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Nano Research 2018, 11(1): 464-476 https://doi.org/10.1007/s12274-017-1654-8
Research Article | Issue | Published: 19 July 2017

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

Show Author's Information Koshonna Brown1,  Ted Thurn1,+,  Lun Xin1,  William Liu1,╪,  Remon Bazak1,‖,  Si Chen2,  Barry Lai2,  Stefan Vogt2,  Chris Jacobsen3,  Tatjana Paunesku1,  Gayle E. Woloschak1( )
Department of Radiation OncologyFeinberg School of MedicineNorthwestern UniversityChicagoIllinois60611USA
X-ray Science DivisionAdvanced Photon SourceArgonne National Laboratory9700 South Cass AvenueArgonneIllinois60439USA
Department of Physics & AstronomyWeinberg College of Arts and Sciences2145 Sheridan RoadEvanstonIllinois60208USA

+Present Address: U.S. Department of State, 2201 C Street, NW Washington, DC 20520, USA

╪Present Address: Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, USA

‖Present Address: Department of Otorhinolaryngology and Head & Neck Surgery, University of Alexandria Medical School, Azarita Medical Campus, Champlollion Street, Khartoum Square, Alexandria 21547, Egypt

Keywords:
TiO2 nanoparticles, anatase, Click reaction, biotin–streptavidin, synchrotron X-ray, fluorescence microscopy
Cite this article:
Brown K, Thurn T, Xin L, et al. Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection. Nano Research, 2018, 11(1): 464-476. https://doi.org/10.1007/s12274-017-1654-8
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Abstract Full text Electronic supplementary material About this article

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 posttreatment 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 alkyneconjugated 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.


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Abstract
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Electronic supplementary material
About this article

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

Show Author's information Koshonna Brown1, Ted Thurn1,+, Lun Xin1, William Liu1,╪, Remon Bazak1,‖, Si Chen2, Barry Lai2, Stefan Vogt2, Chris Jacobsen3, Tatjana Paunesku1, Gayle E. Woloschak1( )
Department of Radiation OncologyFeinberg School of MedicineNorthwestern UniversityChicagoIllinois60611USA
X-ray Science DivisionAdvanced Photon SourceArgonne National Laboratory9700 South Cass AvenueArgonneIllinois60439USA
Department of Physics & AstronomyWeinberg College of Arts and Sciences2145 Sheridan RoadEvanstonIllinois60208USA

+Present Address: U.S. Department of State, 2201 C Street, NW Washington, DC 20520, USA

╪Present Address: Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, USA

‖Present Address: Department of Otorhinolaryngology and Head & Neck Surgery, University of Alexandria Medical School, Azarita Medical Campus, Champlollion Street, Khartoum Square, Alexandria 21547, Egypt

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 posttreatment 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 alkyneconjugated 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.

Keywords: TiO2 nanoparticles, anatase, Click reaction, biotin–streptavidin, synchrotron X-ray, fluorescence microscopy

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Publication history
Copyright
Acknowledgements

Publication history

Received: 30 December 2016
Revised: 27 April 2017
Accepted: 29 April 2017
Published: 19 July 2017
Issue date: January 2018

Copyright

© Tsinghua University Press and Springer-Verlag GmbH Germany 2017

Acknowledgements

Acknowledgements

This research was supported by the National Institutes of Health (Nos. CA107467, EB002100, U54CA119341 and GM104530). Implementation of the Bionanoprobe is supported by NIH ARRA (No. SP0007167). Confocal optical imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. Confocal microscopy was performed on a Nikon A1R multiphoton microscope, acquired through the support of NIH 1S10OD010398-01. Work at the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences contract No. DE-AC02-06CH11357. Metal analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by NASA Ames Research Center (No. NNA06CB93G). Use of the Simpson Querrey Institute Analytical BioNanoTechnology Equipment Core (ANTEC) facility was supported by the U.S. Army Research Office, the U.S. Army Medical Research and Materiel Command, and Northwestern University funding received from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF NNCI-1542205). Cryo-TEM work was performed at the Northwestern University Biological Imaging Facility by Imaging Specialist Charlene Wilke. The authors thank Dr. Teng-Leong Chew for his valuable discussion and advice.

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