2) Alexa488 Model Immunoassay

We  performed a model immunoassay on glass and SACS substrates using an  Alexa488 fluorescent probe. Emission spectra (Fig. 9) show more than  tenfold enhancement on SACS compared to the glass. The fluorescence  signal from a control (non-specific binding) was about 10% of the sample  signal in both glass and SACS substrates. The distributions of  intensities and lifetimes on SACS substrate are shown on Fig. 10. Again,  higher intensity corresponds to shorter lifetime, and in the less  bright spots lifetimes are longer. Although the average enhancement is  not high (see Fig. 9), in the local “hot spots” (Fig. 10, top), the  brightness is about 100-fold higher than in the absence of silver a  nanostructure.

The Alexa488 lifetime  distributions on glass and SACS are shown in Fig. 11. The average  lifetime on SACS is a few times shorter than on glass. This observation  was confirmed in lifetime measurements with a high-resolution  fluorometer (not shown). Finally, we compared the photostabilities of  Alexa488 immunoassays on glass and SACS (Fig. 12). The shorter lifetimes  are responsible for higher photostabilities on metallic surfaces. The  photodegradation occurs mostly in the excited state, and near metallic  nanostructure molecules can emit many photons before they are bleached.

注意事项

The surface preparation is crucial in MEF and has to be done very carefully.

For detection purposes in macroscopic devices SIFs surfaces perform  well, providing a uniform enhancement in large areas. In the case of  microscopy measurements, like in single molecule detection (SMD), the  SACS substrates are more useful because they offer “hot spots” with  unprecedented fluorescence enhancements. Observation of “hot spots”  enables a significant reduction of the excitation power. In turn, this  reduces background which often makes SMD measurements difficult or  impossible.