Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

Abstract

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Multifunctional fluorescent titania nanoparticles: green preparation and applications as antibacterial and cancer theranostic agents

“…Although this fluorescent molecule binds well to the TiO 2 NPs surface, but its fluorescent yield is small. Other labelling approach to stain TiO 2 NPs are complicate and come with challenges [7].…”

“…Up to now, the efficacy of TiO 2 NPs has been studied for delivery of various anticancer drugs [5,6]. However, the techniques to fluorescently labelled TiO 2 NPs are limited and come with several challenges including: small fluorescent yield, non-specific and high cost [7].…”

Subcellular Chemical Imaging: New Avenues in Cell Biology

“…But overall, synchrotron X-ray Fluorescence (S-XRF) provides arguably the best combination of high spatial resolution capabilities (down to few 10 nm) and high sensitivity (sub-ppm) to light and heavy elements ( Figure 2, Box 1) [10,11]. S-XRF has allowed mapping and quantification of metals, such as Fe, Zn, Cu in microalgal and human cells [12][13][14], as well as silica, drugs, organometallic molecules, and titanium oxide nanoparticles in cancer cells [2,[15][16][17][18][19]. In combination with XRF imaging, X-ray Absorption Spectroscopy (XAS) can be performed in order to reveal the chemical speciation of a target element.…”

Intracellular Localization of an Osmocenyl‐Tamoxifen Derivative in Breast Cancer Cells Revealed by Synchrotron Radiation X‐ray Fluorescence Nanoimaging

“…To minimize element leakage, cells were fixed as quickly as possible and immediately dried upon completion of aldehyde fixation, as suggested by Jin and co‐workers and as recently used for the intracellular study of TiO 2 nanoparticles . While most cells were scanned at 50 nm step size, some SR‐XRF maps were also recorded at 35 nm step size as shown in Figure C,E.…”