur. These devices, also known as micro-total analysis systems (µTAS), can be mass produced in silicon by techniques similar to those used in the semiconductor industry, or, for even lower cost, made out of plastics by using casting, cutting, and stamping techniques. They 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. 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.
A true Lab-on-a-chip will perform all analytical functions, including sampling; sample pretreatment, separation, dilution, and mixing steps, chemical reactions; and detection in an integrated microfluidic circuit. 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.
Separation of particles from soluble components is usually done by filtering or centrifugation. None of these methods are suitable for integrated microsystems. Efficient centrifugation requires that the sample be placed at some distance from the center of rotation in order to achieve a high enough centrifugal force, which is usually not practical in a microfluidic system. Filtering requires membranes that can clog, and are difficult to mass-manufacture as part of a microfluidic circuit.
In channels in which either width or height is less than~200 µm (typical of most microfluidic devices), liquids that flow slowly follow predictable laminar paths characteristic of low Reynolds numbers (The Reynolds number is a non-dimensional parameter relating the ratio of inertial to viscous forces in a specific fluid flow configuration.). This allows two or more layers of fluid to flow next to each other without any mixing other than by diffusion of their constituent molecular and particulate components. Aqueous flow streams in a microchannel are hydrodynamically similar to very viscous oil-like streams in a significantly larger channel.
Microfluidic systems have distinctive properties inherent to their small dimensions:
- (i) aqueous flow is generally laminar, not turbulent;
- (ii) diffusion is an efficient process for mixing the dissolved contents of two or more fluids; and
- (iii) particles can also be separated by diffusion according to their size.
For example, at room temperature in an aqueous solution, a spherical molecule with a molecular weight of 330 (e.g., a small organic dye molecule) takes 0.2 s to diffuse 10 µm, whereas a particle with a diameter of 0.5 µm (e.g., a small bacterium) would require about 200 s to cover the same distance.
The so-called T-Sensor, a mTAS component that combines separation and detection functions, is based on these properties. A T-Sensor system has been designed in which a sample solution, a receptor/indicator solution, and a reference solution are introduced in a common channel (see Figure 1). The fluids interact during parallel flow until they exit the microstructure. Large particles such as blood cells do not diffuse significantly within the time the flow streams are in contact. Small particles such as H+, Na+, and small molecules diffuse rapidly between streams, whereas larger polymers diffuse more slowly and equilibrate between streams further from the point of entry to the device. As interdiffusion proceeds, interaction zones are formed in which sample and reagents may bind and react. Typically, an indicator changes color or fluorescence intensity upon interdiffusion and reaction with analyte molecules. While intensity, width, and shape of the interaction zone between sample and indicator may yield information about analyte concentration, usually the ratio of a property such as fluorescence of both interaction zones (sample/indicator and indicator/reference) is used to determine the concentration of the analyte. Such a ratio can be largely free of cross-sensitivities to other sample components and instrumental parameters and enables essentially calibration-free sample analysis without the need for external sample preprocessing or blood cell removal. If an indicator solution is used in the detection solution, the diffusion interaction zones will be optically detectable. The positional variation in intensity of that signal is a complex function of the concentration of the indicator and analyte. However, it is straightforward to calibrate the optical response to analyte concentration.
Figure 2 shows a micrograph in which control, indicator, and sample solutions are introduced as adjacent streams in a T-Sensor; they display the behavior outlined in Figure 1.
Figure 3 shows images of the diffusion interaction zones in a T-Sensor during a series of albumin assays with increasing concentrations of that analyte. The left line is the interaction zone between reference and detection solutions, and the right line shows the interaction between indicator and sample solutions.
Single-frame video or CCD images of the detection channels for each sample are digitized and processed. A wealth of data can be gained from determining the absorption or fluorescence profile across one or more portions of the detection channel. The cross-sections typically are divided into several zones, including reference background, reference interaction zone intensity, detection solution background, sample interaction zone intensity, and sample background. By applying custom algorithms comparing intensities and positions of diffusion interaction zones, these parameters yield essentially calibration-free analytical concentration values independent of variations in experimental conditions. It is possible to compensate for effects such as variations in flow cell geometry, temperature dependent reaction kinetics, light source stability, instabilities in the optical system and detection electronics, as well as fluid parameters such as turbidity, color, concentration of detection chemistry, cross-sensitivities to other sample components, viscosity, and flow speed.
Signal strength is an inherent problem of all optical detection in microchannels due to typically very small optical pathlengths. Since all flow in a T-Sensor is laminar, and reagents and sample are constantly renewed, images can be integrated over time for greater sensitivity without fear of photobleaching, reagent degradation, separation membrane clogging, and other problems typical of traditional sensor systems.
Referenced versus non-referenced data for a typical T-Sensor experiment (Figure 4) illustrate the significantly enhanced accuracy of the sample concentration determination when the sample fluorescence intensity is normalized to that of the reference channel.
To date, T-Sensor assay feasibility has been demonstrated for a variety of clinical parameters such as blood pH and oxygen, electrolytes, proteins, enzymes and drugs, by using detection methods ranging from fluorescence and absorption to voltammetry. Of particular interest are also novel immunoassay formats that utilize the diffusion separation feature of T-Sensors to isolate and detect bound and unbound antibody-antigen complexes.
In addition, monitoring signal intensities along the T-Sensor detection channel (in flow direction) provides a means for looking at the kinetics of a reaction, thus allowing kinetic diagnostic reactions to be measured not as a function of time but of distance from the starting point of the diffusion interaction.
For more information, please see Micronics' Web site at http://www.micronics.net, or the following original research articles:
(1) Weigl, B.H., Yager, P., Microfluidic diffusion-based separation and detection, Science, p 346, Vol 283, 1999
(2) Weigl, B.H., Yager, P., Silicon-microfabricated diffusion-based optical chemical sensor. Sensors and Actuators B (Chemical). vol. B39, no.1-3. pp. 452-7, 1997
(3) Weigl, B.H., Holl, M., Schutte, D., Brody, J.P., Yager, P., Diffusion-based optical chemical detection in silicon flow structures. Analytical Methods & Instrumentation, µTAS96 edition, 1996
(4) Galambos, P., Forster, F.K., Weigl, B.H., A method for determination of pH using a T-sensor, Transducers 97, International Conference on Solid-State, Sensors and Actuators. Digest of Technical Papers (Cat. No.97TH8267). Chicago, IL, USA. pp. 535-8 vol.1, 1997
(5) Weigl, B.H., Kriebel, J., Kelly, M., Bui, T., Yager, P., Whole blood diagnostics in standard and in micro-gravity using microfluidic structures (T-SensorsTM), Microchim. Acta, Special MEMS Editon, accepted, 1998.
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