Ultrasensitive surface-enhanced Raman scattering flow detector using hydrodynamic focusing

Abstract

Label-free, chemical specific detection in flow is important for high throughput characterization of analytes in applications such as flow injection analysis, electrophoresis, and chromatography. We have developed a surface-enhanced Raman scattering (SERS) flow detector capable of ultrasensitive optical detection on the millisecond time scale. The device employs hydrodynamic focusing to improve SERS detection in a flow channel where a sheath flow confines analyte molecules eluted from a fused silica capillary over a planar SERS-active substrate. Increased analyte interactions with the SERS substrate significantly improve detection sensitivity. The performance of this flow detector was investigated using a combination of finite element simulations, fluorescence imaging, and Raman experiments. Computational fluid dynamics based on finite element analysis was used to optimize the flow conditions. The modeling indicates that a number of factors, such as the capillary dimensions and the ratio of the sheath flow to analyte flow rates, are critical for obtaining optimal results. Sample confinement resulting from the flow dynamics was confirmed using wide-field fluorescence imaging of rhodamine 6G (R6G). Raman experiments at different sheath flow rates showed increased sensitivity compared with the modeling predictions, suggesting increased adsorption. Using a 50 ms acquisition, a sheath flow rate of 180 μL/min, and a sample flow rate of 5 μL/min, a linear dynamic range from nanomolar to micromolar concentrations of R6G with a limit of detection (LOD) of 1 nM is observed. At low analyte concentrations, rapid analyte desorption is observed, enabling repeated and high-throughput SERS detection. The flow detector offers substantial advantages over conventional SERS-based assays such as minimal sample volumes and high detection efficiency.

Figure 1

Schematic representation of a COMSOL simulation (a) and a wide-field fluorescence image (b) showing the analyte eluting from the sample capillary under the influence of the surrounding sheath flow. COMSOL simulations (left panels) showing analyte concentration with corresponding wide-field fluorescence images (right panels) in the xy-plane are depicted at sheath flow rate to capillary flow rate ratios of 36:1 (c) and (d), 10:1 (e) and (f), and ~0 (g) and (h), respectively. The capillary flow rate was held constant. The dimensions and parameter ratios were kept identical for the fluorescence experiments and the COMSOL simulations. The concentration intensity scales from zero concentration (blue) to 1 mM concentration (red) in the COMSOL simulations. Scale bar = 75 micrometers.

Figure 2

(a) The flow cell and its components in the xz-plane, normal to the SERS substrate, are shown schematically. COMSOL simulations show the confinement and the predicted analyte concentration at different sheath flow to capillary flow rates. The sheath flow rate to capillary flow rate ratios depicted are: (b) 10:1, (c) 36:1, and (d) 72:1, respectively. The capillary flow rate was held constant. The concentration intensity scales from zero concentration (blue) to 1 mM concentration (red). Scale bar = 75 micrometers.

Figure 3

The areas of the Raman bands at 1174, 1306, 1357, 1506, and 1648 cm⁻¹ observed in the SERS spectrum of R6G are plotted as a function of sheath flow rate in the range from 0 to 360 μL/min. The capillary flow rate was held constant at 5 μL/min. Each data point represents the average area of a band taken from 1500 spectra consecutively acquired at 50 ms intervals. Error bars represent the standard deviation.

Figure 4

Spectra obtained with (A) and without (B) hydrodynamic focusing show the increased detection limit achieved with sample confinement. (a) Single SERS spectrum of a 10⁻⁵ M R6G solution, (b) average SERS spectrum of a 10⁻⁹ M R6G solution, and (c) background SERS spectrum collected using a 50 ms spectral acquisition and a sheath flow rate of 180 μL/min. (d) Single SERS spectrum of a 10⁻⁵ M R6G solution, (e) average SERS spectrum of a 10⁻⁶ M R6G solution, and (c) background SERS spectrum collected using a 50 ms spectral acquisition and by flowing the analyte in the flow cell at a flow rate of 150 μL/min. SERS spectra shown in (b) and (e) are averages of 10 individual, 50 ms SERS spectra. The dashed vertical lines in denote the five R6G bands used for analysis.

Figure 5

Log-log plot of the integrated SERS intensity as a function of R6G concentration in the range from 10⁻⁴ to 10⁻⁹ M at each Raman shift frequency. The lines are the fit to an exponential for the two different intensity profile. The inset (plotted in linear scale) shows a linear concentration dependence in integrated SERS intensity for R6G concentrations between 10⁻⁹ and 10⁻⁶ M. Each data point represents the average area of a band taken from 1500 spectra consecutively acquired using a 50 ms acquisition and a sheath flow rate of 180 μL/min. Error bars represent the standard deviation.

Figure 6

(A) The heatmap shows the observed SERS intensity at each Raman shift as a function of acquisition time for a 10⁻⁵ M R6G solution during an experiment where the analyte (1) travels through the capillary, (2) is eluted onto the SERS substrate, (3) analyte flow is stopped and a 0.1M NaOH solution is exchanged for the sample, (4) residual analyte in the capillary elutes, and (5) the NaOH solution is eluted. (B) The SERS intensity profile of a single Raman band (1357 cm⁻¹) as a function of acquisition time is shown, along with the on (Δt_m) and off (Δt_−m) times at which adsorption and desorption of R6G take place during the course of the experiment. Of note, the length of segments 1 and 4 are similar, illustrating the time to displace the capillary volume. Spectra were recorded using a 50 ms acquisition and a sheath flow rate of 180 μL/min.