High-resolution spectral analysis of individual SERS-active nanoparticles in flow

Abstract

Nanoparticle spectroscopic tags based on surface enhanced Raman scattering (SERS) are playing an increasingly important role in bioassay and imaging applications. The ability to rapidly characterize large populations of such tags spectroscopically in a high-throughput flow-based platform will open new areas for their application and provide new tools for advancing their development. We demonstrate here a high-resolution spectral flow cytometer capable of acquiring Raman spectra of individual SERS-tags at flow rates of hundreds of particles per second, while maintaining the spectral resolution required to make full use of the detailed information encoded in the Raman signature for advanced multiplexing needs. The approach allows multiple optical parameters to be acquired simultaneously over thousands of individual nanoparticle tags. Characteristics such as tag size, brightness, and spectral uniformity are correlated on a per-particle basis. The tags evaluated here display highly uniform spectral signatures, but with greater variability in brightness. Subpopulations in the SERS response, not apparent in ensemble measurements, are also shown to exist. Relating tag variability to synthesis parameters makes flow-based spectral characterization a powerful tool for advancing particle development through its ability to provide rapid feedback on strategies aimed at constraining desired tag properties. Evidence for single-tag signal saturation at high excitation power densities is also shown, suggesting a role for high-throughput investigation of fundamental properties of the SERS tags as well.

Figure 1

(a) Top-view schematic of Raman full-spectral flow cytometer experimental apparatus with grating-spectrograph dispersive element. Flip mirrors are used to allow selection of krypton or argon laser excitation and 488 nm or diode laser (532 or 635 nm) for the trigger (TRG) laser. DCSP: dichroic short pass filter, SSC: side scatter PMT detector, FSC: forward scatter photodiode detector. (b) Sideview schematic of flow cell showing spatial separation of trigger and interrogation lasers. Sample stream (dark central region) is hydrodynamically focused by the surrounding sheath flow (lighter colored region). (c) Diagram of the high-sensitivity cytometer optics breadboard, flow cell, and sample holder. H, half wave plate; P, polarizer; S, shutter; M, mirror; FL, focusing lens; CL, collection lens; F1, filter 1; F2, filter 2; PMT photomultiplier tube and APD avalanche photodiode.

Figure 2

(a) Bulk spectra of oxazine 170 (Ox170), styryl 9M and safranine (Saf) SERS-tags taken with 0.5 mW of 514 nm or 488 nm (safranine only) excitation, 10 s integration times. (b) Analogous spectra taken in flow on a single SERS-tag particle with 50 mW of 514 nm or 488 nm (safranine only) excitation, 20 μs integration times.

Figure 3

Plot of spectra from 1000 single SERS-tags, taken with 50 mW of 514 nm excitation from: (a) Oxazine 170 tags; (b) Styryl 9M tags; (c) safranine tags (with 50 mW of 488 nm excitation). Integration time of 20 μs for each spectrum. A background spectrum from PBS has been subtracted from each single-tag spectrum.

Figure 4

Pixel-by-pixel percent standard deviations of particle spectral variability for each of the three SERS-tag populations of Figure 3. Plots are offset for clarity.

Figure 5

Correlated Raman intensities at different frequencies for 1000 individual SERS-tag particles. (a) Oxazine 170, 1638 cm⁻¹ peak compared to 1706 cm⁻¹ background (blue) and 595 cm⁻¹ peak compared to 642 cm⁻¹ background (red); (b) Safranine, 1541 cm⁻¹ peak compared to 1583 cm⁻¹ background (blue) and 598 cm⁻¹ peak compared to 534 cm⁻¹ background (red); (c) Safranine, 1541 cm⁻¹ peak compared to 598 cm⁻¹ peak. Small subpopulations in behavior are highlighted by the dashed rectangles. Peak intensities are background-subtracted. Data extracted from the spectra of Figures 3a and 3c.

Figure 6

Bivariate histograms of (a) ~100 nm beads and (b–d) silver nanoparticle aggregates. Each histogram represents roughly 10,000 individual particles. Color contours represent particle counts, with red corresponding to fewer/individual particles and blue corresponding to greatest number of particles. (a) 100 nm fluorescent microspheres. (b) Oxazine 170. (c) Styryl 9M. (d) Safranine. Samples were interrogated at 532 nm, with 1 mW of power, and 250 μs integration time per particle.

Figure 7

Bivariate histogram of approximately 10,000, 100 nm silver nanoparticle aggregates from independent preparations. Color contours represent particle counts as per Figure 6. (a) Unconjugated. (b) With Safranine dye attached to surface of nanoparticle. Excitation with 1 mW at 532 nm, 250 μs integration time per particle.

Figure 8

Univariate histograms of: (a) Side scatter intensity; and (b) SERS intensity for approximately 8,000 safranine-labeled SERS-tags as a function of three different laser power levels: 1 mW (black), 1.5 mW (red), 3 mW (blue). For comparison, bivariate histograms for the same particle sets showing light scatter versus Raman signal for: (c) 1 mW laser power; (d) 3 mW laser power. Excitation at 532 nm, 250 μs integration time per particle. Color contours represent particle counts as per Figure 6.