The objective of this doctoral research is the development and the implementation of SERS-active substrates with biological samples. The substrates consist in coated silver nanoparticles synthesized by chemical reduction of a silver salt. The biological samples are the anionic chromophore bilirubin and two heme protein, the cationic cytochrome-c and the anionic protein cytochrome b5.
In the first part of this work, positively charged nanoparticles were prepared by coating citrate-reduced silver nanoparticles with the cationic polymer poly-L-lysine and were employed with bilirubin in the experiments listed below:
detection of nanomolar bilirubin concentrations in aqueous solutions, showing that the SERS intensity increases linearly with concentration in a range from 10 nM to 200 nM, allowing quantitative analysis of bilirubin aqueous solutions.
indirect quantification of bilirubin cellular up-take, demonstrating the ability to detect the bilirubin also in a buffer solution suitable for cell growth with pH 7.4. Since the bilirubin quantification at this pH is no longer possible, the poly-L-lysine was substituted by two polymers with a quaternary nitrogen atom.
bilirubin measurement in serum, but TEM images highlights the formation of a albumin layer around the nanoparticles, blocking the interaction between bilirubin and the nanoparticles. Hence the citrate-reduced silver nanoparticles were coated with an hydrophobic capping and re-dispersed in hexane, to avoid the albumin layer around the nanoparticles.
In the second part of this doctoral thesis, silver nanoparticles were prepared via seed growth method and subsequently coated with chitosan or silica in order to obtain positively or negatively charged nanoparticles respectively. Such substrates enhance the spectrum of the cytochrome-c and cytochrome-b5 on polished silver electrode without directly interact with the protein. Thanks to the presence of chitosan or silica coated nanoparticles, the cytochrome-c and cytochrome-b5 can be detected on a gold substrate.
