RESUMEN
The concept of plasmonic "hotspots" is central to the broad field of nanophotonics. In surface-enhanced Raman scattering (SERS), hotspots can increase Raman scattering efficiency by orders of magnitude. Hotspot dimensions may range from a few nanometers down to the atomic scale and are able to generate SERS signals from single molecules. However, these single-molecule SERS signals often show significant fluctuations, and the concept of intense, localized, yet static hotspots has come into question. Recent experiments have shown these SERS intensity fluctuations (SIFs) to occur over an extremely wide range of timescales, from seconds to microseconds, due to the various physical mechanisms causing SERS and the dynamic nature of light-matter interaction at the nanoscale. The underlying source of single-molecule SERS fluctuations is therefore likely to be a complex interplay of several different effects at different timescales. A high-speed acquisition system that captures a full SERS spectrum with microsecond time resolution can therefore provide information about these dynamic processes. Here, we show an acquisition system that collects at a rate of 100,000 SERS spectra per second, allowing high-speed characterization. We find that while each individual SIF event will enhance a different portion of the SERS spectrum, including a single peak, over 10s to 100s of microseconds, the SIF events overall do not favor one region of the spectrum over another. These high-speed SIF events can therefore occur with relatively equal probability over a broad spectral range, covering both the anti-Stokes and the Stokes sides of the spectrum, sometimes leading to anomalously large anti-Stokes peaks. This indicates that both temporally and spectrally transient hotspots drive the SERS fluctuations at high speeds.
RESUMEN
Surface-enhanced Raman scattering (SERS) is a sensitive, widely used spectroscopic technique. However, SERS is perceived as poorly reproducible and insufficiently robust for standard applications in analytical chemistry. Here, we demonstrated that reliable SERS immunoassay quantification at low concentrations (pM range) can be achieved by careful experimental design and appropriate data analysis statistics. A SERS-based immunoassay for IgG in human serum (3.1-50.0 ng mL-1 or 20.6-333 pM) was developed as a proof of concept. Calibration curves were created using the population median of the band area, centered at 592 cm-1, of a SERS reporter (Nile Blue A). Histograms of 7200 SERS spectra show lognormal distributions. SEM images of the sensor platform confirm a correlation between the number of SERS probes (ERLs) at the surface and the SERS intensity response. The IgG immunosensor reported here presented a limit of detection of 1.11 ng mL-1 or 7.39 pM and a limit of quantification of 9.04 ng mL-1 or 60.30 pM, within a 95% confidence level. The % error of the predicted versus the actual response of a quality control (QC) sample was 0.13%. The percent error of the QC sample decreases exponentially with the number of measurements. Randomly selected spatially separated measurements provided lower QC % error than a larger number of measurements that were closely spaced. We propose that it is necessary to describe the measured populations using an appropriate sample size for good statistics and consider the interrogation of sufficiently large and well-separated areas of the sensor surface to achieve a reliable sampling.
