Silver colloids were produced for surface-enhanced Raman scattering (SERS) experiments using hydroxylamine hydrochloride as the reduction agent. The roles of hydroxylamine hydrochloride and bulk solution pH values in the formation of functional groups on the surface of silver colloids and in determining the dimensions of silver colloids were examined using Raman, Fourier transform infrared (FT-IR) and ultraviolet–visible (UV-Vis) spectroscopy, atomic force microscopy (AFM), and zeta-size measurements. The spectrum of hydroxylamine hydrochloride reduced silver colloids was compared with the spectrum of sodium borohydride reduced colloids. The effect of colloid solution pH on SERS results was demonstrated using analyte molecules with biological significance, such as ribonucleic acid, egg albumin, L-<i>α</i>-phosphatidylcholine, and glucose. In general, it was shown that at high pH values the SERS effect was more pronounced due to the surface functional groups and colloid dimensions, and sharp, high spectral intensity values were obtained. At low pH values, protonation and rapid aggregation of colloids occurred and the surface chemistry was different. Depending on the analyte, bands were shifted, broadened, and/or the enhancement effect was reduced. Using <i>Pseudomonas aeruginosa</i> PAO1 and <i>Streptococcus mutans</i> it was also shown that by changing the solution bulk pH value, it was possible to enhance the response from different molecular groups in the bacteria and obtain different spectra from the same bacteria strain and the process was reversible. It was concluded that it is possible to produce site- or molecule-specific metal colloids and to tune the SERS effect to certain functional groups of analytes by means of the pH of colloidal suspension.
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