Machine learning enabled multiplex detection of periodontal pathogens by surface-enhanced Raman spectroscopy.

Periodontitis is a chronic inflammation of the periodontium caused by a persistent bacterial infection, resulting in destruction of the supporting structures of teeth. Analysis of microbial composition in saliva can inform periodontal status. Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Streptococcus mutans (Sm) are among reported periodontal pathogens, and were used as model systems in this study. Our atomic force microscopic (AFM) study revealed that these pathogens are biological nanorods with dimensions of 0.6-1.1 µm in length and 500-700 nm in width. Current bacterial detection methods often involve complex preparation steps and require labeled reporting motifs. Employing surface-enhanced Raman spectroscopy (SERS), we revealed cell-type specific Raman signatures of these pathogens for label-free detection. It overcame the complexity associated with spectral overlaps among different bacterial species, relying on high signal-to-noise ratio (SNR) spectra carefully collected from pure species samples. To enable simple, rapid, and multiplexed detection, we harnessed advanced machine learning techniques to establish predictive models based on a large set of raw spectra of each bacterial species and their mixtures. Using these models, given a raw spectrum collected from a bacterial suspension, simultaneous identification of all three species in the test sample was achieved at 95.6 % accuracy. This sensing modality can be applied to multiplex detection of a broader range and a larger set of periodontal pathogens, paving the way for hassle-free detection of oral bacteria in saliva with little to no sample preparation.

Role of photobleaching process of indocyanine green for killing neuroblastoma cells.

Indocyanine green (ICG) is an FDA-approved near infrared (NIR) imaging agent for diagnosis and imaging guided surgery. It also exhibits phototoxicity under high-dose NIR irradiation, expanding its application as a photo-therapeutic agent. Since ICG's efficiency as a type II photosensitizer has been controversial due to its low triplet state yield, other mechanisms have been explored. While claims of toxic decomposition products, accompanied by irreversible ICG photobleaching, were proposed as the main mechanism, evidences from systemic studies are lacking. In this work, we aimed to unravel the factors affecting ICG photobleaching and the associated photo-killing effect on neuroblastoma, one of the most common pediatric tumors but often escapes therapy. Specifically, we examined how albumin-induced ICG stabilization affects the ICG photobleaching process, and the effect of photobleached ICG on cell proliferation and viability of neuroblastoma cells. It was found that ICG photobleaching was significant only under aerobic conditions and was more efficient in solutions with higher concentration ICG monomers, which were stabilized from aggregates by the presence of BSA while increasing photobleaching and associated oxygen consumption. Photobleached ICG inhibited cell proliferation, indicating another effect of tumor treatment by ICG. Taken together, while enhanced photobleaching by BSA-bound ICG monomers may reduce the photodynamic effect targeting cellular components, the photoproducts directly contribute to tumor growth inhibition and assist in a secondary mechanism to stop tumor growth.

Combined application of Indocyanine green (ICG) and laser lead to targeted tumor cell destruction.

PURPOSE: Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. METHODS: Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. RESULTS: Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. CONCLUSIONS: Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.