Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-04-16
2002-04-23
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S604000, C422S105000, C436S180000
Reexamination Certificate
active
06375817
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to apparatus and methods for sample analysis. More specifically, the invention is directed to apparatus and methods for sample plug formation and subsequent separation and/or analysis.
BACKGROUND OF THE INVENTION
Methods for conducting and analyzing microscale chemical reactions often require multiple steps and extensive handling of reagents. These methods typically are labor intensive and involve complex combinations of instrumentation, e.g., pipettes, pumps, syringes, valves, tubing, reagent vessels, and reaction chambers. Such complexities may contribute to inaccuracies, increased costs and reduced reaction yields.
Analytical techniques typically require a high degree of labor and the use of complex apparatus. Moreover, many laboratory and industrial chemical processes involve the use of relatively large volumes of reagents and multiple laboratory instruments. Typical large scale immunoassays, e.g., require the use of pipettes, reagent vessels, and reaction chambers. See, e.g., Mattiasson et al.,
Proc. Int. Symp. on Enzyme
-
Labeled Immunoassay of Hormones and Drugs
, (Pal, S., Ed., Walter de Gruyter, Berlin (1978), p. 91). Such processes, regardless of the size of the reaction, also may require multiple steps. Accordingly, there is a potential for reduced accuracy due to the introduction of impurities, volumetric inaccuracies, and low reproducibility. These problems especially are acute in microscale diagnostic applications in which biological samples are analyzed, such as, e.g., immunoassays, polynucleotide amplifications, or hybridizations.
Recently, efforts have been made to streamline chemical processes to reduce costs, increase accuracy, and improve reaction yields. For example, capillary electrophoresis techniques have been proposed to increase resolution in immunoassays. Various attempts have been made to enhance other common analytical techniques, such as the polymerase chain reaction (PCR). For example, U.S. Pat. No. 5,273,907 reports a capillary pre-loaded with PCR reagents which is used to deliver a sample to the reagents for DNA amplification. Similarly, International Patent Publication WO 93/22058 describes a micro-scale device for performing PCR In this case, PCR reagents from a first chamber are mixed with sample in a second chamber by movement of materials through channels in a microchip.
Recently, efforts have been made to streamline chemical processes to reduce labor and complexity. One such effort involves the use of microchip assemblies. A microchip assembly typically consists of a thin silica substrate or other polymeric substrate onto which channels are etched. The channels serve as means for reagent transport and/or as the reaction chambers themselves. Microchip assemblies for performing micro-scale chemical reactions may comprise a series of interconnected channels. For example, channels may be etched onto the surface of a microfabricated solid. Reagents in solution then are placed into the channels, and allowed to react with, e.g., reagents already in the channels. Voltage gradients may be used to control sample flow and mixing. See, e.g., International Publication WO 96/04547. Hydrogen and oxygen gas often results from use of voltage gradients to control sample flow. Electrolysis products also may accumulate near the electrode surface.
Microchip assemblies which do not require voltage gradients to inject the samples and solutions have been designed. See, e.g., U.S. Pat. No. 5,304,487; International Publication WO 93/22053; and International Publication WO 93/22054 (describing microchip assemblies for sample analysis). In such systems, a steady flow of liquid is pumped through a series of channels etched on a microchip. Sample is introduced through one of the channels and is mixed with the flow of liquid. These systems are used to detect the presence of a sample component or the presence of some biological entity (a bacterium or virus, e.g.) by measuring the variance in flow rate between liquid and liquid mixed with sample as each flows through the microchip.
There remains a need in the art for methods and devices which will decrease the time, labor, costs, biohazard exposure and complexity currently involved in the performance of chemical analysis of microscale biological samples. More particularly there exists a need for apparatus and methods which efficiently and economically form and deliver a sample plug to a separation channel and/or analytical device.
SUMMARY OF THE INVENTION
Apparatus and methods have been developed for rapid, automated analysis of microscale samples using pressure differentials. A sample plug formation device of the invention generally comprises two intersecting channels, an introduction channel and a separation channel. A sample is introduced through an opening in a first channel, referred to herein as a sample introduction channel. The sample moves through the sample introduction channel by vacuum, pressure, capillary action, or a combination thereof. At a distance from the point of sample introduction, the sample introduction channel forms a juncture or junction with (i.e., intersects) a second channel, referred to herein as a separation channel. Through the use of pressure and/or vacuum applied to the separation channel and/or the sample introduction channel, a portion of the sample is transported into the separation channel as the bulk sample crosses the junction between the sample introduction and separation channels. With the proper control, a discrete plug of sample reproducibly may be formed in the separation channel and subjected to separation techniques and/or analysis. Subsequent to the formation of the sample plug, the portion of sample which does not form the sample plug typically is moved to a waste outlet.
Formation of the sample plug at the channel junction is controlled by application of pressure differentials in and between the sample introduction and separation channels. A first pressure differential is applied so as to induce sample flow through the introduction channel to the juncture. Subsequently, at least a second pressure differential is applied to move a portion of the sample into and along the axis of the separation channel. A plug of sample generally is formed in the separation channel at the junction when pressure is increased axially along the separation channel relative to the sample introduction channel. The frequency and size of plug formation is controlled by controlling the pressure differentials.
The sample introduction and separation channels may be capillaries formed to intersect at a junction. A preferred structure defining the sample introduction and separation channels is a microfabricated solid, such as a microchip. Typically, channels are etched directly into the microchip. In a preferred embodiment, the microchip comprises a series of sample introduction and separation channels etched onto its surface. Such channels preferably have cross-sectional dimensions of between about 0.1 &mgr;m and about 1000 &mgr;m.
In a preferred embodiment, the separation channel comprises a longitudinal axis containing a medium, e.g., water, an electrolyte, or a polyacrylamide solution or gel, which aids the separation of components suspected to be in the sample. Thus, a sample plug that is formed at the junction of the sample introduction and separation channels migrates axially along the separation channel where it may be separated into its components. Preferably, the sample introduction and separation channels contain a buffer that is compatible with the sample and separation medium, if present. The device may further comprise a voltage generator for applying a voltage axially along the longitudinal axis of the separation channel. The application of a voltage along the separation channel may aid in the separation of components of the sample, e.g., when the separation is accomplished by electrophoresis.
Also in a preferred embodiment, a device of the invention comprises a detector. The detector is placed in proximity to the separation channel f
Carson William W.
Koutny Lance
Taylor Todd A.
PerSeptive Biosystems Inc.
Starsiak Jr. John S.
Testa, Huwitz & Thibeault, LLP
Warden Jill
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