Microanalytical device and use thereof for conducting...

Details Microanalytical device and use thereof for conducting... Microanalytical device and use thereof for conducting...

2000-02-11

2003-10-21

Nguyen, Nam (Department: 1753)

Chemical apparatus and process disinfecting, deodorizing, preser

Chemical reactor

C422S198000, C422S199000, C422S307000, C435S283100

Reexamination Certificate

active

06635226

ABSTRACT:

TECHNICAL FIELD
This invention relates generally to the field of miniaturized devices for conducting chemical processes, and more particularly relates to novel microanalytical devices for conducting chemical processes such as separation (e.g., chromatographic, electrophoretic or electrochromatographic separation), screening and diagnostics (using, e.g., hybridization or other binding means), and chemical and biochemical synthesis (e.g., DNA amplification conducted using the polymerase chain reaction, or “PCR”).
BACKGROUND
In sample analysis instrumentation, smaller dimensions generally result in improved performance characteristics and at the same time result in reduced production and analysis costs. Miniaturized separation systems, for example, provide more effective system design, result in lower overhead, and enable increased speed of analysis, decreased sample and solvent consumption and the possibility of increased detection efficiency.
Accordingly, several approaches have been developed in connection with miniaturization of devices for use in chemical analysis, particularly in micro-column liquid chromatography (&mgr;LC), wherein columns with diameters of 100 to 200 microns are used, in capillary electrophoresis (CE), wherein electrophoretic separation is conducted in capillaries on the order of 25 to 100 microns in diameter, and in microchannel electrophoresis (MCE), wherein electrophoresis is carried out within a microchannel on a substantially planar substrate. The conventional approach in miniaturization technology as applied to CE and &mgr;LC involves use of a silicon-containing material, i.e., a capillary fabricated from fused silica, quartz or glass. With MCE, an attractive method that is useful in conjunction with high throughput applications and enables reduction in overall system size relative to CE, miniaturized devices have been fabricated by silicon micromachining or lithographic techniques, e.g., microlithography, molding and etching. See, for example, Fan et al. (1994)
Anal. Chem
. 66(1):177-184; Manz et al., (1993)
Adv. in Chrom
. 33:1-66; Harrison et al. (1993),
Sens. Actuators
, B B10(2):107-116; Manz et at. (1991),
Trends Anal. Chem
. 10(5):144-149; and Manz et at. (1990)
Sensors and Actuators B
(
Chemical
) B1(1-6):249-255. The use of micromachining techniques to fabricate miniaturized separation systems in silicon provides the practical benefit of enabling mass production of such systems, and there are a number of techniques that have now been developed by the microelectronics industry for fabricating microstructures from silicon substrates. Examples of such micromachining techniques to produce miniaturized separation devices on silicon or borosilicate glass chips can be found in U.S. Pat. No. 5,194,133 to Clark et al., U.S. Pat No. 5,132,012 to Miura et al., U.S. Pat. No. 4,908,112 to Pace, and U.S. Pat. No. 4,891,120 to Sethi et al.
Use of silicon-containing substrates such as fused silica, quartz and glass in microanalytical devices is problematic in a number of ways. For example, silicon dioxide substrates have high energy surfaces and strongly adsorb many compounds, most notably bases. Silicon dioxide materials also dissolve to an appreciable extent when used with basic solutions. Furthermore, when used in electrophoretic applications, the internal surface of a silica capillary or microchannel will be negatively charged at basic pH as a result of deprotonation of surface silanol groups (i.e., they are in the form of anionic, Si—O
−
, groups). The surface charge on the interior of the capillary or microchannel not only exacerbates the problem of unwanted adsorption of solute, but also modulates the velocity of electroosmotic flow (also termed “electroendoosmotic flow” or EOF) on an unmodified surface, in turn affecting the sensitivity and reproducibility of the chemical analysis conducted. (That is, the EOF velocity is a function of zeta potential &zgr;, which is essentially determined by surface charge.) Microfabrication using silicon per se is similarly problematic insofar as a silica surface will form on a silicon substrate under even mildly oxidizing conditions.
For the foregoing reasons it would be desirable to fabricate microanalytical devices from materials that are not silicon-based, e.g., using inexpensive and readily available polymeric materials. It would also be desirable to extend the utility of microanalytical devices beyond electrophoretic and chromatographic separation techniques to other types of chemical processes, processes that may involve high temperatures, extremes of pH, harsh reagents, or the like. The present invention provides such microanalytical devices.
One area with which the present invention is particularly useful is in bioanalysis. An important technique currently used in bioanalysis and in the emerging field of genomics is the polymerase chain reaction (PCR) amplification of DNA. As a result of this powerful tool, it is possible to start with otherwise undetectable amounts of DNA and create ample amounts of the material for subsequent analysis. The technique is described in U.S. Pat. No. 4,683,195 to Mullis et al. and related U.S. Pat. Nos. 4,683,202, 4,800,159 and 4,965,188 to Mullis et al. Automated systems for performing PCR are known, as described, for example, in U.S. Pat. Nos. 5,333,675 and 5,656,493 to Mullis et al. PCR uses a repetitive series of steps to create copies of polynucleotide sequences located between two initiating (“primer”) sequences. Starting with a template, two primer sequences (usually about 15-30 nucleotides in length), PCR buffer, free deoxynucloside tri-phosphates (dNTPs), and thermostable DNA polymerase (commonly Taq polymerase), one mixes these components, and then heats to separate the double-stranded DNA. A subsequent cooling step allows the primers to anneal to complementary sequences on single-stranded DNA molecules containing the sequence to be amplified. Replication of the target sequence is then accomplished by the DNA polymerase which produces a strand of DNA that is complementary to the template. Repetition of this process doubles the number of copies of the sequence of interest, and multiple cycles increase the number of copies exponentially.
Since PCR requires repeated cycling between higher and lower temperatures, PCR devices must be fabricated from materials capable of withstanding such temperature changes. The materials must be mechanically and chemically stable at high temperatures, and capable of withstanding repeated temperature changes without mechanical degradation. Furthermore, the materials must be compatible with the PCR reaction itself, and not inhibit the polymerase or bind DNA. To date, however, there remain many problems with performing PCR in microdevices. One problem involves the low thermal stability of many materials. That is, many types of materials, e.g., polymeric materials, cannot withstand the cycling temperatures used in PCR, typically in the range of about 37° C. to 90° C., without significant or complete loss of mechanical integrity. In addition, contaminants may be present on or leach out of a substrate surface, affecting the precise balance of appropriate ingredients (metal ions, salts, buffering systems, oligonucleotides, primers, and polymerases) required for PCR, in turn resulting in unsuccessful amplification reactions. Also, the polymerase enzyme or any of the components involved in the PCR reaction may bind to or become adsorbed on a microchannel surface. Contact between the polymerase and a substrate surface will generally result in irreversible denaturation. These types of “biofouling” are especially problematic with capillaries or microchannels of micron or submicron dimensions because of the very high surface area to volume ratio.
SUMMARY OF THE INVENTION
The present invention addresses the aforementioned needs in the art, and provides a novel microanalytical device in which chemical and biochemical reactions can be conducted. In its simplest embodiment, the microanalytical device comprises:
a substrate having first and

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