Modern metal nanoparticle (NP) synthesis techniques have enabled the development of nano-scale materials with unique optical properties for a wide range of devices and applications, including biomolecule detection1–4
and cancer therapeutics.5,6
A barrier to exploiting the unique structure-dependent optical properties of NPs comes from difficulty in controlling particle size and shape. NP characterization techniques are diverse and include spectroscopy, imaging, and structural measurements, and most often a number of complementary techniques are used to correlate NP structure and functional properties. These techniques typically fall into one of two categories: ensemble techniques, which are quick, but unable to distinguish subpopulations within a sample, and single NP characterization techniques, which reveal sample heterogeneity but are typically slow and labor intensive.
Single particle characterization is particularly important for applications that use NP tags for biosensing, where “dim” tags can reduce sensitivity by occupying binding sites while contributing negligibly to the detectable signal. Dark field microscopy, which has excellent spectral resolution and sensitivity, has enabled the measurement of optical properties of single NPs arrayed on a substrate and has become a popular characterization tool,4,7–10
although the serial measurement of enough particles for a useful statistical analysis is very slow, with analysis rates on the order of 1 particle per minute. Recently, a faster implementation of this approach has been presented that increases the speed with which single particles are analyzed by 1–2 orders of magnitude,9
while still requiring deposition of particles on a substrate. This technique allows analysis rates of up to 1–2 NPs per second with ~1.5% coincidence,9
which occurs when two or more particles are located within a diffraction-limited spot and the individual NP spectra cannot be resolved. The throughput of this technique depends upon both sample integration time and sample preparation, with more populated samples resulting in higher throughput, but also in a higher frequency of coincidence.
Another approach uses correlation spectroscopy to measure Rayleigh and Raman scattering of single NPs in solution at specific wavelengths as they diffuse into and out of a laser beam.11
This method characterizes selected optical properties as well as NP translational and rotational diffusive properties11–13
but offers limited spectral resolution as band-pass filters are used to select a spectral window and the intensity of the spectral band measured using avalanche photodiodes. This technique allows solution phase NP analysis, but the event rate and collection time depend upon the speed at which the sample diffuses into and out of the laser spot. This may inadvertently predispose the analysis toward faster diffusing particle populations and requires changing the medium viscosity to reduce NP diffusivity and increase sample integration time.12
Thus, while both dark field and correlation spectroscopy can be extremely useful for specific applications such as monitoring NP growth9
and chemical reactions that occur on a NP surface10
(dark field microscopy), or simultaneously monitoring NP optical and diffusive properties11–13
(correlation spectroscopy), a general method for making high throughput and high resolution spectral measurements of individual nanoparticles is still lacking.
We are interested in the development of surface-enhanced resonant Raman scattering (SERRS) tags for cytometry,14–16
where spectrally distinct tags enable multiplexed and multiparameter cell measurements.17–19
Fluorescent tags are most often used to label antibodies or other molecules, but the broad emission spectra of fluorophores limit the number of different labels that can be used simultaneously. The narrow spectral features of Raman scattering present the potential for highly multiparameter measurements and are attractive for many applications including flow and image cytomety14,20–23
and microarray-based sensing.1,24
Surface-enhanced Raman scattering (SERS) is a special case of Raman scattering where the interactions of a Raman-active compound (usually a chromophore) with the high electric fields of a plasmon resonant NP or roughened metal surface result in Raman scattering intensity enhancements of many orders of magnitude.25,26
Maximum enhancements are expected when the excitation laser is coresonant with both the chromophore absorption and the NP plasmon resonance, a case known as surface-enhanced resonant Raman scattering (SERRS). The engineering of SERS and SERRS tags has been a subject of much interest,27–29
with a number of groups making significant progress.16,20,30–36
However, given the number of criteria which can effect SERRS tag optical properties (including NP size, shape, number of particles per tag, and plasmon resonance frequency, among others), it is not uncommon to observe significant Raman scattering intensity variations within a sample. While analyzing thousands of tags from multiple preparations might take hours or days using dark field microscopy, here we demonstrate a high throughput single particle characterization technique capable of measuring thousands of NPs in just a few minutes.
The high throughput Raman flow spectrometer employs hydrodynamic focusing of the sample in a sheath flow cuvette to make simultaneous measurements of NP Rayleigh and Raman light scatter. NPs in the sample pass through the center of the ~4 pL probe volume defined by the dimensions of the laser beam, sample stream, and focal spot of the collection optics (). Using a small probe volume provides advantages such as reducing the background signal from system autofluorescence and the probability of having multiple nanoparticles in the probe volume simultaneously. The transit time of a NP within the beam depends primarily upon the laser beam spot height and the sheath fluid linear flow rate. Elastically scattered light is measured using a PMT and triggers acquisition of Raman scattering spectra by an EMCCD attached to an imaging spectrograph (). By using a highly efficient light collection setup, an EMCCD for spectral collection, and a sensitive PMT, we are able to collect high resolution Raman spectra of individual SERRS tags with signal integration times of only 300 μs ().
Figure 1 Single nanoparticle flow spectrometer. (a) Sample is hydrodynamically focused to flow through a focused laser beam. Note that the figure is not to scale. (b) Schematic of the Raman flow spectrometer setup. Refer to the Materials and Methods section for (more ...)
The NP analysis rate depends upon NP concentration, sample and sheath stream flow rates, and the response time of the detector. In conventional flow cytometry using PMTs, signal integration times of a few microseconds enable particle analysis rates of tens of thousands per second. Our system uses a CCD detector that can capture spectra from single particles with exposure times as short as 10 μs;37
however, the readout speed of the CCD limits the event rate to few hundred events per second at most. Beyond these hardware limitations, the single particle analysis rate is limited by particle concentration and, ultimately, coincidence from NPs that just happen to pass through the probe volume simultaneously.
The expected frequency of coincidence can be calculated from the laser beam height and the particle concentration (Supporting Information, Figures S1 and S2
), and the contribution of coincidence can be measured through a simple sample dilution experiment. Data collected from NP samples at a variety of dilutions (Supporting Information, Figure S3
) demonstrated that at high concentrations the mean scattering intensity was elevated due to a significant number of coincident events, which can be identified as a shoulder on the Rayleigh scattering frequency histogram. Lower concentration samples exhibited minimal coincidence, evidenced by the absence of a histogram shoulder and consistent scattering intensity measured across a range of dilutions. Because the sample volumetric flow rate through the measurement probe volume is known, the event rate can be used to calculate the NP concentration in the sample and to validate theoretical estimates of expected coincidence. For the data presented here, samples were analyzed at concentrations that resulted in event rates of 5–6 NPs/s and coincidence rates of less than 0.5% for the sheath and sample flow rates and laser beam height used. In many cases, especially where coincident events can be accurately identified (for example, on the basis of Rayleigh scatter) and excluded from analysis, higher concentrations and analysis rates can be employed.
Flow spectroscopy offers many of the benefits of dark field microscopy (control of signal integration time, high spectral resolution) while offering the ability to interrogate samples in solution and easily control the measurement rate. Low resolution flow-based methods have been used for NP Rayleigh scattering38
and size analysis,39
and NPs have been used as Rayleigh and Raman scattering tags on beads and cells for low40
resolution flow spectroscopy methods. However, to the best of our knowledge, this work is the first to report a high throughput high spectral resolution flow spectroscopy instrument with single NP sensitivity.
We are using this single NP approach to optimize the preparation of SERRS scattering tags for cellular and molecular measurement applications. As an example, we have prepared SERRS tags for 647 nm excitation from gold nanoshells composed of a 200 nm SiO2
core and an approximately 30–40 nm gold layer. Nanoshells have highly tunable resonances41
and high electric-field enhancements, making them ideal SERS substrates.42
Nanoshells were incubated with Raman-active chromophores oxazine 170, thionin, or malachite green, prior to depositing silver on the nanoshell surface using a commercially available silver enhancement kit. Finally, bovine serum albumin was added to encapsulate the tags and promote stability. An ensemble SERRS spectrum from oxazine 170 SERRS tags is presented in .
Figure 2 Ensemble and flow spectroscopy analysis of nanoshell SERRS tags. (a) Ensemble oxazine 170 SERRS tag Raman spectrum from tags prepared with 2 μL of silver enhancement solution. Spectrum was collected from a 0.9 pM nanoshell solution (5.5 × (more ...)
Flow spectroscopy enables the simultaneous measurement of Rayleigh and Raman scattering from individual tags and, unlike bulk spectroscopy, allows us to distinguish SERRS tag preparations that produce a high percentage of Raman-active tags from those that produce a small number of extremely bright tags. Data were collected from approximately 5000 individual tags for seven oxazine 170 SERRS tag preparations. displays Rayleigh and Raman (background-corrected integrated intensity from 559 to 615 cm−1) scattering data from a single SERRS tag preparation binned in frequency histograms plotting scattering intensity versus event frequency, as well as in a bivariate plot in which correlated Rayleigh and Raman scattering signals from individual tags are displayed. Using this method, tag subpopulations can be distinguished based upon their optical properties. In this example, we can define three subpopulations: (I) tags that do not exhibit Raman scattering; (II) Raman-active tags that have Rayleigh scattering intensities similar to bare nanoshells; and (III) Raman-active tags that exhibit significantly increased Rayleigh scattering and may be multishell clusters. Four representative single tag spectra from each population are displayed, and as expected, spectra from subpopulation I display no Raman scattering, while spectra from subpopulations II and III display Raman scattering of varying intensities. Comparison of the spectra from subpopulations II and III implies a positive correlation between Rayleigh and Raman scattering intensity, further suggesting that the tags in subpopulation III (<2% of all tags) may be multishell clusters.
Having demonstrated that flow spectroscopy can be used as a high throughput tool for the characterization of individual NPs, we used this method to guide the development of bright and uniform SERRS tags. SERRS tags were prepared with varying volumes of silver enhancement solution to produce tags with silver layers of varying thickness. The Rayleigh scattering intensities of the SERRS tags and bare nanoshells were found to be similar, suggesting that the preparation technique causes minimal aggregation, a hypothesis that was confirmed using transmission electron microscopy (TEM, Supporting Information, Figure S4
). displays overlaid Raman scattering intensity histograms from selected SERRS tag preparations with silver layers of varying thickness, controlled by varying the volume of silver enhancement solution (0, 3, or 5 μL) used during the silver plating process. The histograms display Raman-active and -inactive populations and demonstrate that, in the absence of silver plating, fewer than 20% of the tags exhibit detectable Raman scattering signals (). Initially, plating of a silver layer increases the number and intensity of the Raman-active tags; however, if the silver layer is too thick due to the addition of too much silver enhancement solution, the percentage of active tags and Raman scattering intensity decrease. Additionally, bivariate density plots, which have been colored to convey information about the number of events at each location, can be used to portray information about the Rayleigh and Raman scattering intensity of each sample (. These plots are a useful way to view the main populations of each tag preparation. The majority of the tags with no silver plating are Raman-inactive and have relatively low Rayleigh scattering intensities, as seen in the majority population (depicted in red) in . The main population of tags prepared with 3 μL of silver enhancement solution have tightly clustered Rayleigh and Raman scattering intensities (represented by the orange/red cluster, ), while tags prepared with 5 μL of silver enhancement solution display increased variation in both Rayleigh and Raman scattering. The tight clustering of the events in demonstrates the relative uniformity of this SERRS tag preparation, making these tags superior for biosensing applications.
Figure 3 SERRS tag population analysis. (a) Histograms of Raman scattering intensity from three SERRS tag preparations prepared with 0, 3, and 5 μL of silver enhancement solution. Illustrations display the relative sizes of the silica core (gray), gold (more ...)
Flow spectroscopy was used to determine the optimal volume of silver enhancement solution by comparing the Raman scattering intensity and percentage of Raman-active tags for seven SERRS tag preparations (). Both the mean Raman scattering intensity and percentage of Raman-active tags varied with the thickness of the silver layer. Tags prepared with 2–4 μL of silver enhancement solution, which have silver layers ranging from approximately 35 to 70 nm thick (as measured by TEM, , and Supporting Information Figure S5
and Table S1
) appear optimal, as they exhibit high Raman scattering intensities and produce greater than 80% Raman-active tags. Variations in SERRS tag brightness and yield may occur due to changes in the effective surface roughness or enhanced electric fields caused as the excitation of the gold nanoshell plasmon induces oscillations of the silver conduction electrons, as previously reported for lithographically templated disks and wires.43
Thicker silver layers may reduce the Raman signal due to changes in the location of the nanoshell resonance or a decrease in surface roughness.
Figure 4 Analysis of SERRS tag preparations. (a) Mean Raman scattering intensity of Raman-active tags (red squares) for each tag preparation and the percentage of Raman-active tags (black circles) were found to vary with the thickness of the tag silver layer. (more ...)
It should not be overlooked that flow spectroscopy can also be used to measure NP concentration. While NP concentrations are typically calculated using an extinction spectroscopy measurement and an estimated molar extinction coefficient, the sensitivity of the plasmon resonance to NP size, shape, surface chemistry, and dielectric medium can introduce errors into these calculations. As demonstrated by the linear relationship between event rate and NP concentration (Supporting Information, Figure S3
), it is possible to explicitly measure NP concentration by counting the number of NPs in a given volume, as long as all NPs have scattering intensities that are above the sensitivity threshold of the instrument.
Progress in the development and application of nanoengineered systems is limited by the speed and precision of quantitative measurement techniques. For the engineering of NP-based systems, single NP measurements are an essential, but rate-limiting, need. The rapid analysis of individual NPs in a flowing sample stream represents a powerful tool to address this need. Like conventional flow cytometry of cells, flow spectroscopy of NPs enables correlated, multiparameter optical analysis of individual particles at high serial measurement rates. Unlike conventional flow cytometry, which uses optical filters and point detectors to measure integrated intensities from selected wavelength ranges, our flow spectroscopy approach enables high spectral resolution suitable for the analysis of Raman scattering spectra. NP-based SERRS tags with narrow spectral features have the potential to enable significantly increased levels of multiplexing for measurements of cells and molecules, but brighter and more uniform tags are required. These systems have an inherent dispersity in their structural and optical properties that require some amount of empirical optimization to minimize. Rapid measurements of individual NP-based SERRS tags will be an essential tool for developing uniform tags, and the flow spectroscopy-based approach presented here provides that tool. Moreover, the high throughput and high resolution NP analysis capabilities of this approach should find application in many other areas of nanoengineering.