Recent interest in the application of surface enhanced Raman scattering (SERS) to flow cytometry1,2
has been spurred by the potential use of SERS in novel optical tags for bioassay and imaging applications.3-12
Flow cytometry is a powerful and versatile approach to high throughput analysis, finding widespread use in clinical diagnostics, fundamental biochemical studies, and the development of pathogen detection and drug discovery applications.13
Currently, flow cytometry approaches to cell marker analysis, immunoassays, evaluation of molecular avidity, etc. are typically assessed primarily by fluorescence labeling and readout. The introduction of multi-color flow cytometry has allowed simultaneous multi-analyte assays and multiple parameter measurements to be performed on individual cells in a sample stream.14
This enhanced capability drives a continuing demand to further expand the number of distinct measurements made on each cell, with a concurrent interest in high resolution instrument development.15-25
However, the degree of spectral overlap between the various fluorophores limits simultaneous multiparameter measurement, and has led to interest in alternate, non-fluorescent, probes.2,26,27
One such alternative involves the use of Raman-based probes. Fluorescence spectra are typically broad and featureless, with emission peak widths in the range of 50 – 60 nm. Furthermore, multi-color applications require multiple excitation and detection channels. In contrast, Raman probes generate highly featured fingerprint spectra consisting of many narrow lines (typically <0.5 nm FWHM), allowing multiple overlapping spectra from different molecules to be easily distinguished, with the further advantage of reducing the instrumentation requirements to include only single source excitation and a single detector. Thus, Raman-based optical probes are inherently suitable for advanced multiplexed analysis. While the use of intrinsic Raman is made difficult by small Raman cross sections, SERS can provide more than sufficient sensitivity based on scattering by ‘tags’ consisting of Raman-active molecules adsorbed on nanostructured gold or silver surfaces.7,28,29
In principle, many types of nanostructures can be employed as SERS-tags, including stabilized colloidal particles,7,28,29
and small nanoparticle aggregates.32-35
The large variety of potentially suitable tag structures has led to a surge in research related to their application in assays and imaging. In flow cytometry applications, individual SERS-tags may serve to both identify and signal the presence of an analyte or the occurrence of a binding event of interest and may also serve as the foundation for encoded capture beads.36
In short, SERS-based detection offers the opportunity to significantly advance in-flow multiplexing. The resultant technique presents a unique potential for ultra-sensitive molecular identification and analysis. However, while many of the fundamental building blocks are now available, there remain significant challenges to realizing in-flow Raman-based multiplexing. Its full exploitation requires effective full spectral data acquisition, which can only be achieved once several interlinked objectives are met. The instrumentation must possess sufficient sensitivity to both capture single nanoparticle SERS-tag spectra and yield the spectral resolution required to allow detailed analysis of all information encoded in a spectrum. Yet this sensitivity must be achieved with rapid analysis times (particles typically transit a flow cytometer’s laser in ~10 μs) in order to provide the high throughput demanded of flow cytometry. This, in turn, requires SERS-tags that are optimized both in terms of spectral brightness, and spectral diversity.
Despite the availability of many potential tag architectures, coupled with an understanding of key factors contributing to SERS signal strength and quality, the ability to batch engineer suitable structures with quantitative and consistent properties remains elusive. This is critical since flow cytometry examines individual tags, and not ensemble properties. Tag-to-tag variability typically includes differences in absolute signal intensity, which will limit applicability to quantitative assays. Peak-to-peak variations within the spectral signature, and features such as changing background intensities, may also disrupt fingerprint patterns. Fidelity must be preserved in these spectral signatures across all tags in a population to maintain confidence in the ability to use the fingerprint features to identify all targeted species in a given assay. Possible contributions to this behavior come from variations in dye loading between tags, as well as from differences in molecular orientation of the bound dyes, which may be due to either chemical or electrostatic variations between binding sites. Additionally, impurity species or photodegradation products from unstable dyes may contribute new features. Beyond chemical binding effects, factors determining the plasmonic response of the nanoparticle architectures also impact variability. These include existence of a size distribution for individual nanoparticles and differences in the relative dimensions of core and shell in multi-component particles. In the case of SERS-tags based on small nanoparticle aggregates, differences in the number of constituent particles and relative aggregation geometries determine the extent of electromagnetic “hot-spots” generated from interparticle interactions. Such geometric factors may also feed back into chemical effects such as different electrostatic behavior at dye binding sites. One recent development addressing this issue takes the approach of first self-assembling polymer-stabilized dimers and small clusters, followed by infusion of the SERS-active molecule.37
To the extent that these contributions to variability in SERS-tag response are controllable synthetically, improvements in SERS-tag synthesis will require a reliable method to obtain quantitative, statistically significant data addressing questions of SERS signal uniformity in ensembles of purportedly identically fabricated SERS substrates. In particular, rapid characterization of individual nanoparticle SERS-tags is required for direct evaluation of particle-to-particle variability. Ideally, such characterization will provide simultaneous correlated multi-parameter measurements of optical and geometric characteristics directly tied to SERS-tag performance, including fully-resolved spectral fingerprints, SERS brightness, and Rayleigh scattering efficiency. The results can be linked to specific preparation conditions to guide future synthesis. Such single-particle evaluation would also be invaluable in providing a meaningful direct comparison of the different nanostructures currently being developed as SERS-tags. Measurements at the single particle level also provide fundamental insight into intrinsic behaviors impacting SERS response (such as particle-dependent plasmon damping38,39
) that are often masked at the ensemble level. Beyond the clear impact such rapid measurements would have on further enabling of the broad range of SERS-tag applications, similar issues related to property variability are also important for a variety of nanostructured materials used in electronic and optical applications.
Current characterization methods for SERS-active nanoparticles are inefficient, normally interrogating immobilized particles one by one via single mode optical, electron, and/or scanning probe microscopies. Most studies present data for only a small number (typically not more than ~100) of nanoparticles, providing limited statistics for addressing issues of synthetic uniformity. Correlation of separate measurements on selected particles typically requires spatially registered sample deposition and serial application of the various measurement techniques. While these methods have provided important fundamental information, the lengthy data acquisition impedes useful feedback into particle development. Reliance on such static samples can also skew statistics by acquiring the combined responses of overlapping particles. While there are faster techniques for morphological characterization, such as dynamic light scattering, they produce at best a skewed average size and lack the ability to perform multiparameter measurements on the per particle basis needed for correlating SERS signal to the size and structure of a SERS-tag.
Recently, Laurence et al40
presented a method that represents a major step forward in solving these analysis problems. This work extends fluorescence23
and Raman correlation spectroscopies41,42
to investigate the scattering characteristics of solution-based SERS-tags diffusing through a confocal laser spectrometer. Histograms can be generated that correlate individual SERS-tag size, polarization response, Raman, Rayleigh, and continuum backgrounds for thousands of tags. While providing powerful information in a short time, the approach has two key unaddressed issues: (a) Detection is performed at only two wavelengths, allowing correlation of SERS intensity to background and scattering rates, but providing no evaluation of the uniformity of spectral signatures. (b) Analysis rates are limited by diffusion, while diffusing particles can theoretically pass through the beam multiple times resulting in potentially skewed statistics. One approach to addressing the latter issue is to perform the measurements in flow.1,2,43
We present here the successful convergence of two significant objectives, representing the realization of both high-throughput Raman-based multiplexed analysis and a method for the rapid nanoparticle characterization required to meet the needs of SERS-tag development. We describe a flow cytometer capable of Raman signal acquisition in analytically relevant rates required of current fluorescence-based cytometers. Instrument sensitivity allows collection of the full Raman fingerprint region with a 14 μs particle residence time. Furthermore, instrument sensitivity is attained without the need to bin data pixels in frequency space to artificially boost signal intensity, thus maintaining the highest possible spectral resolution. Significantly, this permits full access to the high information content encoded within the SERS-tag spectrum, while enabling assessment of the variability of not just tag brightness, but also fidelity of the spectral signature. Additionally, we demonstrate the capability of this instrument to obtain correlated multiparameter measurements on individual SERS-tags, which are used to characterize SERS-tag populations. The SERS signatures of several different tag types are evaluated over thousands of particles to correlate brightness and spectral variability with background and scattering behavior. Certain preparations show relative standard deviations in spectral response as low as 5%. In more variable populations, the correlated information provides clear discrimination between subpopulations of tags generated within a single preparation batch. We thus demonstrate an approach that enables both the quantitative statistical evaluation of SERS signal uniformity based on synthesis conditions and evaluation of SERS nanoparticles for use in cytometry. Furthermore, the flexibility of the resulting capability will be of general interest for extension to direct comparisons of competing SERS-tag architectures, exploration of a range of fundamental properties of plasmonic particles, and application to other nanoparticle types.