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

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.

Self-assembly approach to multiplexed surface-enhanced Raman spectral-encoder beads.

We present a strategy for the synthesis of multiplexed spectral encoder beads based on combinations of different surface enhanced Raman (SERS) signatures generated by dye-functionalized Ag nanoparticle tags. A key problem in SERS-based multiplexing arises in balancing the competitive binding of different signal generating dyes to the nanoparticle surfaces, which leads to difficulty in generating final summation spectra by design. We avoid this complication by decoupling the formation of individual tags from multiplexing of their spectra by self-assembly of different tag combinations onto SiO(2) microbead supports via biotin-avidin binding. Linear combinations of individual nanoparticle tag spectra are generated in precursor solutions and are found to directly translate to the final encoder bead fingerprint spectrum in a 1:1 binding stoichiometry that preserves the original solution ratios. The result is an ability to multiplex spectral signatures in both frequency and intensity space to generate a large number of unique encoder signatures from a limited number of initial tag spectra. Raman microscopy of 75 individual beads shows that spectral response is highly uniform from bead-to-bead, making the encoder assemblies suitable for highly multiplexed bioassay applications and as model systems for cellular surface labeling studies for imaging and immunoassays.

Spectral measurements of large particles by flow cytometry.

Flow cytometers designed to analyze large particles are enabling new applications in biology and chemistry. Similarly, flow spectroscopy approaches are extending the capabilities of the flow cytometry platform. Here, we report on the adaptation of a commercial large particle analyzer to measure fluorescence and Raman spectra of individual particles at high speeds. We modified a Union Biometrica COPAS Plus instrument to allow red excitation and optical fiber-based light collection and spectral analysis using a spectrograph and CCD array detector. These modifications did not compromise the ability of the instrument to resolve different sized particles based on their extinction and time of flight signals. The modified instrument has the sensitivity and spectral resolution to measure the fluorescence and Raman signals from individual particles with signal integration times of 10 usec. The high speed spectral analysis of individual particles in flow will enable new applications in biological and chemical analyses.

A flow cytometer for the measurement of Raman spectra.

Multiparameter measurements in flow cytometry are limited by the broad emission spectra of fluorescent labels. By contrast, Raman spectra are notable for their narrow spectral features. To increase the multiparameter analysis capabilities of flow cytometry, we investigated the possibility of measuring Raman signals in a flow cytometry-based system. We constructed a Raman Spectral Flow Cytometer, substituting a spectrograph and CCD detector for the traditional mirrors, optical filters, and photomultiplier tubes. Excitation at 633 nm was provided by a HeNe laser, and forward-angle light scatter is used to trigger acquisition of complete spectra from individual particles. Microspheres were labeled with nanoparticle surface enhanced Raman scattering (SERS) tags and measured using the RSFC. Fluorescence and Raman spectra from labeled microspheres were acquired using the Raman Spectral Flow Cytometer. SERS spectral intensities were dependent on integration time, laser power, and detector pixel binning. Spectra from particles labeled with one each of four different SERS tags could be distinguished by either a virtual bandpass approach using commercial flow cytometry data analysis software or by principal component analysis. Raman flow cytometry opens up new possibilities for highly multiparameter and multiplexed measurements of cells and other particles using a simple optical design and a single detector and light source.

A controlled and reproducible pathway to dye-tagged, encapsulated silver nanoparticles as substrates for SERS multiplexing.

Silver nanoparticles tagged with dyes and encapsulated within a silica layer, offer a convenient potential substrate for performing multiplexed surface-enhanced Raman scattering (SERS) analysis. In contrast to our earlier work with gold particles, aggregation of silver particles is found to be mostly independent of dye addition, allowing for a reproducible preparation in which aggregation is actively induced by the addition of NaCl. Separating the aggregation step eliminates competitive binding between the dyes and silica-coating reagents, enabling the efficient use of a wide variety of weakly binding dyes to conveniently generate robust, high-intensity SERS substrates at a variety of excitation frequencies.

Optimization of the preparation of glass-coated, dye-tagged metal nanoparticles as SERS substrates.

Dye-tagged metal nanoparticles are of significant interest in SERS-based sensitive detection applications. Coating these particles in glass results in an inert spectral tag that can be used in applications such as flow cytometry with significant multiplexing potential. Maximizing the SERS signal obtainable from these particles requires care in partitioning available nanoparticle surface area (binding sites) between the SERS dyes and the functionalized silanes necessary for anchoring the glass coating. In this article, we use the metal-mediated fluorescence quenching of SERS dyes to measure surface areas occupied by both dyes and silanes and thus examine SERS intensities as a function of both dye and silane loading. Notably, we find that increased surface occupation by silane increases the aggregative power of added dye but that decreasing the silane coverage allows a greater surface concentration of dye. Both effects increase the SERS intensity, but obtaining the optimum SERS intensity will require balancing aggregation against surface dye concentration.

Reversible fluorescence quenching in carbon nanotubes for biomolecular sensing.

Biosensing applications of single-walled carbon nanotubes have been demonstrated in solid-state device structures. Bioanalyte sensing schemes based on coupling of reversible nanotube fluorescence quenching to redox reactions paired to enzymatic peroxide generation have also been pursued. Here we show a new approach to highly sensitive nanotube-based optical sensing. Single-walled carbon nanotubes interacting with dye-ligand conjugates--a redox-active dye molecule that is covalently bound to a biological receptor ligand (such as biotin in this case)--showed fluorescence quenching. Further interaction between the receptor ligand on the conjugates and target analytes (avidin in this case) induced the recovery of the quenched fluorescence, forming the basis of the sensing scheme. Nanomolar sensitivity was attained with high specificity for the target analyte. This is a versatile approach because a wide range of conjugation possibilities exists between the potential receptors and redox quenchers.

Thiol-functionalized, 1.5-nm gold nanoparticles through ligand exchange reactions: scope and mechanism of ligand exchange.

Ligand exchange reactions of 1.5-nm triphenylphosphine-stabilized nanoparticles with omega-functionalized thiols provides a versatile approach to functionalized, 1.5-nm gold nanoparticles from a single precursor. We describe the broad scope of this method and the first mechanistic investigation of thiol-for-phosphine ligand exchanges. The method is convenient and practical and tolerates a surprisingly wide variety of technologically important functional groups while producing very stable nanoparticles that essentially preserve the small core size and size dispersity of the precursor particle. The mechanistic studies reveal a novel three-stage mechanism that can be used to control the extent of ligand exchange. During the first stage of the exchange, AuCl(PPh3) is liberated, followed by replacement of the remaining phosphine ligands as PPh3 (assisted by gold complexes in solution). The final stage involves completion and reorganization of the thiol-based ligand shell.