the silanes with primary amines present in antibody Fab fragments. Antibodies are selected that bind the drug 4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]-benzonitrile, an inhibitor of aromatase enzyme activity. This molecule has two chiral centers, yielding four possible isomers: RR, SS, SR, and RS. Fab fragments (anti-RS) of this antibody that selectively bind the RS relative to the SR form of the drug are used to modify membranes.

A racemic mixture of the drug molecule is placed on one side of a U-tube permeation cell and the flux of each species is monitored as a function of time by periodically monitoring the concentration of each enantiomer present in the permeate solution with a chiral chromatographic method. A selectivity of 2 was obtained for the RS relative to the SR enantiomer, indicating that the membranes transport the RS form twice as fast as the SR form. A facilitated transport mechanism was determined to be responsible for transport in these membranes. As in the case of the apoenzyme-modified membranes, by decreasing the pore diameter the selectivity coefficient is increased to 4.5 (at the expense of lower total flux). It was also found that by adding dimethyl sulfoxide (DMSO) to the feed and permeate solutions in concentrations from 10% to 30%, the rate of transport for the RS form of the drug could be regulated. This occurs because DMSO weakens the affinity of the anti-RS Fab fragment for the RS enantiomer. Thus, at 30% DMSO content the relative transport rates for the RS and SR enantiomers were essentially equal. Because antibodies can be developed for a wide variety of species of biochemical interest, this method should be highly adaptable to a wide variety of targets. Separation of Nucleic Acids

We have also used nanotube membranes to perform separation of DNA with single-base mismatch selectivity [18]. In these experiments, 6 mm thick polycarbonate membranes with 30 nm diameter pores are coated with gold using electroless deposition. The diameter of the pores after gold deposition is determined to be 12 + 2 nm. Linear DNA or hairpin DNA are used as the molecular recognition agent in these experiments. DNA hairpins contain complementary sequences at each end of the molecule, and under appropriate conditions form a stem-loop structure. As a result of this structure, hybridization of complementary DNA is very selective, in optimal cases a single-base mismatch will not hybridize. A 30 base DNA hairpin with a thiol modification at the 5' end allowed facile chemisorption of the molecular recognition agent to the gold-coated nanotubes. The six bases at each end of the DNA strand were complementary, forming the stem, with the loop comprised of the remaining 18 bases in the middle of the DNA strand. The thiol-modified linear DNA molecular recognition modifiers used the same 18 bases in the middle of the molecule, but the six bases at each end were not complementary, thus these linear sequences do not form the stem-loop structure. DNA molecules to transport are 18 bases long and are either perfect complements to the bases in the loop or contained one or more mismatches.

DNA-modified membranes are mounted in a U-tube permeation cell and molecules to transport are added to the feed side of the membrane. Transport is monitored by measuring the UV-vis absorbance of the permeate solution as a function of time. These systems also demonstrated a facilitated transport mechanism for complementary sequences of DNA. In the case of linear DNA, the selectivity coefficient for perfect complement DNA (PC-DNA) versus single-base mismatch DNA is 1, that is to say there was no selectivity. PC-DNA versus a seven-base mismatch showed a selectivity coefficient of 5. In the case of hairpin DNA-modified membranes, transport plots of PC-DNA through a modified and unmodified membrane are shown in Figure 9.20. In Figure 9.20A, the flux of DNA through an unmodified membrane is significantly lower than transport through the membrane modified with a perfectly complementary hairpin DNA. In Figure 9.20B, the Langmuirian shape characteristic of facilitated transport is observed for the PC-DNA, whereas diffusive flux is observed for the membrane with no DNA modification. In the case of hairpin DNA molecular recognition elements, a selectivity coefficient of 3 is obtained for a PC-DNA sequence versus a single-base mismatch sequence. A selectivity coefficient of 7 is obtained for a PC-DNA sequence versus a seven-base mismatch.

Nanotube membranes have shown the ability to separate an amazingly diverse field of biochemical species, from DNA to proteins to drug molecules. The selectivity in each of these separations is governed by the inherent selectivity in the immobilized biochemical species used to effect recognition or through physical properties of the nanotubes themselves.

0 0

Post a comment