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4-Mercaptopyridine (4-Mpy) is a pH reporter molecule commonly used to functionalize nanoprobes for surface-enhanced Raman spectroscopy (SERS) based pH measurements. However, nanoprobes functionalized by 4-Mpy alone have low pH sensitivity and are subject to interference by halide ions in sample media. To improve nanoprobe pH sensitivity and reliability, we functionalized gold nanoparticles (AuNPs) with both 4-Mpy and bromide ion (Br - ). Br - electrostatically stabilizes protonated 4-Mpy, thus enabling sensitive SERS detection of the protonation state of 4-Mpy as a function of pH while also reducing variability caused by external halide ions. Through optimization of the functionalization parameters, including suspension pH, [4-Mpy], and [Br - ], the developed nanoprobes enable monitoring of pH from 2.1 to 10 with high SERS activity and minimal interference from halide ions within the sample matrix. As a proof of concept, we were able to track nanoprobe location and image the pH distribution inside individual cancer cells. This study provides a novel way to engineer reliable 4-Mpy-functionalized SERS nanoprobes for the sensitive analysis of spatially localized pH features in halide ion-containing microenvironments.

Production of gold nanoparticle (AuNP) surface-enhanced Raman spectroscopy (SERS) nanoprobes requires replicable aggregation to produce multimers with high signal intensity. Herein, we illustrate a novel, yet simple, approach to produce SERS nanoprobes through control of co-solvent composition. AuNP multimers were produced by mixing AuNP monomers in water : ethanol co-solvent for variable periods of time. By varying the water : ethanol ratio and the amount of 4-mercaptobenzoic acid (4-MBA) present, the aggregation rate can be systematically controlled. Thiolated poly(ethylene glycol) was then added to halt the aggregation process and provide steric stability. This approach was used to produce pH nanoprobes with excellent colloidal stability in high ionic strength environments and in complex samples. The pH probe exhibits broad pH sensitivity over the range 6-11 and we calculate that a single AuNP dimer in a 35 fL volume is sufficient to generate a detectable SERS signal. As a proof-of-concept, the probes were used to detect the intracellular pH of human prostate cancer cells (PC-3). The internalized probes exhibit a strong 4-MBA signal without any interfering bands from either the cells or the culture media and produce exceptionally detailed pH maps. pH maps obtained from 19 xy surface scans and 14 yz depth scans exhibit highly consistent intracellular pH in the range of 5 to 7, thus indicating the greater reliability and reproducibility of our pH probes compared with other probes previously reported in the literature. Our water : ethanol co-solvent production process is fast, simple, and efficient. Adjustment of solvent composition may become a powerful way to produce SERS tags or nanoprobes in the future.

A SERS-based method for the concentration and detection of Giardia lamblia cysts in finished drinking water is reported. In this method, samples are concentrated with a membrane filter and then cysts captured on the filter surface are labeled with immunogold SERS labels and quantified via Raman spectroscopy. Anodisc((R)) membrane filters, silver membrane filters, and electroless gold-coated polycarbonate track etched (PCTE) membrane filters were investigated for their compatibility with the SERS based detection strategy. The largest pore size Anodisc((R)) membrane commercially available was too small for the proposed method because they led to physical retention of immunogold. When silver membrane filters were employed, cysts were difficult to distinguish from nonspecifically bound labels and cyst recovery from distilled water samples was only approximately 12.3%. With gold-coated PCTE membranes, however, cysts were readily detectable and cyst recovery was approximately 95%. This Raman based method simplifies Giardia detection and has potential to be extended to the simultaneous detection of numerous pathogenic organisms. To our knowledge, this is the first report coupling the use of membrane filters for the concentration and detection of organisms from water samples with a SERS based detection strategy.