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The development of oligonucleotide-based microarrays (biochips) is a major thrust area in the rapidly growing biotechnology industry, which encompasses a diverse range of research areas including genomics, proteomics, computational biology, and pharmaceuticals, among other activities. Microarray experiments have proved to be unique in offering cost-effective and efficient analysis at the genomic level. In the last few years, biochips have gained increasing acceptance in the study of genetic and cellular processes. As the increase in experimental throughput has posed many challenges to the research community, considerable progress has been made in the advancement of microarray technology. In this review, chemical strategies for immobilization of oligonucleotides have been highlighted with special emphasis on post-synthetic immobilization of oligonucleotides on glass surface. The major objective of this article is to make the researchers acquainted with some most recent advances in this area.

In the last few years, DNA-microarray technology has emerged as a powerful tool for gene discovery, detection of mutations, and mapping. Here, we have developed a simple and efficient single-step method for immobilization of phosphoryl-, mercaptoalkyl-, thiophosphoryl- and aminooxyalkylated oligonucleotides onto an epoxylated glass surface. It resulted in higher immobilization and hybridization efficiency in comparison to those obtained with the standard method. The probes immobilized were found to be stable over a range of temperature and pH, suggesting that the chemistry could be used in integrated PCR/microarray devices. Also the immobilized spots were found to be of superior quality in terms of spot morphology, homogeneity and signal reproducibility. The constructed microarrays were successfully used for the discrimination of nucleotide mismatches and detection of bacterial diseases.

In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.

We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.