Multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) offer the potential for rapid molecular phenotyping of tissues, thereby enabling accurate disease detection as well as patient stratification to guide personalized therapies or to monitor treatment outcomes. The clinical success of molecular diagnostics based on SERS NPs would be facilitated by the ability to accurately identify tissue biomarkers under time-constrained staining and detection conditions with a portable device. In vitro, ex vivo and in vivo experiments were performed to optimize the technology and protocols for the rapid detection (0.1-s integration time) of multiple cell-surface biomarkers with a miniature fiber-optic spectral-detection probe following a brief (5 min) topical application of SERS NPs on tissues. Furthermore, we demonstrate that the simultaneous detection and ratiometric quantification of targeted and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to nonspecific variations in NP concentrations.

The biological investigation and detection of esophageal cancers could be facilitated with an endoscopic technology to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and investigation through the sensitive and multiplexed detection of cell-surface biomarkers. Here, we demonstrate that the topical application and endoscopic imaging of a multiplexed cocktail of receptor-targeted SERS NPs enables the rapid detection of tumors in an orthotopic rat model of esophageal cancer. Antibody-conjugated SERS NPs were topically applied on the lumenal surface of the rat esophagus to target EGFR and HER2, and a miniature spectral endoscope featuring rotational scanning and axial pull-back was employed to comprehensively image the NPs bound on the lumen of the esophagus. Ratiometric analyses of specific vs. nonspecific binding enabled the visualization of tumor locations and the quantification of biomarker expression in agreement with immunohistochemistry and flow cytometry validation data.

Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20–50%. While imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients.

Abstract:The early detection and biological investigation of esophageal cancer would benefit from the development of advanced imaging techniques to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and the investigation of cancer progression through the sensitive and multiplexed phenotyping of cell-surface biomarkers. Here, a miniature endoscope featuring rotational scanning and axial pull back has been developed for 2D spectral imaging of SERS NPs topically applied on the lumenal surface of the rat esophagus. Raman signals from low-pM concentrations of SERS NP mixtures are demultiplexed in real time to accurately calculate the concentration and ratio of the NPs. Ex vivo and in vivo experiments demonstrate the feasibility of topical application and imaging of multiplexed SERS NPs along the entire length of the rat esophagus.

In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.