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Freeze-resistant Cadmium-free Quantum Dots for Live-cell Imaging Suman Mallick, Prashant Kumar, and Apurba Lal Koner ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02231 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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ACS Applied Nano Materials

Freeze-resistant Cadmium-free Quantum Dots for Live-cell Imaging Suman Mallick, Prashant Kumar and Apurba Lal Koner* Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462 066, Madhya Pradesh, India. KEYWORDS: Cadmium free quantum dots, Live-cell imaging, Freeze-resistance, Tetrathiolate ligand, Bioconjugation

ABSTRACT: A new strategy has been developed to provide freeze-resistant, watersoluble, and cadmium free quantum dots made of CuInS2/ZnS, which can easily be conjugated with bio-molecules. A new class of multi-functional and tetra-coordinating ligands has been synthesized based on lipoic acid, starting from lysine. The synthesized ligands not only provide facile ligand-exchange of CuInS2/ZnS quantum dots but also offer an exceptional colloidal stability upon freezing, robust photophysical properties in various aqueous media across a wide pH range and buffer composition with a tunable surface charge for the quantum dots. The quantum dots after ligand-exchange is easily amenable to covalent modification with biotin binding traptavidin protein exclusively by employing simple carbodimide coupling chemistry. The quantum dots also showed

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excellent biocompatibility and low non-specific binding to the cells, thus suitable for live-cell imaging of cell-surface receptors.

Quantum dots (QDs) have gained enormous popularity among the scientific community as a fluorescent marker for its unique intrinsic photophysical properties such as high brightness and quantum yield (QY), excellent photostability, broad absorption and narrow emission, size and composition-based tuning of emission properties ranging from visible to near-IR spectrum.1-6 QDs have been extensively utilized in various research fields such as biological imaging, determining the interaction between bio-molecules, production of efficient solar cells, sensing, etc.1, 7-8 A general route for QD synthesis from organometallic precursors involve the use of high boiling long-chain hydrophobic molecules e.g., Trioctylphosphine, Trioctyl phosphine oxide, Oleyl amine, Oleic acid, Dodecanethiol, etc.9-11 These molecules, also act as surface passivating ligand, are attached to the outer surface of the QDs and essential for their colloidal stability in the solution. Due to the presence of hydrophobic surface ligands, they are only soluble in non-polar solvents like hexane, toluene, chloroform, etc. So, making the water-soluble QDs is essential for biological applications.12-13 A variety of strategies has been introduced already to make the QDs soluble in aqueous media like encapsulation in micelles, coating with silica, and ligand-exchange (LE) with small molecule, etc.14 Among all these methods, LE of the hydrophobic ligands with hydrophilic ligands containing thiols, amines, pyridines, histidines and phosphine derivatives as the coordinating ligand are well documented.15-16 Among these ligands,


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thiol and di-thiol-based ligands originating from lipoic acid (LA) have shown to be the most promising candidate by providing enhanced stability of the QDs in aqueous media by strong coordination with the ZnS coating of the QDs.17-18 LA-based ligands decorated with a poly ethylene glycol (PEG) spacer provide the advantage to introduce different functional groups at the end of the PEG chain to conjugate the QDs with biomolecules of interest with superior water solubility.19-20 To improve colloidal stability and water-solubility of QDs, polymeric ligands decorated with free thiols have been introduced.21 However, this approach leads to large hydrodynamic radius for the QDs, which limits their use in biology.21 CdSe/ZnS QDs, have been introduced earlier for imaging important bio-molecules due to their intrinsic excellent photophysical properties.22 However, recent studies reveal the heavy metal toxicity effects of these materials on biological systems due to the leaching of Cadmium from QDs core.23 Recently, to avoid hazardous toxic effects from the CdSe/ZnS QDs, non-toxic QDs composed of CuInS2/ZnS is a much popular choice to the researchers across the world.24 Though there are still very few reports for using CuInS2/ZnS QDs with appropriate ligands with long-term colloidal and photo-chemical stability and efficient bio-conjugation.19, 25-27 To overcome aforementioned problems, multi-coordinating and multi-functional ligands are essential for providing long-term colloidal stability, robust photophysical properties and facile bioconjugation of these QDs, so that they can be used in biology as a potential fluorescent probe.


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5: Amine LIgand

N3 S

S S

S

Scheme 1: Synthesis of tetrathiol ligand for QDs surface passivation and bioconjugation

Herein, we report the synthesis of a group of lipoic acid-based tetra-thiolated multifunctional ligands containing a PEG spacer, starting from lysine with a variety of functional groups. The synthesis of the ligands were scalable to gram amount with good to excellent yields. These ligands were used successfully for LE of native CuInS2/ZnS QDs to provide water-solubility. After LE QDs showed exceptional colloidal and photophysical stability over a long-time in solution along with over storage in frozen condition. LE of the QDs with our synthesized multi-functional ligands enabled us to conjugate the QDs with biotin binding protein Traptavidin (Tr). The Tr conjugated QDs showed very low non-specific binding to mammalian cells, as shown by live-cell imaging of biotinylated cell surface.

The key feature of our ligand design is the choice of lysine as the central precursor, consisting of three functional groups i.e., two primary amines and one carboxylic acid. The presence of two different type of functional groups in lysine i.e., amine as well as carboxylic acid group helped us significantly to design our multi-functional ligands


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easily. The conventional ligands used for capping QDs surface and to solubilize them in water are achieved by employing thiol-containing compounds, like the reduced LA derivatives attached with polyethylene glycol chain. In our case to reinforce the capping ability of the ligands, which is essential for improving colloidal stability of the QDs in solution for long-term storage, we have designed ligands with multi-coordinating ability. For introducing multi-coordinating ability, we have attached two LA units, which essentially would provide four thiol groups per ligand in the reduced state. Water solubility in the presence of different functional groups was achieved by introducing the modified PEG unit. The carboxylic acid unit of BOC-protected lysine,28 was first used to introduce suitable bi-functional PEG unit,14 by simple carbodiimide chemistry while amine units were kept protected. Next, after deprotection of amine groups, they were used to couple with N-hydroxy ester of LA,16 to achieve the multicoordinating units in our ligands. Later, the desired functional groups were introduced by modification of the terminal of the PEG moiety. Thus, using only carbodiimide chemistry and protection-deprotection of amine groups, a series of tetra-coordinated and multi-functional ligands was synthesized in gram scale with good to excellent yield (scheme 1, see SI). We have synthesized CuInS2/ZnS QDs following two different protocols reported in the literature which exhibits emission maxima around 550 nm (QD-1)29 and 660 nm (QD-2)9, and size was measured by TEM (Figure 1a, S1) and matches well as reported. The QD-2 mainly consists of dodecanethiol as the passivating ligand and the QD-1 consists of oleic acid, oleyl amine, along with dodecanethiol as the passivating ligands after synthesizing from respective organometallic precursors.


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Firstly, we have assessed the effect of LE on the ground state and excited state photophysical properties of the aforementioned QDs. The detailed protocol of LE is provided in experimental section. The absorption and emission spectra of the two different QDs after LE with our synthesized ligands showed identical photophysical properties (Figure 1b) with good QY as high as 64%, and their other photophysical characteristics remain unaltered. Next, we have investigated their long-term photophysical stability after LE in aqueous media. Our results show that the emission intensity of both QDs after LE was decreased only ~ 20 % after 45 days while incubation at room temperature (298 K, Figure 1c, S2) without any sign of precipitation. These results confirm the preservation of photophysical and colloidal stability of the QDs after LE. Along with that, the excitedstate lifetime of the QDs in water after 45 days of LE, were also comparable with the native QDs in organic solvents (Figure S3-4). These results prove that after LE the surface of the QDs is well protected from the bulk. Inspired by these results, we wanted to check the potential of our ligands to retain the photophysical and colloidal stability of the QDs after LE followed by storage at low temperature. Generally, bio-molecules labeled QDs should be stored at low temperature for their long-term stability.


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Figure 1. (a) TEM image of QD-2. Inset shows HRTEM picture of QD-2 (b) UV and fluorescence spectra of QD-1 and QD-2, solid lines show the spectra of the native QDs in hexane, dotted lines shows the spectra of the QDs in water (c) The time-dependent fluorescence intensity of QD-1 in water (d) The fluorescence intensity of QD-1 on multiple freeze cycle with time.

We have investigated the emission properties of the QDs after LE over multiple freeze cycles (25 C to -20 C) up to 30 days (Figure 1d, S5). The results show that the fluorescence intensity only decreased around 20 % over that period without


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aggregation. LE of the QDs results into the presence of different functional groups on the surface of the QDs, which is very essential for their use in bio-conjugation. Now the nature of the functional group present on the outer surface of the QDs plays a crucial role for their colloidal stability in the aqueous solution. The colloidal stability also influence directly the optical properties of the QDs. Generally, in bio-conjugation chemistry depending on the nature of the reaction, researchers choose different pH values for the optimum reaction. But very often, this different pH values leads to the loss of colloidal stability as well as the optical properties of the QDs used. Our aim in this project was to develop robust QDs in terms of colloidal and optical stability in aqueous solution by LE, using our synthesized ligands. Thus, we have checked the fluorescence intensity of the QDs after LE in water with different pH values. The results showed that the QDs can retain their optical properties nicely when their outer surface is decorated with our synthesized ligands containing different functional groups over the range of the pH (Figure 2a-b).


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Figure 2. pH-dependent fluorescence intensity of QDs passivated with different surface ligands (a) QD-1 (b) QD-2. Fluorescence intensity of QDs with in presence of NaCl for (c) QD-1 and (d) QD-2

The colloidal stability as well as the optical properties of the QDs in water also highly depends on the ionic strength of the solution. Thus, we have also checked the stability of the QDs after LE in water by varying the ionic strength. For this, we have checked the fluorescence intensity of the QDs after LE, in presence of different concentrations of sodium chloride. The results show that the fluorescence intensity was retained more than 80% even in presence of very high concentration (2M) of sodium chloride (Figure 2c-d). For imaging of biological samples, the fluorescence intensity of the QDs after LE in different buffer composition and different pH of the medium is very important. We have tested the fluorescence intensity of the QDs with different ligands consisting of different functional groups in varieties of buffer and in water at different pH (Figure 3a, S6).


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Figure 3. (a) Fluorescence intensity of QD-1 in different buffers, bicarbonate (pH 8.5), HEPES (pH 7.4), Borate (pH 8.2), MES (pH 6.5), PBS (pH 7.4), TAE (pH 8.3), Tris (pH 7.3) (b) Plot of zeta potential of QD-1 with different surface ligands in PBS (c) Image of QD-1 with different surface ligands, only amine, 20 % acid + 80 % OMe, only acid after agarose gel electrophoresis (d) MTT assay of MCF-7 cells with QD-1 with different surface ligands concentration

The results show that the QDs retain their fluorescence intensity very well irrespective of different buffer composition and pH of the medium with different functional particularly important for their use to specifically labeled groups at their surface. Surface functionalization of the QDs is important for conjugation of bio-molecules of


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interest. The presence of different functional groups on the surface of QDs was confirmed by the zeta potential values of the QDs after LE. The amine containing ligands shown to have a significant positive charge (QD-1, +21.43 mV) on their surface while the QDs containing carboxylic acid shown to have a significant negative charge (QD-1, –22.63 mV) on their surface in PBS buffer (Figure 3b). The methoxy (OMe) functional QDs have only a negligible negative charge. Similar results were also obtained for QD-2 (Figure S7). The agarose gel electrophoresis shows that the amine containing QDs moved towards the cathode and the carboxylic acid containing QDs moved towards the anode, while the OMe QDs did not move at all for both the QDs (Figure S8-9). We have also validated the differential mobility of the QD-1 with mixture of ligands (20 % COOH and 80 % OMe, Figure 3c). These results are in the good agreement with the zeta potential values measured for the respective QDs.

Next, we have assessed QDs cytotoxicity after LE prior using them for live-cell imaging. For this, we have checked the cytotoxicity of the QDs with different functional groups by varying their concentration on a breast cancer cell line, MCF-7, using MTT assay. The results show that the QDs with different functional groups on their surface were nontoxic up to 300 nM concentration (Figure 3d, S10). From the literature, it is already known that the QDs containing only amine or acid functional group tend to attach the cell surface non-specifically.16 Thus, we proceeded for the live-cell imaging with QD-1 containing 20% carboxylic acid functional group along the rest of the surface was covered with OMe functional group, which minimises the non-specific interaction with


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the cells. The MCF-7 cells were biotinylated prior the imaging by chemical biotinylation. The QDs containing 20% carboxylic acid group was attached with Tr30 using carbodiimide chemistry, followed by incubation with biotinylated cells. The Tr-biotin interaction helps to attach the QDs specifically on the biotinylated cell surface (Figure 4a). While no non-specific interaction towards the Tr-conjugated QDs was observed in a control experiment without chemical biotinylation of the cells (Figure 4b).

Figure 4. Specific cellular targeting of 20% COOH QD-1. MCF-7 cells incubated with 20 nM QD-1-Tr (a) with chemical biotinylation (b) without chemical biotinylation


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We have also performed another control experiment to ensure the non-specific nature of interaction of the QDs with the cells. In this case at first we have biotinylated the cell surface similarly as described above. In the same time the Tr conjugated QDs were also incubated with excess amount of biotin (1 mM) externally to ensure that all the Tr present on the QD surfaces will be already attached with biotin. Next we have incubated the biotinylated cells with the Tr conjugated QDs (already incubated with excess biotin). In this case, we didn't observe any fluorescence from the cell surfaces (Figure S11), which confirms the non-specific nature of the QDs. In summary, we have demonstrated the synthesis of a series of tetra-coordinating and multi-functional ligands starting from a single-precursor and their applications to provide water-soluble, freeze-resistant CuInS2/ZnS QDs, which are readily available for bioconjugation. These QDs with the new ligands showed excellent colloidal stability and photophysical properties over a long-time both in solution and in frozen condition. QDs decorated with the synthesized ligands with a variety of functional groups are essentially non-toxic in nature and show very low nonspecific interaction towards the cells when attached with a specific bio-molecule of interest. We strongly believe that the freeze-resistant photophysical properties along with low non-specific binding of CuInS2/ZnS QDs will be a highly demanding marker for live-cell fluorescence imaging. Currently, we are involved in applying these QDs for single-particle tracking. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website.


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A detail synthesis protocol for ligand synthesis, along with results associated with photophysical measurements, freeze-resistance study, pH tolerance, effect of buffer composition, cell culture, zeta-potential, mobility-shift assay using agarose gel electrophoresis, cytotoxicity assay and characterization of QDs by TEM and ligands by NMR, and mass spectrometry. (PDF) AUTHOR INFORMATION Corresponding Author * ALK: [email protected] ORCID Suman Mallick: 0000-0002-3203-1026 Prashant Kumar: 0000-0001-5136-5411 Apurba Lal Koner: 0000-0002-8891-416X

Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENT SM thanks, UGC India, and PK thanks CSIR India for their doctoral fellowship. ALK would like to thank Department of Science of Technology, India for Inspire (CH-77), TEM facility via FIST support from Department of Science and Technology, India and IISER Bhopal for generous financial and infrastructural support.


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28. Zuravka, I.; Roesmann, R.; Sosic, A.; Göttlich, R.; Gatto, B., Bis-3chloropiperidines Containing Bridging Lysine Linkers: Influence of Side Chain Structure on DNA Alkylating Activity. Bioorg. Med. Chem. 2015, 23, 1241-1250. 29. Zhang, J.; Xie, R.; Yang, W., A Simple Route for Highly Luminescent Quaternary Cu-Zn-In-S Nanocrystal Emitters. Chem. Mater. 2011, 23, 3357-3361. 30. Chivers, C. E.; Crozat, E.; Chu, C.; Moy, V. T.; Sherratt, D. J.; Howarth, M., A Streptavidin Variant with Slower Biotin Dissociation and Increased Mechanostability. Nat. Methods 2010, 7, 391-393. Freeze-resistant Quantum Dots

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O

S S

R=OH, NH 2, -COOH, OMe

1 st Cycle

1 2 3 4 5 6 7 8 9 10 11

O

14 th Cycle

LIpoic acid

HN

QD1 QD2

-20 ºC

25 ºC ACS Paragon Plus Environment Freeze-resistant ZnS Quantum Dots

CuInS 2

Freeze-Resistant Cadmium-Free Quantum Dots for ... - ACS Publications

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