Kindopp, A. | bioRxiv Science

bioRxiv2025

Enhancing Optical Properties and Stability of DNA-Functionalized Carbon Nanotubes with Cryoprotectant Mediated Lyophilization

The long-term optical performance and stability of single-walled carbon nanotubes (SWCNTs) functionalized with single-stranded DNA are critical for their application in near-infrared (NIR) fluorescence biological sensing and imaging. However, the aggregation of such DNA-SWCNTs during storage presents a significant challenge. Here, we explored the use of lyophilization combined with various cryoprotectants to enhance the long-term stability and reconstitution of DNA-SWCNTs at room temperature. Five conventionally used cryoprotectants, including glucose, sucrose, mannitol, polyethylene glycol (PEG), and polyvinyl alcohol (PVA), were evaluated for their ability to maintain desired optical properties and prevent aggregation of SWCNTs through the process of lyophilization and reconstitution. Our results indicated that glucose and PEG, particularly in an 80:20 ratio by weight, provided the best performance, preserving NIR fluorescence and ensuring consistent reconstitution without significant aggregation. Further, in vitro studies using murine macrophages demonstrated that lyophilized SWCNTs with glucose-PEG protectants and then held at room temperature before subsequent reconstitution maintained stable intracellular optical performance, supporting their potential for long-term storage, ease of transport, and use in biomedical applications. These findings suggest that the optimized lyophilization protocol with specific cryoprotectant combinations can significantly improve the shelf life and reproducibility of SWCNT-based sensors, paving the way for their broader application in biological and clinical settings.

bioengineering↗

bioRxiv2024

Machine Learning Assisted Spectral Fingerprinting for Immune Cell Phenotyping

Spectral fingerprinting has emerged as a powerful tool, adept at identifying chemical compounds and deciphering complex interactions within cells and engineered nanomaterials. Using near-infrared (NIR) fluorescence spectral fingerprinting coupled with machine learning techniques, we uncover complex interactions between DNA-functionalized single-walled carbon nanotubes (DNA-SWCNTs) and live macrophage cells, enabling in situ phenotype discrimination. Through the use of Raman microscopy, we showcase statistically higher DNA-SWCNT uptake and a significantly lower defect ratio in M1 macrophages as compared to M2 and naive phenotypes. NIR fluorescence data also indicate that distinctive intra-endosomal environments of these cell types give rise to significant differences in many optical features such as emission peak intensities, center wavelengths, and peak intensity ratios. Such features serve as distinctive markers for identifying different macrophage phenotypes. We further use a support vector machine (SVM) model trained on SWCNT fluorescence data to identify M1 and M2 macrophages, achieving an impressive accuracy of > 95%. Finally, we observe that the stability of DNA-SWCNT complexes, influenced by DNA sequence length, is a crucial consideration for applications such as cell phenotyping or mapping intra-endosomal microenvironments using AI techniques. Our findings suggest that shorter DNA-sequences like GT6 give rise to more improved model accuracy (> 87%) due to increased active interactions of SWCNTs with biomolecules in the endosomal microenvironment. Implications of this research extend to the development of nanomaterial-based platforms for cellular identification, holding promise for potential applications in real time monitoring of in vivo cellular differentiation. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/583608v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11e2c60org.highwire.dtl.DTLVardef@1f3553forg.highwire.dtl.DTLVardef@61dcb9org.highwire.dtl.DTLVardef@e216b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗