Fluorescence-Raman Endoscopic System for In Vivo Multiplexed Molecular Diagnostics

Abstract

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows non-/minimally invasive real-time detection of cellular structural deformations as well as pathological conditions on the surface of hollow organs. Particularly, by integrating the endoscopic imaging techniques with the molecular probes which are able to specifically target toward biomolecules, the in vivo and intraoperative molecular diagnostics could be achieved

it allows for early cancer detection, personalized medicine, and image-guided therapy by identifying tumor-related protein expression. Among the various kinds of molecular targeting probes, the fluorescence dyes have been widely utilized to visualize a specifically targeted lesion in extensive area

the surface enhanced Raman scattering (SERS) active nanoparticles (NPs) are also used for multiplexed molecular diagnostics due to its high sensitivity and multiplexing capability. As a targeting agent, antibody conjugated NPs have a great potential in diagnostic and therapeutic applications due to their high sensitivity and specificity for bio-targets, as well as their wide applicability. Unfortunately, these features are significantly affected by antibody conjugation methods in terms of conjugation efficiency, orientation of the target binding site in the antibody, and denaturation during chemical conjugation reactions. Furthermore, the number of conjugated antibodies on each NP and the overall targeting efficacy are critical factors for a quantitative bioassay with antibody conjugated NPs. For these reasons, I developed a versatile and oriented antibody conjugation method using copper-free click chemistry. Moreover, the number of conjugated antibodies and their binding capacity were quantitatively and experimentally evaluated using fluorescently-labeled antibodies and antigens. The strong binding capability of antibody-conjugated NPs prepared using the copper-free click chemistry-based conjugation strategy was 8 times superior to the binding capability seen following the use of the EDC/NHS-coupling method. To characterize pathological condition of suspicious lesions in a multiplexed way during endoscopic procedure, I developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots) as targeting agents. By utilizing the FRES along with antibody conjugated F-SERS dots, real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals in breast and colorectal cancer (CRC) xenograft models. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), as tumor cell targeting biomarkers, on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way. As a clinical diagnostic model, in CRC xenograft models, epidermal growth factor receptor (EGFR) for targeting tumor cells and vascular endothelial growth factor (VEGF) for identifying tumor microenvironment were successfully identified using the FRES via colonoscopic examination procedure. Additionally, the relative quantification ability of the FRES was demonstrated in in vitro and ex vivo condition using Raman intensity corresponding to the targeted F-SERS dots. These features illustrated the potential of FRES as a molecular diagnostic tool that enables real-time characterization of tumor cell receptors and tumor microenvironment during routine endoscopic procedures, allowing an early cancer detection and tailored therapy.