Development of PNA microarrays for gene diagnostics with TOF-SIMS

We have used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to examine different immobilization processes of peptide nucleic acid (PNA) and their influence on the hybridization efficiency of unlabeled DNA fragments to complementary PNA. Two different approaches have been used to immobilize PNA onto Au surfaces. One method was to immobilize thiolated PNA in a single step reaction to the Au surface via an Au–S bond. The other method was to crosslink the N-terminal end of the PNA to a preformed functionalized self-assembly monolayer (SAM). The influence of length and type of spacer molecules, which increase the distance between the biosensor surface and the PNA sequence, on the hybridization efficiency was also investigated. It was found that a greater spacer length leads to an increased hybridization efficiency. DNA could readily be identified by detecting PO2 and PO3 or other phosphate-containing molecules from the DNA backbone. Using TOF-SIMS a very good discrimination between complementary and non-complementary hybridized DNA sequences was achieved. It can be concluded that TOF-SIMS is a very useful technique for identifying unlabeled hybridized DNA on PNA biosensor chips. This method has the potential for providing a rapid method for DNA diagnostics and is also very suitable for studying the complexity of the immobilization and hybridization processes. Both the absence of a labeling procedure and the increase in the number of phosphate signals with increasing sequence length makes this technique especially useful for directly analyzing genomic DNA. # 2004 Elsevier B.V. All rights reserved.