Multiplexing potential of NIR resonant and non-resonant Raman reporters for bio-imaging applications

Abstract

Multiplexed imaging, which allows for the interrogation of multiple molecular features simultaneously, is vital for addressing numerous challenges across biomedicine. Optically unique surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to serve as a vehicle to achieve highly multiplexed imaging in a single acquisition, which is non-destructive, quantitative, and simple to execute. When using laser excitation at 785 nm, which allows for a lower background from biological tissues, near infrared (NIR) dyes can be used as Raman reporters to provide high Raman signal intensity due to the resonance effect. This class of imaging agents are known as surface-enhanced resonance Raman scattering (SERRS) NPs. Investigators have predominantly utilized two classes of Raman reporters in their nanoparticle constructs for use in biomedical applications: NIR-resonant and non-resonant Raman reporters. Herein, we investigate the multiplexing potential of five non-resonant SERS: BPE, 44DP, PTT, PODT, and BMMBP, and five NIR resonant SERRS NP flavors with heptamethine cyanine dyes: DTTC, IR-770, IR-780, IR-792, and IR-797, which have been extensively used for biomedical imaging applications. Although SERRS NPs display high Raman intensities, due to their resonance properties, we observed that non-resonant SERS NP concentrations can be quantitated by the intensity of their unique emissions with higher accuracy. Spectral unmixing of five-plex mixtures revealed that the studied non-resonant SERS NPs maintain their detection limits more robustly as compared to the NIR resonant SERRS NP flavors when introducing more components into a mixture.

Fig. 1

Scheme of SERRS NP preparation.

Fig. 2

Optical properties of 10 SERS-active NP flavors. Raman spectra of SERRS NP flavors, each bearing spectral features of DTTC, IR-775, IR-780, IR-792, and IR-797, with respective DFT-calculated Raman spectra of the NIR dyes (in gray). Chemical structures and optical properties (excitation and emission) with the identification of ${\lambda }_{\mathsf{ex}}$ 785 nm used for Raman measurements. Raman spectra of SERS NP flavors, each bearing spectral features of BPE, BMMBP, PODT, PTT, and 44DP, with respective DFT-calculated Raman spectra of the non-resonant Raman reporters (in gray). The DFT spectra were simulated for the Raman reporters attached to a Au₂₀ cluster with B3LYP/6-311++G(d,p) level of theory. For the baseline corrected Raman spectra of SERRS and SERS NP flavors with spectral background subtracted, see ESI Fig. S2-S6.

Fig. 3

Multiplexing compatibility of SERS and SERRS NPs. (a) Correlation matrices built from the spectra of SERS NPs (a) and SERRS NPs, (b) demonstrating similarity among each type of flavors. The color bar indicates the level of fitting signals, where 1 (yellow) means 100% spectral overlap of two flavors and 0 (dark blue) means 50% spectral overlap of two flavors. (c) The lowest (well-conditioned subsets) and the highest (ill-conditioned subsets) condition numbers for different plexities of SERS and SERRS NP flavors (see ESI Table S8†). A lower condition number is preferred to achieve easier unmixing of the NP subsets and higher plexity imaging. Notice how both the ill- and well-conditioned subsets of NP mixtures maintain a low condition number for the SERS NPs as opposed to the SERRS NPs. For deriving the condition number for each combination, we used the normalized reference spectra of SERS and SERRS NPs without baseline subtraction.

Fig. 4

Sensitivity comparison of SERRS versus SERS NPs in Raman imaging experiments. The average Raman spectra with standard deviation (shaded area, n = 500) for (a) 37.5 pM SERRS NPs (labeled with DTTC, IR-775, IR-780, IR-792, or IR-797) and (b) 37.5 pM SERS NPs (labeled with BPE, BMMBP, PODT, PTT, or 44DP). Raman imaging channels for the dilution series of SERRS NPs (a) and SERS NPs (b) starting from 150 pM with 2× dilution steps. Scale bars represent 5 mm. Scheme of nanoparticle concentrations applied onto the coffee filter paper substrate (c). Limits of detection for SERS and SERRS NPs (d).

Fig. 5

Spectral unmixing accuracy. The estimated ratios of the five-color SERS (a) and SERRS NPs (b) from the spectral unmixing, which were normalized to the average concentration of the BPE-labeled and DTTC NPs, respectively, and the average Raman spectra of the nanoparticle mixtures with standard deviation (shaded area, n = 500). The SERS NPs were mixed with a 0 : 1: 2 : 3 : 4 ratio of PODT, BPE, 44DP, PTT, and BMMBP-labeled NPs, respectively. The SERRS NPs were mixed with a 0 : 1 : 2 : 3 : 4 ratio of IR-780, IR-792, DTTC, IR-775, and IR797-labeled NPs, respectively. (c) Raman imaging channels for the dilution series of DTTC-labeled NPs in the presence of 37.5 pM IR-775, IR-792, IR-780, and IR-797-labeled NPs, and BPE-labeled NPs in the presence of 37.5 pM BMMBP, PODT, PTT, and 44DP-labeled NPs starting from 150 pM with 2× dilution steps. Scale bars represent 5 mm. The average Raman spectra of the nanoparticle mixtures with standard deviation (shaded area, n = 500) for BPE- and DTTC-labeled NPs, both at 37.5 pM concentration. (c) Unmixed weight values for BPE-labeled NPs in the presence of 37.5 pM BMMBP, PODT, PTT, and 44DP-labeled NPs and DTTC-labeled NPs in the presence of 37.5 pM IR-775, IR-792, IR-780, and IR-797-labeled NPs, along with the LOD concentration for each NP flavor in 1-plex, which can be used as a ground rule for assessing the impact of multiplexing.