Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles

Liu JTC (2013) Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles. PLoS ONE 8(4): e62084. doi:10.1371/journal.pone.0062084 Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles Steven Y. Leigh 0 Madhura Som 0 Jonathan T. C. Liu 0 James P. Brody, University of California, Irvine, United States of America 0 Stony Brook University (SUNY), Department of Biomedical Engineering , Stony Brook, New York , United States of America Surface-enhanced Raman scattering (SERS) nanoparticles have been engineered to generate unique fingerprint spectra and are potentially useful as bright contrast agents for molecular diagnostics. One promising strategy for biomedical diagnostics and imaging is to functionalize various particle types (''flavors''), each emitting a unique spectral signature, to target a large multiplexed panel of molecular biomarkers. While SERS particles emit narrow spectral features that allow them to be easily separable under ideal conditions, the presence of competing noise sources and background signals such as detector noise, laser background, and autofluorescence confounds the reliability of demultiplexing algorithms. Results obtained during time-constrained in vivo imaging experiments may not be reproducible or accurate. Therefore, our goal is to provide experimentalists with a metric that may be monitored to enforce a desired bound on accuracy within a user-defined confidence level. We have defined a spectral reliability index (SRI), based on the output of a direct classical least-squares (DCLS) demultiplexing routine, which provides a measure of the reliability of the computed nanoparticle concentrations and ratios. We present simulations and experiments to demonstrate the feasibility of this strategy, which can potentially be utilized for a range of instruments and biomedical applications involving multiplexed SERS nanoparticles. - Funding: This research was supported by the Office of the Vice President for Research at Stony Brook University, the United States National Institutes of Health (NIH)/NIBIB Pathway to Independence award R00EB008557 (J.T.C. Liu) and R21EB015016 (Liu) from NIH/NIBIB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The field of biomedical optics has traditionally approached disease detection by deducing tissue status through the measurement of optical signals generated either intrinsically by tissue constituents [112] or extrinsically by targeted probes with known signatures [1323]. In particular, diagnostic approaches involving both intrinsic and extrinsic Raman scattering have seen much success owing to the sharp separable features of Raman spectra. Intrinsic Raman spectroscopy has been demonstrated to identify malignant tissues with high sensitivity and specificity [2,6,8,10] and has also enjoyed the benefit of expedited regulatory approval since no external contrast agent is necessary. [8,9,11,12]. However, the spectral features of diagnostic value in intrinsic Raman detection are generated by highly conserved chemical constituents such as hydrocarbon, lipid, or nucleic acid bonds, which may be difficult to relate to pathological or clinical variables. Additionally, the acquisition time for intrinsic Raman spectroscopy is necessarily long given the low efficiency of Raman scattering and therefore presents a practical challenge for clinical diagnostics and imaging. Recently, a number of groups [1319,21,23] have explored the use of surface-enhanced Raman-scattering (SERS) nanoparticles that are engineered to emit bright distinct spectra (Fig. 1). These Raman-coded nanoparticles are available as different flavors, each emitting a characteristic spectral signature that may potentially be targeted against various molecular biomarkers for highly multiplexed molecular imaging. However, determining the reliability of particle concentrations or concentration ratios computed by spectral demultiplexing algorithms is a non-trivial challenge facing experimentalists since other signals such as detector noise, incomplete removal of excitation photons (laser background) and autofluorescence background degrade the accuracy of demultiplexing routines, especially as spectral overlap becomes a concern with increasing numbers of flavors competing within a limited spectral bandwidth. Therefore, in this report, we propose a general method for quantifying the reliability of particle concentrations and ratios that are computed from a least-squares demultiplexing algorithm. First, this method is developed through numerical simulations of various mixtures of SERS nanoparticles in the context of noise and background signals. We define a metric, the spectral reliability index (SRI), which serves as a predictor of error in single- and multi-flavor applications. We further provide results from wellcontrolled experiments to assess the feasibility and accuracy of our approach. While initial experiments are intentionally simplified to verify the accuracy and reproducibility of these methods, our strategy could potentially be of value for a range of technologies that utilize targeted SERS-based nanoparticles to provide multiplexed measurements of molecular biomarkers both in vitro and in vivo. Figure 1. Surface-enhanced Raman scattering (SERS) nanoparticles. (a) Multiple flavors of nanoparticles exist where each nanoparticle contains a gold core coated with a Raman-active layer, encased in a silica shell. (b) Raman spectra of five nanoparticle flavors. (c) Example result from a least-squares routine showing the ability to demultiplex two different nanoparticles from a mixture under noisy conditions. doi:10.1371/journal.pone.0062084.g001 Since our ultimate goal is to provide experimentalists with a reliable measure of nanoparticle concentrations or concentration ratios, we set out to simulate spectral measurements of SERS nanoparticles under the varying noise and background conditions that may be encountered experimentally. Simulated spectra were generated first for single-flavor applications, then for two-particle applications where the relative concentrations between particle flavors ranged from 1:1 to 5:1, and finally for two-flavor ratios within three-flavor mixtures. The rationale for a 5:1 maximum range of relative nanoparticle concentrations is based on the observation that signal contrast between tumor and normal tissues rarely extends beyond a factor of five for in vivo preclinical and clinical molecular imaging studies [14,24,25]. It bears mentioning that actual measurements of SERS nanoparticles in cells and tissues can include endogenous Raman background signals from tissues as well as variable autofluorescence and laser background components. With this in mind, our study ma (...truncated)