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

Abstract

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.

Comprehensive spectral endoscopy of topically applied SERS nanoparticles in the rat esophagus

“…While our previous studies have utilized three or more multiplexed NP flavors [30,41], this study demonstrates the initial feasibility of our technologies with the simplest case of two NP flavors topically applied on cylindrical tissue phantoms and on fresh rat esophagus tissues (ex vivo and in vivo). Future studies using this device will include in vivo multiplexed molecular imaging of rat esophageal cancer models with targeted SERS NPs, such as the NPs recently described by us [30].…”

“…A customized spectrometer (Bayspec Inc.) is used to disperse the light collected from the 36 multimode collection fibers onto a cooled deep-depletion spectroscopic CCD (Andor Newton, DU920P-BR-DD) with a spectral resolution of ~2 nm (~30 cm −1 ). Additional details about the detection system have been described previously [41].…”

“…To calculate the concentration and ratio of SERS NPs from a raw spectrum, this study employed a commonly used direct classical least squares (DCLS) demultiplexing method described previously [18,41,43]. Generally, other than sources of random noise, it is assumed that each measured spectrum consists of a linearly weighted sum of fixed nanoparticle spectra (F n ) and broadband background signals (B i ).…”

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

“…However, it should be noted that the receptor concentrations estimated using both pairs of imaging agents for the two cell lines in all conditions, matched well with expected levels of EGFR. These agents have also been used extensively and validated in other work, where EGFR concentrations matched well with gold standard and expected levels (Leigh et al , 2013; Wang et al , 2016; Wang et al , 2015; Wang et al , 2014b; Tichauer et al , 2012a; Tichauer et al , 2012b; Samkoe et al , 2014). This body of work suggests that both non-specific binding and internalization are relatively negligible, at least within 1 h of initial administration of the imaging agents.…”

Transparency of Reporting in Molecular Diagnostics

“…MDx has become more than just analysis of genetic content for disease information, and makes use of a range of techniques including isothermal amplification methods [9], nucleic acid sequencing [10], high resolution melt analysis [11], DNA microarrays [12], tissue arrays [13], mass spectrometry [14], nanoparticles [15] and fluorescence in situ hybridisation [16]. Perhaps the most ubiquitous molecular technique is the real-time quantitative polymerase chain reaction (qPCR) [17], which can detect DNA, RNA (reverse transcription (RT)-qPCR) and proteins (proximity ligation/extension assay).…”