Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-12-22
2002-06-11
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287200, C435S288500, C204S545000, C204S547000, C204S403060, C204S409000, C204S643000, C422S081000, C422S082010
Reexamination Certificate
active
06403367
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to devices and methods for performing active, multi-step sample preparation and molecular diagnostic analysis of biological materials. More particularly, it relates to integrated, compact, portable devices for self-contained sample to answer systems. Specifically, this invention relates to a device and method for performing multi-step sample preparation and assay on either two or even a single microchip. Examples of applications for this integrated system include food and/or quality monitoring, diagnosis of infectious diseases and cancers, bone marrow plastesis (e.g., stem cell separation and analysis), and genetics-based identification of individuals for forensic purposes.
BACKGROUND OF THE INVENTION
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
Generally, analysis of biological-derived sample materials cannot occur until the sample is processed through numerous pre-analysis steps. Often, the preparation process is time consuming and laborious. For example, many immuno and molecular-biological diagnostic assays on clinical samples, such as blood or tissue cells, require separation of the molecules of interest from the crude sample by disrupting or lysing the cells to release such molecules including proteins and nucleic acids (i.e., DNA and RNA) of interest, followed by purification of such proteins and/or nucleic acids. Only after performing processing steps can analysis of the molecules of interest begin. Additionally, protocols used for the actual analysis of the samples require numerous more steps before useful data is obtained.
For example, charged and uncharged microparticles in solution (such as cellular material or crude extracts of protein or nucleic acids thereof) may be separated by dielectrophoresis. On a microscale, dielectrophoresis can be performed using a glass slide-based device having exposed, i.e., naked, interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. With such a device, cells, proteins, and nucleic acids can be separated based on their respective dielectric properties by using separation buffers having appropriate conductivity and an AC signal with a suitable amplitude and frequency. Such devices, however, have several problems including the nonspecific binding of both separated and unseparated cells to exposed portions of the glass surface and the electrodes. Such devices are also problematic in that the flow chamber volume (several hundred microliters) is so large that thermal convection can disturb and push materials such as cells and large molecules initially attracted to and retained by the electrodes off of the electrodes. Additionally, undesired cells and molecules are not easily washed off the surface without disturbing and loosing the desired cells as such cells and molecules can interfere with fluidic flow and, hence, block the flow during wash steps.
Conventional methods to disrupt whole cells for the release of proteins and nucleic acids have employed the use of a series of high voltage DC pulses in a macrodevice, as opposed to a microchip-based device. Such conventional electronic lysis techniques have several problems. For example, some commercial macro-devices use lysis conditions that do not release high molecular weight (larger than 20 Kb) nucleic acids because the high molecular weight molecules can not fit through pores created in the cell membranes using such methods. Additionally, released nucleic acids are often lost due to their non-specific binding to the surface of the lysis chamber. Such loss of material, especially when molecules of interest are in low concentration, is further compounded by the fact that the dielectrophoretic cell separation macro-device systems are stand alone systems allowing for loss of sample in the transfer of material from one device to the other as sample preparation is carried forward.
Processing of the crude lysate often requires chemical reactions to remove undesired cellular components from the specifically desired ones. These reactions typically include subjecting the lysate to enzymatic reactions such as proteinase K and restriction enzymes or nucleases. Processing can also include enhancing the presence of desired molecules, particularly nucleic acids, by performing amplification reactions such as by strand displacement amplification (SDA) or polymerase chain reaction (PCR) methodologies. These reactions are also carried out in stand-alone processes. Only after these sample preparation and processing steps can assaying for data retrieval begin. Because of the numerous steps between sample collection and assay, many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility.
Attempts have been made to use dielectrophoresis to separate and identify whole cells. For example, U.S. Pat. No. 4,326,934 to Pohl discloses a method and apparatus for cell classification by continuous dielectrophoresis. With such method cells are separated by making use of both the positive and negative dielectrophoretic movement of cell particles. Separated cells are allowed to be characterized and/or classified by viewing the characteristic deflection distance of cells moving through the positive and negative electrodes.
In another example, U.S. Pat. No. 5,344,535 to Belts et al. discloses a method and apparatus for the characterization of micro-organisms and other particles by dielectrophoresis. In this system, cells are characterized by matching their signature dielectrophoretic collection rates.
In yet another example, U.S. Pat. No. 5,569,367 to Belts et al. discloses a method and apparatus for separating a mixture of cells using a pair of energized interdigitated electrodes comprised of interweaved grid-like structures arranged to obstruct flow of cells through the apparatus and cause differentiation of cell types into fractions by applying a non-uniform alternating field.
In addition, other attempts have been made to combine certain processing steps or substeps together. For example, various microrobotic systems have been proposed for preparing arrays of DNA probes on a support material. Beattie et al., disclose in “The 1992 San Diego Conference: Genetic Recognition”, November, 1992, use of a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample wells on a glass substrate.
Various other attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. A. Manz et al., in “Miniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensing”,
Sensors And Actuators,
B1(1990), pp. 244-248, describe a ‘total chemical analysis system’ (TAS) that comprises a modular construction of a miniaturized TAS. In that system, sample transport, chemical reactions, chromatographic separations and detection were to be automatically carried out.
Yet another proposed integrated system by Stapleton, U.S. Pat. No. 5,451,500, a system for automated detection of target nucleic acid sequences is described. In this system multiple biological samples are individually incorporated into matrices containing carriers in a two-dimensional format.
Various multiple electrode systems are also disclosed which purport to perform multiple aspects of biological sample preparation or analysis. Pace, U.S. Pat. No. 4,908,112, entitled “Silicon Semiconductor Wafer for Analyzing Micronic Biological Samples” describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodes are positioned in the channel to activate motion of liquids through the conduit.
Cheng Jing
Diver Jonathan
Heller Michael J.
Jalali Shila
O'Connell James P.
Lyon & Lyon LLP
Nanogen Inc.
Redding David A.
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