Christopher M. Strohsahl

Preparation and use of metal surface-immobilized DNA hairpins for the detection of oligonucleotides

The following protocol describes the experimental steps used to prepare arrayable and label-free biological sensors that are based on the fluorescence unquenching of DNA hairpins immobilized on metal surfaces. This two-part protocol describes both the creation of gold-coated substrates and the oligonucleotide surface self-assembly process that transforms the substrates into reporters for detecting DNA. Using this procedure, one can create sensors for oligonucleotides that are highly sensitive and have demonstrated an exceptional specificity to single nucleotide polymorphisms. From start to finish, the entire procedure can be accomplished in 24–30 h.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) using the NanoLantern Biosensor

Staphylococcus aureus is a leading cause of human illness, and has developed the remarkable ability to resist the bactericidal capabilities of many of the worlds leading antibiotics (i.e. MRSA). In an effort to enable rapid detection and treatment of MRSA infections, we have developed a DNA detection technology termed the NanoLantern(TM). The NanoLantern(TM) biosensor technology is based on the simple immobilization of a fluorophore-terminated DNA hairpin onto a gold chip. This produces a label-free sensor that allows for a positive response to be obtained without extensive processing of the sample, saving cost and increasing accuracy. We will also discuss a newly developed method of partial gene analysis, used to develop a DNA hairpin probe that is capable of detecting the presence of the mecR gene, a gene necessary for methicillin resistance to be present in S. aureus, with 100% sequence specificity. The successful incorporation of this probe into the NanoLantern(TM) platform, along with the concomitant development of the paired PCR assay has allowed for the successful detection of methicillin-resistance directly from a culture of S. aureus. These results represent an important step forward in terms of developing the ability to rapidly and effectively detect the presence of antibiotic resistance in bacterial infections.