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A number of hetero- and homobifunctional reagents have been reported to immobilize biomolecules on a variety of supports. However, efforts are on to search for a method, which is relatively simple, involving minimum of steps, cost effective, easy to reproduce, and that produces stable oligonucleotide arrays. Two new reagents, viz., [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine], and [N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide] have been designed considering the above points. These reagents contain different functional groups at their two ends. In [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine], one end (triethoxysilyl) is capable of binding to the virgin glass surface and the other one consists of trifluoroethanesulfonate (tresyl) function specific toward aminoalkyl and mercaptoalkyl functionalities, which are easy to introduce at the 3'- or 5'-end of oligonucleotides. Likewise, in [N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide], one end consists of photoactivatable moiety (anthraquinone) capable of reacting to a C-H containing surface and the tresyl function at the other end reacts specifically with aminoalkyl and mercaptoalkyl functionalities in modified oligonucleotides. These reagents have successfully been utilized to construct a number of oligonucleotide arrays and subsequently used for the detection of mismatches.

Synthesis of a new heterobifunctional reagent, N-(3-triethoxysilylpropyl)-6-(N-maleimido)-hexanamide (TPMH), for the preparation of oligonucleotide microarrays is described. Its triethoxysilyl function is specific toward virgin glass surface and maleimide function undergoes conjugate addition to 3'- or 5'-thiol-modified oligonucleotides. The construction of microarrays using TPMH has been realized via two routes. In Route A, TPMH was reacted first with 3'- or 5'-thiol-modified oligonucleotide under microwaves, thereby producing oligonucleotide-triethoxysilyl conjugate in 15min, which was then brought in contact with virgin glass microslide, resulting in immobilization of an oligonucleotide sequence. In Route B, immobilization involves generation of maleimide functions on virgin glass surface by the reaction with TPMH, followed by coupling with thiol-modified oligonucleotides under microwaves in 15 min to produce surface-bound oligonucleotides. The microarrays constructed using both routes were analyzed by hybridizing with tetrachlorofluorescein-labeled complementary oligonucleotide. Subsequently, these microarrays were successfully used in the discrimination of single and double nucleotide mismatches based on fluorescence intensity.

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.