Highly efficient detection of single fluorophores in blood serum samples with high autofluorescence

Single molecule detection (SMD) is usually performed on surface-immobilized molecules with diffraction-limited observation volumes, typically with confocal optics to suppress background from the sample and instrument. In this paper we consider the more difficult task of detecting single fluorophores in the presence of a substantial fluorescence background. We determined that for a readily accessible macroscopic observation volume of 1 pL that the background from undiluted blood serum was approximately equal to 2700 Cy5 molecules, and the background from whole blood equal to about 14 000 Cy5 molecules in whole blood. These high backgrounds appear to preclude the possibility of SMD of Cy5 molecules. However, we show that the signal-to-noise ratio (SNR) in high background samples can be increased dramatically by reduction of the observed volume. We were able to detect single surface-bound Cy5-labeled DNA (Cy5-DNA) oligomers in diluted blood serum with an SNR near 40. We also examined freely diffusing Cy5-DNA in blood serum. The data showed that single Cy5-DNA molecules could be detected even in the undiluted serum. We further investigated the SNR on silver island films. We found that the fluorescence signal was greatly enhanced in the presence of metallic nanostructures showing a larger SNR in the application tested. These results suggest the possibility of clinical assays based on SMD in blood serum and possibly whole blood. Increased SNR near metallic nanostructure could probably overcome the need for diffraction-limited volumes and enhance our ability to do in situ SMD.