Lyophilizing of liquid drops having arbitrary edges and...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S007100, C435S174000, C435S283100, C435S287200, C422S050000, C422S051000, C422S068100, C422S105000, C436S518000, C530S300000, C530S350000, C536S023100

Reexamination Certificate

active

06605438

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to the stabilization of a drop of liquid comprising a solvent and solute, so that upon drying of the solvent to form a spot, the solute is not preferentially concentrated at the edges of the residual spot, and more particularly relates to the preparation of microarrays of spots containing biological reagents.
BACKGROUND OF THE INVENTION
It is a common procedure to prepare arrays of small areas (i.e., spots) of biological reagents on a surface. Generally the biological reagent is dissolved or suspended in a solvent, and a small drop thereof is applied to a prepared surface along with other drops containing other reagents or reagent concentrations to form an array. The array is then dried to remove the solvent, leaving spots of probe reagents. A dried spot is typically not uniform, but dries to form a ring, with the reagent concentrated at the spot edge and almost absent from the center of the spot. This concentration gradient is undesirable, as it reduces the readability of the spots, and degrades measurements thereof. It would therefore be advantageous to provide a spot with a concentration gradient that is more uniform.
SUMMARY OF THE INVENTION
The present invention provides an array of spots containing reagents prepared by lyophilization of drops of liquid comprising a solvent and varying dissolved, dispersed, or emulsified solutes, with an improved concentration gradient of the remaining solute within individual spots after removal of the solvent. Microarrays of biological reagents are often created by applying fluid drops of solvent/solute to a frosted or smooth substrate surface of glass, metal or polymer, and allowing the solvent to evaporate. The substrate is often pre-coated (e.g., with poly-L-lysine or aminosilane) to insure that the reagent adequately adheres to the substrate. Drops of liquid (solvent/solute) applied to the surface typically have a random edge, which is herein defined as the line of contact on a substantially flat portion of the substrate where gas (generally air), substrate, and liquid meet. By “substantially flat”, it is meant that there is no ledge or other constraining substrate geometry directly abutting or constraining the random edge.
The fluid drop is typically characterized by, a random edge which is pinned to the surface, that is, as the solvent contained within the drop evaporates from the entire liquid/gas interface, the edge does not substantially retreat as liquid flows in from the central region of the drop to replenish that which has been lost by evaporation at or near the pinned random edge. Drying of the drop during formation of the spot thus involves a continuous transport of solvent and solute outwards towards the pinned random edge, ultimately resulting in the dilution or even complete disappearance of solute in the center of the resultant spot, and the concomitant excess concentration at the location of the pinned random edge. Such an effect may be commonly observed when drops of coffee dry on a saucer or countertop. It is a robust phenomenon, little affected by choice of solvent or solute.
In the present invention, it has been found that this undesired concentration gradient can be eliminated by first freezing the fluid drops before substantial evaporation has occurred, and then subliming the solvent. In this lyophilizing process, the reagent is substantially immobilized, and the final concentration gradient of the spot substantially mirrors the thickness of the drop at the time of freezing. If the spot is hemispherical, the concentration will be greatest at the spot center, while if the spot is substantially flat, the concentration gradient will also be substantially flat.
Freezing of the entire array may be accomplished after the array is prepared. Alternatively, the substrate may be pre-cooled so that freezing occurs during or shortly after deposition of the spots. The solvent may comprise water, alcohols, hydrocarbons, or any other solvent compatible with the solute, but is preferably water. The solute may be liquid and/or solid, and may comprise, for example, nucleic acids such as RNA, DNA and cDNA, amino acids and proteins, salts, denaturing buffers, and cryoprotectants such as carbohydrates or sugars. The array may alternatively comprise a growth medium such as agar on a slide or petri dish substrate, with individual spots comprising varying levels of nutrients, mutagens, or other reagents. When drops comprising nutrients such as amino acids are spotted and later plated with bacteria, various forms of auxotrophs may be distinguished; and similarly, the effects of mutagens and other reagents on bacteria may be compactly and conveniently assessed on one slide, dish, or other suitable substrate.
A second liquid drop may be placed upon a first frozen or lyophilized drop, with the second drop having the same or different solvent and/or solute. The areal concentration may be built up with a number of drops sequentially placed on top of previously frozen or lyophilized drops, and a spot having chemically distinct lamina may be created. Such a layered spot would be useful for storage of spots wherein the reagents in different layers would otherwise react prematurely if applied from a common solute. Similarly, a film may be applied over an array of lyophilized drops, e.g., for protection. A lyophilized array may be kept frozen for extended shelf life, and may be sealed in an inert atmosphere, e.g. argon or nitrogen.
While spots have been described that are substantially circular, they may in fact be of any size or shape without departing from the scope of the invention.
It is an object of at least one embodiment of the present invention, therefore, to provide a method to modify or eliminate undesirable reagent concentration gradients within spots by lyophilizing of liquid drops having pinned edges.
It is another object of at least one embodiment of the invention to provide a method of drying a fluid drop comprising a solvent and solute on a substrate without forming a ring substantially enriched in solute.
It is another object of at least one embodiment of the invention to provide a method for creating a dried spot of reagent on a substrate having a center reagent concentration that is equal to or greater than the reagent concentration at the spot edge.


REFERENCES:
patent: 5192503 (1993-03-01), McGrath et al.
patent: 5727333 (1998-03-01), Folan
patent: 5807522 (1998-09-01), Brown et al.
patent: 2001/0049148 (2001-12-01), Wolk et al.
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