Vipolymeric Supports For Dna Immobilization

DNA-immobilized sorbents have been widely used in the management of some autoimmune diseases involving the removal of anti-DNA antibodies from plasma [19-25]. Complexation of phosphate segments of DNA with the amine

FIG. 6 Albumin adsorption isotherms derived at pH 5.0 and 25°C, with the poly-(PEGMA-co-DVB) gel beads prepared with different PEGMA/DVB feed ratios. (Reprinted with permission from Ref. 18, Copyright © 2000, Springer-Verlag GmbH & Co. KG.)

groups of the support materials is a commonly preferred route for the immobilization of DNA on the solid supports. For this reason, the support materials carrying primary and secondary amine functionalities have been used for DNA immobilization. A magnetic sorbent based on amine-functionalized agarose was successfully tried for DNA immobilisation. Kato and Ikada proposed plasma-treated poly(ethylene terephthalate) (PET) fibers as carriers for the immobilization of DNA [20]. Elaissari et al. developed a sorbent material for RNA adsorption based on the thermosensitive poly(N-isopropylacrylamide) particles carrying cationic groups [21]. In this study, the conditions providing the maximal RNA adsorption was investigated and an isolation protocol based on the successive adsorption/desorption of RNA was developed [21]. Core shell magnetic latex particles bearing poly(N-isopropylacrylamide) in the shell were also used for the nucleic acid extraction purification and concentration [22].

FIG. 7 Representative optical micrographs of the plain and CBF3G-A attached gel beads prepared with the PEGMA/DVB feed ratio of 5.0/0.5 mL/mL. (Reprinted with permission from Ref. 18, Copyright © 2000, Springer-Verlag GmbH & Co. KG.)

We also attempted to synthesize DNA-carrying support materials based on the poly(p-chloromethylstyrene) (PCMS) particles. As described in the literature, a chloromethyl functionality is a reactive group against primary and secondary amines [23,24]. For this reason, polyamine- or polyimine-type ligands are suitable candidates for the immobilization on the polymeric support materials carrying chloromethyl groups. In such a case, the primary and secondary amine groups not utilized for the binding of polymeric ligand onto the support material should exhibit an ionic interaction affinity against the phosphate segments of DNA molecules. In our studies, we selected polyethyleneimine (PEI) as the ligand having an interaction ability with DNA [23,24]. Hence, PEI was covalently immobilized onto the PCMS particles via the reactions between chlo-romethyl groups and primary or secondary amine groups in PEI [23,24]. Owing to the higher reactivity of chloromethyl group against secondary amine groups, the formation possibility of the first reaction should be higher [23].

In our study, the DNA-sensitive ligand PEI was covalently attached to the PCMS-based particles (average size 186 pm) produced by suspension polymerization [23]. Comparison of surface morphologies of the plain and PEI-attached PCMS particles is given in Fig. 8. As seen here, the roughness of the bead surface almost disappeared as a result of by PEI binding [23]. Hence, a tailor-made particle structure composed of an inner PCMS-based core surrounded by

FIG. 8 Comparison of surface morphologies of the plain and PEI-attached PCMS particles. (Reprinted with permission from Ref. 23, Copyright © 2000, Elsevier Science.)

a PEI layer probably forming flexible polycation chains in the aqueous medium was obtained [23].

The variation of equilibrium DNA adsorption capacity for plain and PEI-attached PCMS beads at pH 7 and at 25°C are given in Fig. 9. The tailor-made structure of the support material provided extremely high DNA adsorption [23]. The possible interaction mechanism between PEI and DNA is given in Fig. 10. As seen here, DNA was adsorbed onto the PCMS beads probably by the H-bond formation between phosphate groups of DNA and primary amine groups of immobilized PEI [23]. The desorption experiments showed that only a fraction lower than 5% (w/w) of adsorbed DNA could be released from the PEI-carrying PCMS beads (23). This result indicated that the ionic interaction between the amine/imine groups of PEI and phosphate groups of DNA was sufficiently strong to obtain a stable DNA coating on the surface of the PCMS particles [23].

FIG. 9 Variation of equilibrium DNA adsorption capacity for plain and PEI-attached PCMS beads. (Reprinted with permission from Ref. 23, Copyright © 2000, Elsevier Science.)

On the other hand, a similar route was also followed for the synthesis of DNA-responsive uniform latex particles. In this study, uniform (1.75-p.m) PCMS beads were obtained by the dispersion polymerization of CMS [24]. PEI (Mr 25.000) was covalently attached to the PCMS particles via the direct reaction between chloromethyl and primary amine or imine groups [24]. The DNA adsorption experiments performed at pH 7.4 and 25°C showed that irreversible DNA binding up to 40 mg DNA/g particles was possible for the PEI-carrying PCMS particles [24]. This interaction resulted in a change in the absorbance of the aqueous latex dispersion containing DNA at a certain concentration in the visible region. Hence, the responsive behavior against DNA concentration was quantified by a spectrophotometric procedure [24]. The variation of absorbance of the aqueous latex dispersion-DNA mixture with the DNA concentration is exemplified in Fig. 11. The aggregation behavior of PEI-carrying PCMS latex is probably due to the binding of some PCMS particles onto the same DNA molecule as shown in Figure 12 [24].

For the immobilization of DNA, another carrier based on monodispersed polycationic gel beads of 3 mm was obtained by the suspension polymerization

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