From the beginning of the science of chromatography, fibers have been employed extensively. Fibers were used as surfaces that were selectively adherent to compounds to be separated. Complex mixtures of molecules would bind to the fibers and be selectively removed by solvent systems. Cellulose is a common fiber comprised of linear polymer of beta 1,4-linked D-glucose, with beat 1,6 crosslinks. Paper chromatography is the classic example, initially used to separate colored molecules as an organic solvent wicked up the paper. The use of chemically derivatized paper fibers, such as cellulose acetate, quickly followed. A powdered form of such derivatized cellulose acetate became the basis of ion exchange and affinity column chromatography in which glass columns were packed with a slurry of cellulose fibers.
While these cellulosic columns were effective in increasing the surface area used to separate compounds, they were sorely deficient in resolving very similar molecules. The use of high flow rates to speed separations, created high back pressure, uneven packing and channelling. These deficiencies were due to the limitations of the physical structure of the fibers themselves. The particles of cellulose were of random length and their orientation in the column was totally random. This resulted in a column having a non-uniform pore size and areas of the column that could form a microcrystalline packing structure, thereby obstructing flow.
One particular application for diagnostic chromatography is DNA strand separation. Heretofore DNA strand separation techniques have been utilized in performing DNA sequence analysis of amplified DNA fragments. Typically, streptavidin, a protein having a very high affinity for binding biotin, is coated (immobilized) onto a solid support, such as for example magnetic microspheres or microtiter plates. When magnetic microspheres are used, the disadvantage is that optimal capture of the biotinylated DNA requires constant mixing of the sample with the streptavidin coated microspheres. Moreover, magnetic collection of the coated microspheres often is incomplete, especially when viscous samples are employed. When microtiter plates are utilized, cross contamination often occurs, and such plates have low binding capacity.
Still another method which utilizes a solid support for the reactive coating is disclosed in our U.S. Pat. No. 5,171,537. This patent teaches the use of a pipette tip containing a glass bead, or the like, which is mounted for limited movement in the tip intermediate its ends. The bead is coated with a receptor, such as a single ligand having an affinity for a specific target molecule in a fluid sample that is to be drawn into the tip by an associated pipetting instrument. While this type of tip is particularly suitable for use in a variety of different test procedures, the quantity of target molecule which can be captured by the receptor element is limited by the surface area of the spherical support.
It is therefore an object of the present invention to provide a preactivated chromatography tip having covalent coupling functionality within a hydrodynamically designed micropipette tip assembly.
It is a further object of the present invention to provide a preactivated chromatography tip through which flow is not restricted and rapid flow rates are possible.
It is a still further object of the present invention to provide a preactivated chromatography tip which enables the detection of proteins including proteases, nucleases, antibodies, antigens and haptens.
These and other objects of the invention will become apparent from the specification and the appended claims.