Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-07-05
2004-05-11
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S004000, C435S006120, C435S174000, C435S180000
Reexamination Certificate
active
06734012
ABSTRACT:
BACKGROUND OF THE DISCLOSURE
The present disclosure relates to composite microarray non-luminescent slides useful for carrying a microarray of biological polymers on the surface thereof and, more particularly, to composite microarray non-luminescent slides having a microporous membrane effectively attached by covalent bonding through a surface treatment to a substrate that prepares the substrate to sufficiently, covalently bond to the microporous membrane formed by a phase inversion process such that the combination produced thereby is useful in microarray applications and, most particularly, to composite microarray non-luminescent slides having a porous nylon membrane covalently bonded to a solid base member, such as, for example, a glass or Mylar microscope slide, such that the combination produced thereby is useful in microarray applications and to a process for producing such composite microarray non-luminescent slides.
A variety of methods is currently available for making arrays of biological macromolecules, such as, for example, arrays of nucleic acid molecules or proteins. One method for making ordered arrays of DNA on a porous membrane is a “dot blot” approach. In this method, a vacuum manifold transfers a plurality, e.g., 96, aqueous samples of DNA from three (3) millimeter diameter wells to a porous membrane. A common variant of this procedure is a “slot-blot” method in which the wells have highly-elongated oval shapes.
The DNA is immobilized on the porous membrane by baking the membrane or exposing it to UV radiation. This is a manual procedure practical for making one array at a timer and usually limited to 96 samples per array. “Dot-blot” procedures are therefore inadequate for applications in which many thousand samples must be determined.
A more efficient technique employed for making ordered arrays of genomic fragments (e.g., PCR products) uses an array of pins dipped into the wells, e.g., the 96 wells of a microtitre plate, for transferring an array of samples to a substrate, such as a porous membrane. One array includes pins that are designed to spot a membrane in a staggered fashion, for creating an array of 9216 spots in a 22×22 cm area (Lehrach, et al., 1990). A limitation with this approach is that the volume of DNA spotted in each pixel of each array is highly variable. In addition, the number of arrays that can be made with each dipping is usually quite small.
Several patents have described the use of microarray slides in microarray applications. These include U.S. Pat. No. 5,919,626 entitled, “Attachment of unmodified nucleic acids to silanized solid phase surfaces”; U.S. Pat. No. 5,667,976 entitled, “Solid supports for nucleic acid hybridization assays” and U.S. Pat. No. 5,760,130 entitled “Aminosilane/carbodiimide coupling of DNA to glass substrate”, the disclosure of each is herein incorporated by reference to the extent not inconsistent with the present disclosure.
Microarray slides are well known in the art. Schleicher & Schuell have attempted to attach nylon membrane to a glass slide using glue or similar adhesive in their commercially available CAST™ slides. However, the layer of glue or adhesive adds additional thickness to the nylon membrane/glass slide combination, and the gluing/adhesive process may require the use of a scrim-reinforced nylon membrane. The extra thickness of the overall nylon membrane/glass slide combination caused by the glue/adhesive and the reinforcing scrim is a disadvantage in microarray applications. Additionally, the scrim makes the surface of the membrane of the nylon membrane/glass slide combination uneven and less than ideal from a cosmetic standpoint. Even further, the chemistry of the glue or adhesive used to attach the nylon membrane to the glass slide is not necessary optimal to effectuate the combination, nor is it necessarily compatible with the biomolecules or analytes for which the product is intended to receive, as it may interfere or react with the analyte.
Similarly, other products known to be currently commercially available include: Modified glass that binds nucleic acids or proteins without the use of a membrane; Corning GAPS Slides, such as, for example CMT-GAPS™ coated slides; Nitrocellulose porous membrane cast onto glass, available from Schleicher & Schuell as FAST™ Slides; Scrim-reinforced nylon glued or adhered to a glass substrate and Schleicher & Schuell CAST™ Slides. Detailed descriptions of these commercially available products are readily available from the respective manufacturer and are known in the art.
However, in microarray applications, binding nucleic acids or proteins directly to a glass substrate has certain disadvantages. Specifically, a considerably smaller surface area for binding the nucleic acids or proteins is available than with a comparably sized microporous membrane/glass slide combination. The larger the binding surface area the, better the signal strength of the biomolecules or analytes, thereby allowing for the detection of smaller samples of biomolecules or analytes. Also, the porous membrane portion of the microporous membrane/glass slide combination naturally adsorbs the biomolecules or analytes and holds them in place on the microporous membrane/glass slide combination, whereas without the microporous membrane portion of the slide, the biomolecules or analytes would just sit on top of a glass surface, as there is no adsorption of the biomolecules or analytes. It is also likely that the efficiency of immobilization of biomolecule on the glass is substantially less than 100%, and may be less than 50%, when compared to immobilization of the target on nylon. This is important, in that the subsequent detection steps require as much of the possible analyte, or target biomolecule, to be available for (in a DNA detection example) hybridization with the labeled probe. Following the immobilization, there are typically several liquid immersion steps including blocking, washing, hybridization buffer exposure, etc. Each step has the potential of removing analyte from the glass surface, and decreasing the potential strength of the signal. Nylon is generally regarded as having the highest biomolecule binding efficiency when compared to other the commercially available polymer or other treated substrates. Nylon is also regarded as providing highest accessibility of the functional groups of the analyte thus bound to the nylon surfaces.
Nylon membranes, a specific species of microporous membrane, formed by a phase inversion process, have some advantages over nitrocellulose membranes in that nylon is naturally hydrophilic. Nylon membranes also have a greater protein and DNA binding capacity than nitrocellulose. This increased binding capacity means better signal strength and lower detection thresholds in assays.
Nylon membrane pore structure is more easily controllable than nitrocellulose membrane pore structure and is more physically robust than the nitrocellulose membranes. Nitrocellulose is more brittle than the nylon membrane, has more pore variability and is extremely flammable. The physical weakness, variability and flammability of the nitrocellulose membranes combine to make nitrocellulose membrane more expensive to manufacture than nylon membrane.
As discussed above, there are at least three main disadvantages to scrim-reinforced nylon glued or otherwise adhered to a glass substrate. First, the glue or adhesive layer adds additional thickness to the combination scrim-reinforced nylon/glass slide. The arraying robots that blot the nylon membranes have narrow spatial tolerances, and any additional thickness represents additional uncertainty about accurate positioning of the combination scrim-reinforced nylon/glass slide relative to the arraying robots. The second, and more important, disadvantage is that the scrim-reinforced membrane on the combination scrim-reinforced nylon/glass slide has an irregular surface on the micro scale. This is an important cosmetic problem since the spot sizes made on the membrane are on a similar scale. Finally, the glue/adhesive and the ana
Amin Murtaza
Andreoli Rita
Chesterson Richard
Meyering Mark
Ostreicher Eugene
Cuno Incorporated
Naff David M.
Payne R. Thomas
Tomich John A.
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