Chemistry: analytical and immunological testing – Automated chemical analysis – With a continuously flowing sample or carrier stream
Reexamination Certificate
1999-11-17
2001-08-21
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Automated chemical analysis
With a continuously flowing sample or carrier stream
C436S053000, C436S172000, C436S177000, C436S180000
Reexamination Certificate
active
06277641
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to diffusion based microsensors and methods for analyzing the presence and concentration of multiple analytes in samples containing both these analytes and, optionally, larger particles. The invention is particularly useful for analyzing complex samples, such as blood, to detect the presence of small particles, such as ions or proteins, in a stream containing larger particles, such as cells.
BACKGROUND OF THE INVENTION
The greater diffusion of small particles relative to larger particles can be used to partially separate the species. Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. 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 any mixing other than diffusion. Moreover, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients.
Using tools developed by the semiconductor industry to miniaturize electronics, it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests.
A process called “field-flow fractionation” (FFF) has been used to separate and analyze components of a single input stream in a system not made on the microscale, but having channels small enough to produce laminar flow. Various fields, including concentration gradients, are used to produce a force perpendicular to the direction of flow to cause separation of particles in the input stream (see, e.g. Giddings, J. C., U.S. Pat. No. 4,147,621; Caldwell, K. D. et al., U.S. Pat. No. 5,240,618; Wada, Y., et al., U.S. Pat. No. 5,465,849). None of these references disclose the use of a separate input stream to receive particles diffused from a particle-containing input stream.
A related method for particle fractionation is the “Split Flow Thin Cell” (SPLITT) process (see, e.g., Williams, P. S., et al. (1992), Ind. Eng. Chew. Res. 31:2172-2181; and J. C. Giddings U.S. Pat. No. 5,039,426). These publications disclose channel cells with channels small enough to produce laminar flow, but again only provide for one inlet stream. A further U.S. patent to J. C. Giddings, U.S. Pat. No. 4,737,268, discloses a SPLITT flow cell having two inlet streams. Giddings U.S. Pat. 4,894,146 also discloses a SPLITT flow cell having two input streams. All these SPLITT flow methods require the presence of more than one output stream for separating various particle fractions.
None of the foregoing publications describe a channel system capable of analyzing small particles in very small quantities of sample containing larger particles, particularly larger particles capable of affecting the indicator used for the analysis. No devices or methods provide simultaneous measurement of more than one analyte.
SUMMARY OF THE INVENTION
The present invention provides a microfabricated sensor capable of rapid simultaneous measurement of multiple analytes in a fluid sample. Reagents can be loaded into the sensor during fabrication and only the sample fluid needs to be introduced for measurement, making it ideal for use outside of the laboratory. The detection apparatus can be as simple as a human eye, a camera, or a voltmeter, which also supports field use. The sensor is sufficiently simple and inexpensive to manufacture that it is practical for disposable use. Multiple analytes can be simultaneously detected with only microliters of sample, a particular advantage with precious fluids such as blood.
The inventor previously provided a channel cell system for detecting the presence of analyte particles in a sample stream also comprising larger particles (U.S. patent applications Ser. No. 08/829,679, filed Mar. 31, 1997, now U.S. Pat No. 5,972,710, and 08/625,808, filed Mar. 29, 1996, now U.S. Pat. No. 5,716,852, both of which are incorporated by reference herein in their entirety). The previous system comprised a laminar flow channel having at least two inlets for conducting fluids into the laminar flow channel. The inlets contained (1) a sample stream containing analyte particles and also containing larger particles and (2) an indicator stream having a substance which indicates the presence of the analyte particles by a detectable change in property. The two streams flow side by side in the channel without turbulent mixing. The analyte particles diffuse into the indicator stream to the substantial exclusion of the larger particles, and their presence is detected by reaction with the indicator substance.
The present invention provides an apparatus and a method which utilize diffusion between layered laminar streams rather than side by side streams. This allows multiple side by side reagent inlets for simultaneous detection of multiple analytes. Additionally, utilizing diffusional separation, the sensor can tolerate fluid samples also containing larger particles.
In this invention, a sample stream and a carrier stream flow in layers, one on top of the other, rather than side by side. A reagent is introduced to the bottom of the carrier stream through either a fluid or a solid reagent inlet. The reagent contains reagent particles which, in the presence of the analyte, have a detectable change in a property. The sample stream can contain larger particles such as cells as well as the analytes of interest. The term “particles” refers to any species, including dissolved and particulate species such as molecules, cells, suspended and dissolved particles, ions and atoms. The analyte diffuses into the carrier stream where it interacts with reagent particles and is detected by optical, electrochemical or other means.
The sample stream may also contain larger particles, which may also be sensitive to the reagent. Because these do not diffuse into the carrier stream, they do not interfere with detection of the analyte. By diffusion of the analyte but not the larger particles, cross-sensitivities of reagents to larger sample components, a common problem, can be avoided. Furthermore, the reagent can be kept in a solution in which it displays its optimal characteristics. For example, cross-sensitivities to pH or ionic strength can be suppressed by using strongly buffered carrier solutions.
A reagent inlet joins the laminar flow channel on the surface abutting the carrier stream. For multiple analyte detection, each inlet is narrower than the flow channel. For single analyte detection the inlet can be as wide as the flow channel. A fluid reagent inlet is a fluid channel. The fluid reagent forms a stream in layered laminar flow with the carrier and sample streams, but it is a relatively thin and narrow layer since the inlet is narrower than the channel and the reagent fluid volume is small relative to the sample and carrier fluids. A solid reagent inlet is a cavity in the laminar flow channel on the side containing the carrier stream, or other means by which a solid or viscous reagent can be immobilized. Flow of the carrier fluid over the solid reagent dissolves or suspends the reagent particles in the carrier stream.
Because the sample and carrier streams are layered rather than side by side, multiple reagent inlets can be positioned side by side in parallel to allow simultaneous detection of multiple analytes. A layered flow of sample and carrier streams is established and reagents are introduced into the carrier stream using parallel inlets which are narrow compared to the width of the carrier stream. The reagent inlets are spaced sufficiently far apart that there need be no undesired inter diffusion between the reagents. Optical measurement of analyte concentrations can be made by multi-wave two-dimensional imaging. Multiple analytes can be detected simultaneously using equipment as simple as a camera. Electrochemical detection can
Greenlee Winner and Sullivan P.C.
Snay Jeffrey
University of Washington
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