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Functional Nanostructured Materials (including low-D carbon)
Semiconductor Nanoplatelets: A New Class of Ultrabright Fluorescent Probes for Cytometric and Imaging Applications Djamila Kechkeche, Edgar Cao, Chloé Grazon, Filippo Caschera, Vincent Noireaux, Marie-Laurence Baron-Niel, and Benoit Dubertret ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07143 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018
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ACS Applied Materials & Interfaces
Semiconductor Nanoplatelets: A New Class of Ultrabright Fluorescent Probes for Cytometric and Imaging Applications Djamila Kechkeche,Γ, ɣ Edgar Cao,ɣ Chloé Grazon,ɣ† Filippo Caschera,ɣ Vincent Noireaux,Շ Marie-Laurence Baron Nielɣ and Benoit DubertretΓ* Γ
LPEM, ESPCI Paris, PSL Research University, CNRS, Sorbonne-Universités, 75005 Paris, France ɣ
Շ
Nexdot, 102 Avenue Gaston Roussel, 93230, Romainville, France
School of Physics and Astronomy, University of Minnesota, 115 Union Street SE, Minneapolis, Minnesota 55455, United States
Keywords:
antibody,
biocompatible,
bioimaging,
ligand-exchange,
protein,
surface
functionalization, zwitterions
Abstract: Fluorescent semiconductor nanoplatelets (NPLs) are a new generation of fluorescent probes. NPLs are colloidal two-dimensional materials that exhibit several unique optical properties, including high brightness, photostability and extinction coefficients as well as broad excitation and narrow emission spectra from the visible to the near infrared spectrum. All these exceptional fluorescence properties make NPLs interesting nanomaterials for biological
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applications. However, NPLs are synthesized in organic solvents and coated with hydrophobic ligands that render them insoluble in water. A current challenge is to stabilize NPLs in aqueous media compatible with biological environments. In this work, we describe a novel method to disperse fluorescent NPLs in water and functionalize them with different biomolecules for biodetection. We demonstrate that ligand-exchange enables the dispersion of NPLs in water while maintaining optical properties and long-term colloidal stability in biological environments. Four different colors of NPLs were functionalized with biomolecules by random or oriented conformations. For the first time, we report that our NPLs have a higher brightness than standard fluorophores, like phycoerythrin (PE) or Brilliant Violet 650™ (BV 650™) for staining cells in flow cytometry. These results suggest that NPLs are an interesting alternative to common fluorophores for flow cytometry and imaging applications in multiplexed cellular targeting.
INTRODUCTION Semiconductor quantum dots (QDs) nanocrystals have attracted a great attention due to their unique photophysical properties that are distinct from traditional organic dyes and fluorescent proteins, including broad excitation, high molar absorption and photostability as well as tunable and narrow emission wavelength.1 Since 1998, QDs have already been used for a multitude of biological applications such as receptor tracking, drug delivery, individual protein monitoring or multicolor immunostaining. 2,3,4 Recently, atomically flat semiconductor colloidal nanoplatelets (NPLs) with 2D geometry were synthesized.5 NPLs are at the center of intense research and development because of their unique physical properties.6,7 A NPL is a single uniform flat nanocrystal synthetized with an atomically controlled precision of their exciton confining direction (the thickness). By controlling the quantum confinement and composition, these colloidal quantum wells offer
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ACS Applied Materials & Interfaces
superior properties compared to QDs.8 Most notably, NPLs can exhibit narrower full width half maximum as low as 7 nm for a photoluminescence signal around 500 nm for core of CdSe. Deposition of a shell can enhance higher quantum yield and better photostability than core only NPLs.9,10 However core-shell structures present line width of the photoluminescence rises between 22-35 nm depending on the nature of the shell and the thickness.11 In comparison to NPLs, QDs and organic fluorophores showed broad FWHM between 25-35 nm and 35-45 nm, respectively.12 Simultaneous photoluminescence detection of different types of NPLs offers the possibility of multicolor analysis with negligible spectral overlap using a single UV-blue excitation source. Another advantage of NPLs is the high brightness of fluorescence due to their high quantum yield and high molar absorption coefficient (1.0×107 cm-1×M-1 value provided at the first excitonic absorption peak (510 nm) of core of NPLs emitting at 513 nm) which is several times higher than organic fluorophores (2.5×105 cm-1×M-1 at 658 nm and 2.5×106 cm1
×M-1 at 405 nm for Cy5 and Brilliant Violet 421™, respectively) and QDs (105 - 106 cm-1×M-1
values provided at the first excitonic absorption peak of different colours of QDs). 12,13 All these unique properties make NPLs a great candidate for ultrasensitive single molecule detection sensors and multiplexed applications. Among the multiple applications of fluorophores in bioimaging, flow cytometry is a popular tool used to analyze the expression of cell surfaces and intracellular proteins. The principle of this technique is to target proteins with specific fluorescent antibodies (Abs). Over the past decades, phycoerythrin (PE) and allophycocyanine (APC) have been the brightest probes used for immunofluorescence applications. However, in complex biological systems, the simultaneous measurement of many proteins is needed and therefore the use of different and distinguishable fluorophores is required. Many organic fluorophores are currently available but, unfortunately,
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their simultaneous use is limited by broad emission spectra leading to overlap of multiple probe emissions and complex compensations. Another limitation with organic fluorophores is the need to use different excitation sources because of their different and narrow absorption spectra. To overcome these limitations, QDs having narrow emission spectra compared to the organic fluorophores offered the opportunity for easier multiplexing experiments.14 Organic fluorophores provided 12-color panel whereas QDs provided 17-colors flow cytometry experiment with minimal compensation requirements (
