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 surface 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 effected 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.