Serendipitously, in one day of experiments, the vesicles were mistakenly not tip sonicated. This prevented unilamellar vesicles from forming, and no bilayer was adsorbed onto the surface. Experiments identical to those in Section 4.5.3 were performed where increasingly high concentrations of fluorescently-labeled IgG were placed in the sample chamber between the prism and coverslip. Remarkably, in the absence of a bilayer, increasing the concentration of the antibody caused a linear decrease in the fluorescence (data not shown). This quenching phenomenon that is responsive to increasing the concentration is noteworthy for two reasons. Primarily, during the fluorescence imaging experiments (Section 4.5.1) the high refractive index substrates produced lower fluorescence intensity than SiO2. It is plausible to deduce that the decreased fluorescence from the bilayers during the imaging experiments is related to the decreased fluorescence with increasing antibody concentration seen here. Secondly, TiO2 is natively luminescent and so a decrease in fluorescence intensity under this situation is unexpected. It is possible that a FRET-like scenario is occurring between the substrate and fluorescent material very near to it. Further experiments are required to deduce the nature of this quenching phenomenon.
4.6 Discussion
The purpose of these experiments was to experimentally pioneer the use of SrTiO3 and TiO2 as total internal reflection elements. The first step in accomplishing this was to reconfirm that phospholipid bilayers can be formed atop these two substrates. This was accomplished as demonstrated in Section 4.5.1. The second step in amending SrTiO3 and TiO to use with TIR technologies was to prove that TIR can be accomplished through these
obtained by TIR-FCS for TiO2. However, more work is required in this area so that the data obtained from total internal reflection atop a TiO2 substrate can be fit to any autocorrelation function and not just simplified versions of the appropriate autocorrelation function. The success achieved thus far in this area provides ample evidence indicating that TiO2 can be used in total internal reflection.
One obstacle that must be addressed is the high background luminescence exhibited by these substrates. The extremely high background luminescence of SrTiO3 eliminated the possibilities for its use with TIR-FCS, and all efforts were focused on TiO2. Although TiO2 exhibited significantly less background luminescence, it is still considerably natively luminescent. This native luminescence is much less a deterrent to obtaining data at 514 nm light. Noisy autocorrelation functions were obtained at this wavelength by utilizing a simplified version of the appropriate autocorrelation function and by taking data at longer times.
Future investigations must also address the quenching phenomenon that has been observed. At high concentrations of fluorophore, it is not uncommon to see self-quenching or self-absorption present in fluorescence data that would cause a negative deviation from linearity in intensity vs. concentration plots (Skoog et al., 1998). Although concentrations in the nanomolar range are not considered high, the lack of a bilayer increases the propensity for nonspecific adsorption which would cause a high surface concentration. Alternatively, perhaps the high background luminescence of the substrate is enough to cause self-quenching or self-absorption. Right now there is not enough evidence to conclusively say if it is self- quenching, self-absorption, a photochemical process, or a FRET-like substrate-fluorophore
interaction. It is clear that if more knowledge is obtained about the luminescent activity seen on TiO2, this information might provide an avenue around these experimental complications. Future studies in this area should concentrate on several important points. First of all, with TiO2, the argon ion laser might not be the most appropriate laser choice. Although there is significantly less background luminescence at 514 nm (Figure 4.5b), moving to a higher wavelength in the visible region might provide better results. Potentially useful lasers in include the Nd:YAG laser (532 nm line) or a Krypton-Argon laser (568 nm or 647 nm laser line).
Secondly, the possibility of a photochemical reaction occurring on TiO2 can be addressed by taking intensity dependent measurements. The possibility of a photochemical interaction occurring is plausible because the data does not fit well to Eq. 4.3 at fast times (less than 0.1 ms). This is the time scale where one would expect to see a photochemical interaction manifest itself. By doubling the laser intensity, obtaining TIR-FCS data, and monitoring the rate of diffusion one could probe this scenario. The rate of diffusion is not intensity dependent, and so large changes in the calculated Re at high intensity would indicate that a photochemical reaction is occurring.
Another approach in determining the problems associated with using TiO2 with TIR- FCS would be to choose a different antibody (with a different radius) to study or change the angle at which the laser is incident on the prism. Both of these situations should produce different Re values according to the theory laid out in chapter 2. The values obtained by
these experiments would provide insight into the rates of diffusion already measured and if the substrates are accurately measuring the rate of diffusion.
In conclusion, TiO2 was proven to be utilizable in TIR-FCS measurements and total internal reflection based techniques. Future experiments are required before TiO2 can be successfully applied in a research study involving TIR-FCS. These investigations should concentrate on eliminating the noise seen in the autocorrelated data. It has also been experimentally verified that phospholipid bilayers can be formed on these substrates. These experiments should provide a foundation for future applications in this area.