We are currently trying to replicate experimental results from a paper by Han, Huang, Zhao and Ozaki (Anal. Chem. 81 3329-3333 (2009)). This seemed like a great place to start trying to understand how to consistently obtain SERS spectra of proteins. Naturally, it isn’t as simple as one might think.

We started by trying to replicate what is in their experimental section – several proteins purchased from standard suppliers. We’re focusing on just one for now – lysozyme. They dissolve the proteins in a phosphate buffer (NaH₂PO₄/Na₂HPO₄, pH = 7.0). Good, we can do that too. They prepare their Ag colloids using the method of Lee and Meisel (AgNO₃ and reduce with sodium citrate), which is how we’ve always made our colloids too.

The novel part is use of sodium sulfate in an acidic solution (pH = 3) to aggregate the colloids. This step is based on work published by Bell and Sirimuthu (JPCA), who showed that sulfate can be used to aggregate citrate reduced silver colloids (CRSC) for anionic or neutral species.

So, what do Han, et al. do? The procedure calls for diluting protein samples 1/10 with the acidified sulfate solution, and then mixing that solution with the colloids in a 1:5 (v/v) ratio. Great! We did that. What did we observe? A beautiful SERS spectrum of citrate, similar to what Bell & Sirimuthu observed! Hmmm. We have some detective work ahead of us. Perhaps we can play with pH a bit. Bell & Sirimuthu (JACS) have obtained spectra of mononucleotides using just MgSO₄ as the aggregating agent.