SERS as a Tool for Detection of Single Molecules

There have been several reports of single-molecule detection using SERS in recent years (4-7,113,136,137). A critical factor in these milestone studies has been the development of exceptional SERS substrates. Most of the reports of single-molecule SERS detection have involved the use of metal colloids in suspensions. For example, Kneipp et al. demonstrated single-molecule detection of crystal violet in both silver (5,6) and colloid suspensions (7). In these studies, an effective cross-section of approx 10-16 cm2/molecule was observed, corresponding to a 1014 enhancement factor. The gold nanoparticles were commercially available but required proper agglomeration through the addition of sodium chloride. These results are promising because these cyanine dyes could be used as bioassay markers.

In some cases, bioassay markers can be resonance enhanced in addition to benefiting from SERS. For example, a DNA marker, 2,5,1',3',7',9'-hexachloro-6-carboxyfluorescein (HEX), has been used to achieve single-molecule detection via SERRS in a silver colloid suspension (113). The HEX signature was observed for 8 x 10-13 M DNA, which corresponded to less than 1 molecule per probed volume at any time required for measurement. Single-molecule detection has also been demonstrated on planar surface-based substrates. For example, enhancements to factors of 1014-1015 have been observed for Rhodamine-6G molecules adsorbed on silver colloids that had been immobilized on a polylysine-coated glass surface (4). Similarly, 1014-1015 factor enhancements have been reported for hemoglobin molecules adsorbed on silver nanoparticles immobilized on a polymer-coated silicon wafer (136). Researchers in this study reported, however, that single-molecule detection was observed only for hemoglobin molecules situated between and adsorbed to two silver nanoparticles.

Fig. 6. Chemistry of dual labeling of SERS-active metal nanoparticles. (A) Formation of SAM of 11-mercaptoundecanoic acid (MUA) on a metal nanoparticle surface; (B) activation of MUA carboxylic acid groups by the carbodiimide, N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC), and formation of NHS ester by N-hydroxy-succinimide (NHS); (C,D) subsequent reaction of activated intermediate with a combination of Raman marker and either streptavidin or amino group-containing biomolecules (e.g., antibodies).

Fig. 6. Chemistry of dual labeling of SERS-active metal nanoparticles. (A) Formation of SAM of 11-mercaptoundecanoic acid (MUA) on a metal nanoparticle surface; (B) activation of MUA carboxylic acid groups by the carbodiimide, N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC), and formation of NHS ester by N-hydroxy-succinimide (NHS); (C,D) subsequent reaction of activated intermediate with a combination of Raman marker and either streptavidin or amino group-containing biomolecules (e.g., antibodies).

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