Nucleic acid testing using surface plasmon resonance fluorescence detection.

Abstract

The completion of the Human Genome Project (1) is likely to accelerate the demand for nucleic acid diagnostics, one of the fastest growing segments of in vitro diagnostics. To meet this demand, fully automated, sensitive, and specific technologies for nucleic acid detection are needed. Surface plasmon resonance fluorescence (SPRF) is an optical detection technology that offers kinetic analysis of biomolecular interactions in real time (2). SPRF technology uses a sensor composed of a dielectric material coated with a thin layer of a noble metal. An evanescent field is generated at the metal-dielectric interface through interaction of p -polarized light with surface plasmons (oscillating electrons) under a resonance condition. The evanescent field excites a fluorophore localized near the metal surface, which in turn emits evanescent waves, generating surface plasmons at the fluorescence emission frequency. These propagating waves (plasmon-mediated emission) radiate through the metal film and dielectric material. This emission emerges as a cone of radiation in the dielectric material. Application of SPRF for nucleic acid testing involves immobilization of synthetic oligonucleotide capture probes on the sensor surface (Fig. 1⇓ ). Biotin-tagged nucleic acid amplicons are hybridized to target-specific capture probes and detected with a streptavidin-fluorophore conjugate by monitoring of the plasmon-mediated emission over time. Because the evanescent field penetrates a short distance into the medium above the metal surface, the hybridized amplicons are selectively excited and detected. This feature of SPRF allows sensitive detection without a wash step to separate the free from the hybridized nucleic acids. The fluorescent emission is collected by a patented optical system, providing higher sensitivity than other similar techniques (2). The rate of …