Author:

Abstract:

Fiber optic (FO) SPR biosensors allow performing complex assays with nanoparticles contrary to traditional SPR systems which are microfluidics based. Efforts by our research group have shown that FO-SPR sensor can be applied in an automated setup for both immuno- and aptamer based bioassays1, which can rival with the ELISA golden standards2. Here, we present application of FO-SPR in real-time monitoring of solid-phase PCR and mutation screening3, which is a major step forward towards cheap integrated DNA biosensors. Moreover, this concept displays some advantages over real-time PCR. While commercial qPCR provides endpoint measurements of fluorescence intensity, our biosensor permits monitoring of the reaction kinetics of every PCR cycle in real time. DNA target concentrations were detected in the femtomolar range using Au nanoparticles (NPs) as signal enhancers. Furthermore, because read-out is Au NP based, possible interference due to primer dimerization does not occur and measurements are possible even in opaque media. Additionally, FO-SPR sensors were used in combination with high-resolution sample heating to monitor the melting behaviour of DNA duplexes. This allowed not only for screening of SNPs (single nucleotide polymorphisms) in real-time but also for designating their location at the specific sequential site. The FO-SPR is therefore ideally suited for fast and sensitive genetic screening of short fragments (ranging from 30-80bp). For instance, we demonstrated that mutations in the p53 gene and several other sequences could be accurately identified in less than 20 minutes.