Toward greener electrochemical synthesis of composition-tunable luminescent CdX-based (X = Te, Se, S) quantum dots for bioimaging cancer cells

Accepted Manuscript Title: Toward greener electrochemical synthesis of composition-tunable luminescent CdX-based (X = Te, Se, S) quantum dots for bioimaging cancer cells Authors: Denilson V. Freitas, S´ergio G.B. Passos, J´essica M.M. Dias, Alexandra Mansur, Sandhra M. Carvalho, Herman Mansur, Marcelo Navarro PII: DOI: Reference:

S0925-4005(17)30795-5 http://dx.doi.org/doi:10.1016/j.snb.2017.04.185 SNB 22273

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

16-12-2016 26-4-2017 27-4-2017

Please cite this article as: Denilson V.Freitas, S´ergio G.B.Passos, J´essica M.M.Dias, Alexandra Mansur, Sandhra M.Carvalho, Herman Mansur, Marcelo Navarro, Toward greener electrochemical synthesis of composition-tunable luminescent CdX-based (X=Te, Se, S) quantum dots for bioimaging cancer cells, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.04.185 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toward greener electrochemical synthesis of composition-tunable luminescent CdX-based (X = Te, Se, S) quantum dots for bioimaging cancer cells Denilson V. Freitasa, Sérgio G. B. Passosa, Jéssica M. M. Diasa, Alexandra Mansurb, Sandhra M. Carvalhob, Herman Mansurb*, and Marcelo Navarroa* a

Department of Fundamental Chemistry, Federal University of Pernambuco, Cidade

Universitária, 50670-901, Recife, PE, Brazil. E-mail: [email protected] b

Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais,

Belo Horizonte, MG, Brazil

*Corresponding author. Tel.: +55 81 21267460; fax: +55 81 21268442. E-mail address: [email protected] (M. Navarro)

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Graphical Abstract

Highlights     

Cd2+ and X2- (X = Te, Se or S) species were simultaneously prepared in a cavity cell for production of QDs. Cadmium sacrificial anode and graphite powder macroelectrode (cathode) in cavity cell methodology. QD one-pot electrochemical synthesis in aqueous medium, RT, and reaction time 16 min. Heat treatment of 60 min furnished nanoparticles of 3.0 nm diameter, pH 9, and quantum yield ca. 10%. HeLa cells (cancer cell detection test) toward CdTe-MPA samples showed no toxicity after 24 h incubation.

Abstract A novel versatile and clean colloidal processing route was developed for synthesizing CdX (X = Te, Se or S) quantum dots (QDs) based on the simultaneous production of the respective precursors via cadmium sacrificial anode electrochemical methodology. The CdX QDs stabilized by 3-mercaptopropionic acid (MPA) were synthesized in onepot electrochemical process using aqueous medium at room temperature with full reaction time of approximately 16 min. The CdX quantum dots conjugates were extensively characterized by UV-vis, PL, FTIR, TEM-EDS, XRD, DLS, XPS, and Zeta potential, evidencing that very stable colloidal quantum dots were produced with uniform narrow-size distributions and average nanoparticle diameter of approximately 3.0 nm and quantum yield (QY) of approximately 10%. The cell viability response of 2

HeLa cells toward the CdX-MPA conjugates clearly demonstrated that they are nontoxic based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) mitochondrial metabolic activity assay for 24 h of incubation with HeLa cervical cancer cells. Moreover, they showed effective fluorescent activity with time-dependent intensities for evaluating continuous endocytosis of cancer cells, thus, offering innumerous possibilities to be used as fluorescent nanoprobes for bioimaging and biosensing of cancer cells. Keywords: green chemistry; quantum dots; cadmium; electrosynthesis; sacrificial anode; cytotoxicity 1. Introduction Colloidal semiconductor nanocrystals, also referred to as ‘‘quantum dots’’ (QDs), are essentially composed of an inorganic core, made up of between a few hundred and a few thousand atoms, surrounded by an organic outer layer of capping molecules (i.e., ligands). Because the nano-size is of the same order of magnitude as the Bohr exciton radius of electron-hole pairs in solids, QDs exhibit quantum size effects. Therefore, quantum dots present unique tunable optical-electronic properties by controlling the size at the nanoscale dimension due to the quantum confinement regime, defined by an increasing bandgap accompanied by the quantization of the energy levels to discrete values. In that sense, their bandgap can be precisely modulated to exhibit distinct luminescence characteristics and electronic properties by varying the size and the chemical composition of the semiconductor crystals usually composed of groups II to VI or III to V elements [1–3]. Based on these unique features, the band gap of QDs can be engineered by using a variety of semiconductor materials, from narrow to wide band gap (e.g., PbS, CdTe, CdSe, CdS, ZnSe, ZnS), which are optically active from the ultraviolet to the near infrared portions of the electromagnetic spectrum. Hence, the size-dependent optical properties associated with the wide range of chemical composition of binary and ternary semiconductor alloys of QDs have been the focus of intense research over the past two decades [4]. The search for new and more efficient QD synthesis methodologies peaked in the 90s, with the development of non-aqueous methodology developed by Murray et al.,[5] where metal precursors were added to coordinating solvents at high temperatures, giving nanoparticles of CdTe, CdSe, and CdS with quantum yields reaching 80%. However, nanoparticles synthesized in organic medium requires 3

structural modifications, such as ligand exchange, or surface functionalization to be applied in biological medium [6]. To minimize this problem, Rogach et al. [7] developed cleaner and biocompatible aqueous medium methodology, free of organometallic compounds used in non-aqueous synthesis, where the QDs are stabilized by short chain thiols, requiring no post-synthesis procedures for obtaining dispersed QDs in an aqueous medium. Since then, some new procedures has been described for obtaining these nanoparticles, basically varying the chalcogenide precursors [8,9]. The electrochemistry is a methodology of synthesis that can fulfill at least 9 out of the 12 postulates of sustainable or green chemistry, which are important criteria needed to develop environmentally compatible processes. Electrochemistry can be used to replace toxic oxidizing or reducing reagents, reduce energy consumption, and can be used for the in situ production of unstable and hazardous reagents [10]. That is why among several QD synthetic procedures, electrochemical methods have been proposed. Electrochemical preparation of chalcogen precursors does not require chemical reducing agents, preventing the formation of residues, and uses in the majority of cases milder reaction conditions. According to literature [9,11], electrochemical methods can be used to prepare gaseous chalcogen precursors, such as H 2S, H2Se or H2Te, which are injected to the cadmium salt/stabilizer solution. Also a twocompartment electrochemical cells, separated by Nafion ® membrane, were used for the CdTe and CdSe preparations, furnishing nanoparticles of high luminescence and low dispersity [12–14]. In this work, we evaluate the reaction parameters for application of a new onepot electrochemical methodology for the CdX (X = Te, Se and S) QDs synthesis based on a cavity cell/cadmium sacrificial anode process, using 3-Mercaptopropionic acid (MPA) as stabilizer (Fig. 1). This methodology rules out chemical reducing agents and post-synthesis treatment steps for the synthesized QDs. Moreover, these nanoconjugates were non-toxic and showed fluorescent activity dependent on the incubation time with HeLa cancer cells offering prospective applications for bioimaging and biosensing in oncology. 2. Experimental 2.1 General

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All chemicals were of reagent grade and were used without further purification. Elemental tellurium (Te0) powder (99.8%, 200 mesh, Aldrich), Elemental selenium (Se0) powder (99.5%, 100 mesh, Aldrich), Elemental sulfur (S0) powder (100 mesh, Aldrich), graphite powder (particle size < 20 m, Aldrich), 3-mercaptopropionic acid (MPA) ( 99%, Aldrich), quinine bisulfate (Aldrich), rhodamine 6G (Aldrich), HClO 4 (70%, Aldrich) and NaOH (97%, Quimex) were used as purchased. The water was of Milli-Q grade. 2.2 Equipment and general procedure for CdX-MPA synthesis (X = Te, Se, S) Controlled-current electrolyses were carried out using an Autolab PGSTAT 30 potentiostat/galvanostat and an electrochemical cavity cell (Fig. 1). The cathodic compartment was prepared compressing 58.5 mg (4.88 mmol) of graphite powder mixed to 6.50 mg (50.9 x 10-6 mol) tellurium powder, or 1.60 mg (50.0 x 10 -6 mol) selenium powder, or 1.97 mg (25.0 x 10-6 mol) sulfur powder, pressed under P = 3.2 kg cm-2 during 10 min. After pressing, a sintered glass with diameter equal to the cavity was placed to avoid the dispersion of the graphite in the aqueous solution, and at the same time allowing the migration of telluride ions (Te2-) formed during the electroreduction. The sacrificial anode was a cadmium rod, which was placed in the anodic compartment of the electrochemical cell, containing 25 mL of 0.2 mol.L -1 NaClO4 solution and MPA, in pH 7. Argon atmosphere was kept during the electroreduction to avoid air oxidation of the chalcogenides produced. The CdX-MPA QDs (X = Te, Se, S) were prepared with the MPA on the ratio: X:Cd:MPA (1:3:6) and the pH was adjusted to 9 and 11 with addition of 1.0 mol.L -1 NaOH solution. Constant current electrolysis (i = -30 mA) was carried out during 984 s. Colloidal solutions of CdX-MPA were generated in the anodic compartment, which were transferred to the reaction flask and heated at 95°C. Four aliquots (6 mL) were taken after 15, 30, 45 and 60 min to follow the particles growing and characterization [9]. 2.3 Characterization of CdX-MPA QDs (X = Te, Se, S) Absorption and emission spectroscopy techniques were performed using a 2 mL aliquot of each CdX-MPA (X = Te, Se, S) colloidal solution, at pH 9 and 11, for the characterization nanoparticles. The quantum yield of luminescence for CdTe was calculated by rhodamine 6G in ethanol as standard (QY = 95%, exc = 488 nm) and for CdSe and CdS quinine bisulfate in H2SO4 0.5 mol.L-1 was used (QY = 54.6%, exc = 366nm) [15]. The optical characterizations (absorption, emission and quantum yield) 5

were performed in triplicate and the average values are shown in Tables S1, S2 and S3 of the supporting information. The UV-Vis (ultraviolet-visible) absorption spectra were registered with a Cary 50/Varian spectrophotometer (Xenon lamp). Emission measurements (Photoluminescence spectroscopy - PL) were obtained on a Shimadzu RF-5301PC fluorimeter (Xenon lamp). The average size of the particles was calculated based on the method described by Yu et al. [16]. Nanostructural characterizations of the QD nanoconjugates were based on the images obtained using a Tecnai G2-20-FEI transmission electron microscope (TEM) at an accelerating voltage of 200 kV. Energy dispersive X-ray spectra (EDS) were collected using the TEM for element chemical analysis. In all of the TEM analyses, the samples were prepared by dropping the colloidal dispersion onto a porous carbon grid. The QD size and size-distribution data were obtained based on the TEM images by measuring at least 200 randomly selected nanoparticles using an image processing program (ImageJ, version 1.50b, public domain, National Institutes of Health). Prior to the X-ray diffraction (XRD) analysis, the QD nanoconjugates were precipitated by addition of isopropanol excess, deposited on a glass slide surface and dried under vacuum. Then, XRD of the solid powder was taken on a Bruker X-ray diffractometer model D8 Advance with a CuK radiation (= 1.5418 Å). CdX-MPA QDs (X = Te, Se, S), at pH 9 and 11, were analyzed by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) method (Thermo Fischer, Nicolet 6700) over the range of 400 - 4,000 cm-1 using 64 scans and with 2 cm-1 resolution. The samples were prepared by placing a droplet of the dispersions onto KBr powder and drying at the temperature of (40 ± 2°C) for 24 h. MPA solutions at the same pH values (9.0 and 11.0) were used as references. Zeta potential measurements (ζ-potential) were performed on QD colloidal media using a Zetaplus instrument by applying the laser light diffusion method (Brookhaven Instruments). This instrument uses the laser Doppler electrophoresis technique (35-mW red diode laser at = 660 nm). All tests were performed using a minimum of three replicates (n = 3), and the values were averaged. Hydrodynamic radius (or diameter, HD) of nanoparticles was evaluated using dynamic light scattering (DLS) technique. Essentially, DLS method evaluates the radius of a hypothetical hard sphere that diffuses in the same way as the system (agglomerates, solvated particles, particles stabilized with molecules and polymers,

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etc.) under examination. DLS is a well-established technique for measuring the size and size distribution of molecules and particles dispersed in a liquid typically in the submicron region. The Brownian motion of the species in suspension causes laser light to be scattered at different intensities. Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size using the Stokes-Einstein relationship. The technique employed in the instrument Brookhaven ZetaPlus, photon correlated spectroscopy (PCS) of quasi-elastically scattered light (QELS), results in a size range of evaluation from 2 nm up to 3 µm with a an accuracy of about 1% to 2% with monodisperse samples. Thus, DLS analyses were performed using a Brookhaven ZetaPlus instrument with a laser light wavelength of 660 nm (35-mW red diode laser) and a thermostat with temperature stabilization. Standard square acrylic cells with a volume of 4.5 mL were used. For the DLS of the QDs, the colloidal solutions (5 mL) were filtered four times through a 0.45 μm aqueous syringe filter (Millex LCR 25 mm, Millipore) to remove any possible dust. Samples were measured at 25 ± 2˚C, and light scattering was detected at 90º. Mean and standard deviation calculation was calculated for size distribution by number assuming a Lognormal distribution. Each measurement required approximately about 3 minutes, and 3 measurements were obtained for each system and averaged. X-ray photoelectron spectroscopy (XPS) analysis was performed on an Amicus spectrometer (Shimadzu) using Mg-Kα as the excitation source. All peak positions were corrected based on C 1s binding energy (284.6 eV). For sample preparation, concentrated QD colloidal medium was dropped onto a glass slide and dried in a vacuum desiccator at room temperature for 48 h. 2.4 Cytotoxicity assays All of the biological tests were performed according to ISO standards 109935:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity; as detailed in the protocol). All experiments were performed using the direct contact methodology. 2.5 Culture of human Epithelioid Cervix carcinoma cells line (HeLa cells). The immortalized human carcinoma-derived (HeLa) cells were provided by Prof. Dr. Z.I.P. Lobato of Department of Preventive Veterinary Medicine, Federal University of Minas Gerais-UFMG. HeLa cells are broadly accepted as a model cancer cell line for the preliminary assessment of biocompatibility of materials and devices. The HeLa

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cells were cultured in DMEM (Dulbecco’s modified eagle medium) with 10% fetal bovine serum (FBS), streptomycin sulfate (10 mg.mL-1), penicillin-G sodium (10 mg.mL-1), and amphotericin-B (0.025 mg.mL-1), all of them were supplied by Gibco BRL (New York, USA), using a humidified atmosphere of 5% CO 2 at 37°C. The cells were used for experiments on passage 89. 2.6 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay HeLa cells were plated (3 × 105 cells/well) in 96-well plates, where the cell populations were synchronized in serum-free media for 24h. After this period, the medium was aspirated and replaced with medium containing 10% FBS. Samples of CdX-MPA nanoconjugates (X=S, Se, Te) were added to individual wells at a concentration of 1%. Controls were used with the cells and DMEM with 10% FBS, the positive control Triton x-100 (1% v/v in phosphate buffered saline, PBS, Gibco BRL, NY, USA) and, as a negative control, chips of sterile polypropylene Eppendorf tubes (1 mg.mL-1, Eppendorf, Hamburg, Germany). After 24 and 72h, the medium was aspirated and replaced with 60 µL of culture medium with serum in each well and photographed using an inverted optical microscope (Leica DMIL LED, Germany). Next, 50 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) medium (5 mg.mL-1) (Sigma-Aldrich, MO, USA) was added to each well and was incubated for 4 h in an oven at 37°C and 5% CO2. Subsequently, 40 µL of the sodium dodecyl sulfate (SDS) solution/4% HCl was placed in each well and incubated for 16 h in an oven at 37 °C and 5% CO2. Then, 100 µL was removed from each well and transferred to a 96-well plate to quantify the absorbance (Abs) using a microplate absorbance reader (i-Mark, Bio-Rad®) with a 595-nm filter. The values obtained were expressed as the percentage of viable cells according to Eq. 1.

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) =

𝐴𝑏𝑠 (𝑠𝑎𝑚𝑝𝑙𝑒𝑠 𝑎𝑛𝑑 𝑐𝑒𝑙𝑙𝑠) 𝑥 100% Abs (control)

(1)

2.7 Fluorescent QD-based nanosensor for bioimaging and detection of cancer cells HeLa cells were plated in cover slips (5 × 104 cells/well) in 6-well plates and synchronized in serum-free media for 24h. The cells were incubated for 4 days in 5% CO2 at 37 °C. The CdX-MPA (X = S, Se, Te) nanoconjugate samples containing 50% of the medium solution was added to the HeLa cells. For the control, HeLa cells were incubated only with DMEM medium with 10% FBS (v/v). Next, the HeLa cells were 8

incubated for different time periods of 15, 30, 60 e 120 min in 5% CO2 at 37 °C. In the sequence, they were washed with PBS and the cells were fixed with paraformaldehyde (4%, v/v in PBS) for 30 min. Lastly, they washed three times with PBS and cover slips were mounted with Hydromount (Fisher Scientific Ltd., Leicestershire, UK). Images were obtained with a Zeiss LSM Meta 510 confocal microscope (Carl Zeiss, Germany) using the water immersion objective with an increase of 63× (1.4 NA). Argon laser was used to excite at =488 nm and emission was collected between 505 and 530 nm. These confocal images were collected in order to evaluate the time-dependence of the cellular uptake by endocytosis and, therefore, as a preliminary study of the potential application of these new CdX QD nanoconjugates as biocompatible fluorescent nanoprobes for the detection and sensing of live cancer cells. 3.

Results and Discussion

3.1 One-pot electrochemical preparation of CdX (X = Te, Se or S) The electroreduction of elemental tellurium in cavity cell has already been described by our work group, where the Te2- formed in the graphite powder cathode is expelled from the cavity due to electrostatic repulsion, reacting with the cadmium salt/stabilizer complex present in the intermediary compartment of the cell [12]. In this work, we describe a new one-pot QD synthetic method, wherein cadmium ions are electrogenerated by the Cd0 rod oxidation, at the same time that electroreduction of the elemental chalcogen, present in the cavity cell (Fig. 1). Therefore, Cd2+ and X2ions (X = Te, Se or S) are simultaneously introduced in the anodic compartment containing 3-mercaptopropionic acid (MPA) aqueous solution. The pH has a direct influence in the chalcogen reduction potential and also over species formed in solution. For electrolysis occurring at pH 7, according to Pourbaix diagrams of the Te, Se and S [17], it should be formed X2- (X = Te, Se or S) species in the cavity (Eq.2) with subsequent formation of the HX- ions in the anodic compartment (Eq. 3) [12]. The pKa of the H2X species are showed in Table 1, demonstrating that NaHX should be the chalcogen precursor for the related synthesis of QDs [18,19]. X0(s) + 2e- → X2-(aq)

(2)

X2-(aq) + H+(aq) → HX-(aq)

(3)

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Simultaneously, the oxidation of the cadmium sacrificial anode is carried out at the anodic compartment of the electrochemical cell, promoting the formation of Cd 2+ ions in solution (Eq. 4). The Cd2+ ions are produced at a constant rate of 0.15 mol.s1,

and can be produced even after total consumption of the chalcogen (X 0) present in

the cavity, generating an excess of 2 mol equivalents of Cd 2+ in solution. It is possible due to the reduction of the water present in the sintered glass and interfaced with graphite powder; hydrogen bubbles can be observed at this step. At pH 7, the MPA (HS(CH2)2COOH, pKa (COOH) = 4.32 and pKa (SH) = 10.20) can be represented as HS(CH2)2COONa, in solution (Eq. 5) [20]. Therefore, Cd2+ species added in solution can be stabilized through the formation of thiol complex [Cd(HS(CH2)2COONa)n]2+, Eq. 6. Cd0(s) → Cd2+(aq) + 2e-

(4)

HS(CH2)2COOH(aq) + NaOH(aq) ⇌ HS(CH2)2COONa(aq) + H2O(l)

(5)

Cd2+(aq) + nHS(CH2)2COONa(aq) ⇌ [Cd(HS(CH2)2COONa)n]2+(aq)

(6)

According to Eq. 3, HX- generated in the anodic compartment reacts with the [Cd(HS(CH2)2COONa)n]2+ complex, furnishing the nanoparticle precursors of the CdX(HS(CH2)2COONa) (Eq. 7). After the complete electrolysis at pH 7, the QD colloidal solution was transferred to a flask and the pH adjusted to 9 or 11, followed by heating at 95°C, aiming the study of the SH group deprotonation effect over the nanoparticle agglomeration process and optical/structural properties of the QDs (Eq. 8) [9,21]. HX-(aq) + [Cd(HS(CH2)2COONa)n]2+(aq) ⇌ CdX(HS(CH2)2COONa)n(aq) + H+(aq)

(7)

HS(CH2)2COONa(aq) + NaOH(aq) ⇌ Na(S(CH2)2COO)Na(aq) + H2O(l)

(8)

3.2 Optical and structural characterization of the QDs Absorption and emission spectra of the CdTe, CdSe, and CdS QD colloidal solutions were recorded after 60 min of heating at 95°C, pH 9 or 11, and are showed

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in Fig. 2. The growth profiles of the same nanoparticles were evaluated through the empirical equation developed by Yu et al. (Figs. 2C, 2F, and 2I) [16]. At pH 9, the absorption spectrum of CdTe-MPA QDs presented the typical first transition peak at abs = 498 nm and the respective emission maximum at em = 551 nm, with the full width at half maximum (FWHM) of 43.9 nm (Fig. 2A). When compared to the same nanoparticle system at pH 11 (Fig. 2B), it can be observed redshifts for the absorption abs = 510 nm and emission em = 567 nm spectra, and the FWHM = 47.4 nm of the emission remains slightly higher (Table S1, supporting information). Therefore, a higher monodispersivity was observed for CdTe nanoparticles after the thermal treatment of conjugates at pH 9. According to the absorption spectra of the CdSe-MPA and CdS-MPA QDs carried out at pH 11 (Figs. 2E and 2H, respectively), it can be observed two absorption peaks in each spectrum (abs = 409 nm and 467 nm for CdSe, and abs = 314 nm and 340 nm for CdS) corresponding to two nanoparticle populations formed at pH 11. The same behavior is not observed for CdSe-MPA (abs = 409 nm) and CdS-MPA (abs = 345 nm) QDs at pH 9 (Figs. 2D and 2G, respectively). FWHM of the emission peaks observed at pH 9 (93.6 nm for CdSe, and 111.7 nm for CdS) are almost the same values observed at pH 11 (93.0 nm for CdSe, and 103.3 nm for CdS) (Table S1, supporting information), indicating a monodispersivity tendency at pH 9, as observed for the CdTe-MPA QDs (Figs. S1 - S6, supporting information). The pH used for the nanoparticle growth also has effect on the size control, and indirectly on the nanoparticle stability (Figs. 2C, 2F, and 2I). Probably, it occurs due to the thiol group deprotonation of the stabilizer molecule, influencing equilibria involved in Eqs. 7 and 8. Gao et al. [22] showed that cadmium ions in presence of thiol stabilizers gives polynuclear complexes in aqueous solution, and the formation of such complexes depends on the solution pH. While Cd2+/MPA complexation is weaker at more acid pH, thiol groups should bind more strongly on the surface of the QDs. Therefore, the nanoparticle growth rate increases simultaneously with the pH increasing. Thus, the formation of a single population at pH 9 for all QDs produced shows that the nanoparticles are better stabilized at this pH condition. High resolution transmission electron microscopy (HRTEM) images were obtained for CdTe, CdSe, and CdS nanoparticles stabilized by MPA at pH 9 (Figs. 3A, 3B and 3C) and pH 11 (Figs. S7A, S8A and S9A). The microscopy showed 11

nanoparticles with crystalline structure dispersed throughout the grid surface, in presence of some agglomerates. The energy dispersive spectroscopy (EDS) analysis of the CdX-MPA nanoparticles show the elemental composition of the system, being observed peaks of Cd and Te attributed to CdTe (Fig. 3D), Cd and Se to the CdSe (Fig. 3E), and Cd and S, to the CdS (Fig. 3F). The sulphur and oxygen peaks observed in all EDS spectra can be assigned to the stabilizer; copper signals are due to the grid surface material (holey carbon-coated copper TEM support grid), and the carbon signal can be attributed to the MPA stabilizer and mostly to the grid surface. [23–25]. Based on 200 nanoparticles randomly selected from TEM images, their average diameters were calculated for pH 9 and pH 11 samples. The average diameters obtained for the CdX-MPA (X = Te, Se or S) QDs are showed in Table 2, and the size distribution histograms are showed in Figs. 3G, 3H and 3I (pH 9), and Figs. S7B, S8B and S9B (pH 11). The trend follows smaller nanoparticle sizes at pH 9 and larger sizes at pH 11, as seen by analyzing the absorption spectra of QDs CdX-MPA (Figs. 2C, 2F and 2I), confirming that the pH has an effect over the stability of the QDs, and the stabilizer have a greater surface interaction on nanoparticles at pH 9 [22]. The interplanar distance of nanoparticles CdX-MPA relating the (111) plane of the cubic crystal structure was calculated (inset Figs. 3A, 3B and 3C): 0.36 nm for CdTe and CdSe and 0.35 nm for CdS, which are consistent with the values found in the literature [26–28]. Hydrodynamic diameter (HD) and zeta potential (ζ) assays were performed (Table 2 and Fig S10). The DLS (dynamic light scattering) technique used for the nanoparticle HD determination take into account the inorganic portion (CdX), the stabilizer shell contribution (MPA), and the water solvation layers [29]. CdTe-MPA showed a smaller nanoparticle HD (10.2 nm at pH 9 and 12.1 nm at pH 11), increasing for CdSe (22.8 nm at pH 9 and 28.0 nm at pH 11) and CdS (30.9 nm at pH 9 and 44.4 nm at pH 11). Such behavior is due to solvation layers present in each QD. Regarding the HD measurements at different pHs, nanoparticles presented minor diameter at pH 9, these results corroborate nanoparticle sizes determined by HRTEM analyses, indicating a stronger interaction between the MPA and the CdX QDs at pH 9 [30].

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A negative charge surface was confirmed for all CdX QDs by zeta potential assays. As the thiol group has a pKa = 10.20 [31], at pH 11 QDs tend to have higher ζ-potential values (i.e., |±ζ|), which is a result of the increased thiol group deprotonation and major concentration of stabilizer molecules on the QDs surface. The quantum yield of luminescence (QY) was calculated for the QDs after 60 min of heating at 95°C. The CdX-MPA prepared at pH 9 showed higher QY than pH 11 (Table 2). There is an inverse correlation between QY and the amount of defects, i.e., the higher QY means a lower number of defects on the QD surface [22]. This is in according with results observed for the CdX-MPA nanoparticles prepared at pH 9, which showed a stronger interaction between the stabilizer and the QDs during nanoparticle growth, decreasing surface defects, thereby resulting in QY rising. More detailed results involving the optical characterizations of CdX-MPA (X = Te, Se and S) are summarized in Tables S1, S2 and S3. Figs. 4A (CdTe), 4B (CdS), and 4C (CdSe) show the Fourier transform infrared spectra (FTIR) of the CdX-MPA systems compared with the reference spectrum of MPA at the pH of synthesis. The changes in the intensities and/or shifts of wavenumber of bands can be mostly attributed to the interactions occurring between the functional groups of the ligand stabilizer and the CdX quantum dots. The main change detected in the spectra was the disappearance of the vibration band of thiol groups (~ 2550 cm 1,

detail in Fig. 4A) suggesting the formation of covalent bonds between –SH species

and Cd+2 at quantum dot surfaces as previously reported in literature for other metal ions [32,33]. At the detail inset, the spectra of Fig. 4(A) are presented in the range of 2700 - 2400 cm-1, where they plotted overlapped instead of stacked using Y-axis offset in order to emphasize the differences among the spectra. Fig. 5 shows the x-ray patterns for CdX-MPA (X = Te, Se and S) synthesized at pH 9 and 6 hours of heat treatment at 95°C. Peaks were observed regarding the plans (111), (220) and (311) for all QDs corresponding to cubic crystal structure of CdTe, CdSe and CdS [28,34,35]. The same X-ray diffraction pattern was observed for QDs at pH 11 (Fig. S11), showing no significant change in the nanocrystal structures, as reported in the literature [36].

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XPS experiments (X-Ray photoelectron spectrometry) were performed for CdTe nanoparticles and the results are showed in Figs. S12A and S12B, respectively. CdTe characteristic peaks were observed for cadmium at 411.6 eV (Cd 3d 3/2) and 404.8 eV (Cd 3d5/2), and for tellurium at 582.6 eV (Te 3d 3/2) and 572.3 eV (Te 3d5/2). The observed energy values are in agreement with literature and confirm the formation of CdTe nanoparticles [37]. Also, peaks were observed at 586.3 eV (Te 3d3/2) and 575.9 eV (Te 3d5/2), referring to the Te-O bond, showing the occurrence of tellurium oxides such as CdTeO3 [37,38]. The deconvolution of the XPS spectra for cadmium and tellurium make possible to calculate the area ratio in the spectra, the Cd/Te proportion found in nanoparticles was found 2.8:1; very close to the amount used in the electrosynthesis reaction, which was 3:1. 3.3 Evaluation of Cytotoxicity of CdX Nanoconjugates by MTT Assay Although colloidal quantum dots have been studied for over 3 decades, the potential toxicity of cadmium-containing quantum dots is always at the center of biocompatibility discussions and

yet

very controversial

[39,40]. The most

comprehensive compiled study so far of Cd-containing QDs has just been published in Nature Nanotechnology by Medintz’s research group [40], where the authors conducted an extensive meta-analysis of cellular toxicity. They concluded that the toxicity response of cells toward Cd-based QDs is indisputably a very complex coupling of attributes and, therefore, poses great challenges for creating a generalized cause– effect relationship. Nonetheless, over 50% of articles published used MTT assay method for assessing the potential in vitro toxicity and cell viability towards QDs. In short, the enzyme-based MTT method relies on a reductive, colored reagent and the presence of dehydrogenase in the viable cells to determine their viability based on mitochondrial function using a colorimetric method. This method is far superior to other similar methods because it is easy-to-use, safe, highly reproducible and widely used in both cell viability and cytotoxicity tests. Hence, in this work the cytotoxicity of CdXMPA conjugates was evaluated by the effect on the cellular metabolic viability through MTT assay and morphological observation of HeLa cells by microscopy analysis after 24 h incubation. Therefore, the mitochondrial metabolic activity results of HeLa cells towards CdS, CdSe and CdTe QD conjugates after 24 h are presented in Fig. 6A. It can be clearly observed that all CdX-MPA samples showed no toxicity with the cell

14

viability responses above 87% (lowest value of CdTe), which are statistically equivalent to the control sample. Thus, these results demonstrated that CdS, CdSe and CdTe QDs capped with MPA ligands are non-toxic nanoconjugates for HeLa cell type according to the ISO standards 10993-5:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity, i.e., cell viability >50%). Moreover, as HeLa cells are the most widely used model cancer cell line in the world derived from cancerous cervical tumor [41], the cytocompatibility of the QD-conjugate produced in this research was confirmed via microscopy images where typical morphological features of viable cells were observed [American Type Culture Collection, ATCC CCL2] and with approximately 100% confluence for the CdX-MPA systems (Fig. 6B). 3.4 Fluorescent QD-Based Nanosensor for Bioimaging and Detection of Cancer Cells In recent years, biosensors have gained enormous attention in nanomedicine and nanotechnology and there is a growing interest for its application in cancer detection and diagnosis. Despite significant progress in the treatment and therapy, cancer remains a great challenge against global public health and a huge burden on society. The main inadequacy of current diagnostic imaging methods is the relatively low sensitivity in the detection of cancer at an early stage, which is considered crucial for better chances for its remission and prognosis in the good quality of life of the patient. In that sense, the major influences of nanotechnology on biosensor development involve nanomaterials, mainly quantum dots, as they can facilitate diagnosis and tracking of cancer cells, allow for more sensitive imaging systems that can detect cancer at an earlier stage [42]. Quantum dots are widely used as biomarkers for cell imaging and sensing as they possess unique strong fluorescent behaviour at narrow emission band and high chemical stability. Therefore, in this study, it is presented as a proof of concept, the use of CdX-MPA conjugates as fluorescent nanoprobes for monitoring the uptake of nanoparticles by HeLa cancer cells using confocal bioimaging correlating with the time of incubation for prospective applications as nanosensors in oncology. Moreover, these bioassays were designed focusing on collecting bioimages at gradual time intervals for assessing the endocytosis activity, which is the key biological process responsible for the internalization of nanoparticles by cells. Figs. S13A, S13B and S13C show the confocal fluorescent images of HeLa cells incubated at different times (i.e., 0, 15, 30 and 60 min) with the CdS-MPA, CdSe-MPA

15

and CdTe-MPA nanoconjugates, respectively. It was detected a significant increase of the fluorescence intensities associated with the increase of incubation time for all CdXMPA conjugates, which demonstrated that these nanoparticulate systems not only effectively penetrated through cancer cell membranes allowing biolabeling but also proved the continuing endocytosis by the cells with further intracellular scattered distribution within the cytoplasmic matrix. These effects were qualitatively referred to as system “OFF”, when no fluorescent signal was detected (Figs. S13A, S13B, S13C: (a)), and “ON”, when fluorescent emissions were detected ((Figs. S13A, S13B, S13C: (b), (c) and (d)), which resembles the behaviour a nanodetector for cancer cells based on fluorescent output signal. It is important to highlight that these are initial qualitative bioimaging results as the fluorescence emission of nanoconjugates composed of semiconductor core and ligand shell (CdX-MPA) is a very complex phenomenon with multiple contributions such as chemical composition of the semiconductor and ligand, density of crystalline defects of QDs, surface states and defects, average size and distribution of QDs, chemical interactions at the nanointerfaces. In order to have a more in-depth investigation of the interactions between the CdXMPA nanoconjugates and HeLa cells, the intensity of the fluorescent emission was correlated to the incubation time and the results are presented in Figs. 7A, 7B and 7C, for CdS, CdSe and CdTe QDs, respectively. A similar trend was observed for all systems, where it was clearly verified a continuous increase of fluorescent emission intensity with the incubation time, which correlated quantitatively, consistent with one of the primary aspects of biosensing. These findings support the hypothesis that the CdX-MPA conjugates underwent cellular uptake through endocytic pathways and transported within the endosomal system. Therefore, these results demonstrated the biocompatibility and fluorescent bioimaging properties of the CdX-MPA nanoconjugates with promising characteristics to be utilized as nanoparticulate-based biosensor for cancer cell tracking, diagnosis and therapy. It is well known by the literature that QDs can be internalized into cells through a number of different pathways, including transfection, peptide-mediated delivery, and endocytosis [43,44]. However, a comprehensive understanding of the actual cellular process of the uptake and subcellular localization and distribution of QDs is very limited [45,46]. Hence, it can be envisioned that these novel CdX-MPA nanoconjugates can be feasibly used as nanosensor for targeting external and intracellular receptors of cancer cells and as fluorescent nanoprobes combined with specific biomarkers for tracking endocytosis pathways because they proved consistent time-dependent internalization via transport across the cell plasma membrane and intracellular distribution inside the cytosol. Nonetheless, further experimental design and new studies are needed for calculating the correlation between the fluorescence response and the cell imaging detection, focusing on the development of a complete biosensor analytical tool (i.e., lower and 16

upper limit of detection, mathematical empirical correlation and function, specificity, selectivity, etc). 4. Conclusion The application of the graphite powder cavity cell and cadmium sacrificial anode was well succeeded for the production of CdX (X = Te, Se, S) QDs by a fully electrochemical method in aqueous medium pH 7. This one-pot methodology proposes a green alternative for existing methodologies in the literature, avoiding chemical reducing agents that correspond to atomic economy and less hazardous chemical syntheses. Furthermore, the proposed method allows the controlled addition of precursors to the anodic compartment of the cell, where each reagent equivalent is simultaneously generated on the anode (Cd2+) and cathode (X2-). The influence of pH was observed during the procedure of heat treatment used for the growth of nanoparticles. Better luminescence quantum yields (~10%) were observed after 60 min of heating at pH 9. The chemical composition of the CdTe was identified by XPS (2.8:1), which is consistent with the initial precursors ratio used in the synthesis 3:1. MTT assays based on mitochondrial metabolic activity of HeLa cancer cells showed no toxicity after 24 h of incubation with CdX-MPA nanoconjugates. Moreover, these electrochemically synthesized nanoparticles with environmentally-friendly and biocompatibility properties were effective as preliminary fluorescent nanosensors for the detection and bioimaging of HeLa cancer cells and monitoring the endocytosis activity with the time of incubation. Acknowledgements The authors acknowledge the financial support from the following Brazilian research agencies: CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROEX-433/2010;PNPD), FACEPE - Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (APQ-1991-1.06/12), FAPEMIG – Fundação de Amparo à Pesquisa do Estado de Minas Gerais (PPM-00202-13;BCN-TEC 30030/12), CNPq - Conselho Nacional de Pesquisa (PQ1B-306306/2014-0, PQ2-302271/2014-7, PDJ168183/2014-5 and UNIVERSAL-457537/2014-0), and FINEP – Financiadora de Estudos e Projetos (CTINFRA-PROINFRA 2008/2010). The authors express their gratitude to the staff at the Microscopy Center at UFMG for their assistance with TEM-EDS analysis. Finally, the authors thank the staff at the Center of Nanoscience, Nanotechnology and Innovation- CeNano2I/CEMUCASI/UFMG for the spectroscopy analyses.

17

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Biographies Denílson V Freitas is bachelor in chemistry (2013) and received his master degree in chemistry (2015) from Federal University of Pernambuco, Brazil. Now he is pursuing for PhD degree in the same University. His research interests are in the electrochemical preparation of quantum dots and applications. Sérgio G B Passos is bachelor in chemistry (2012) from Católica University (Recife, Brazil) and received his MSc degree (2015) in chemistry from Federal University of Pernambuco, Brazil. Now he is pursuing for PhD degree in the same University. His research interests are in the electrochemical preparation of quantum dots and applications. Jéssica M M Dias is bachelor in chemistry (2009), master in chemistry (2011) and received her PhD degree in chemistry (2015) from Federal University of Pernambuco, Brazil. Now she is pursuing her post doctoral stage in the same University. Her research interests are in the preparation of quantum dots and applications. Alexandra A P Mansur: Civil Engineer (1997), master in Materials Chemistry (2002) and PhD in Materials Chemistry of Nanostructures (2007) from Federal University of Minas Gerais (Brazil). Post doctoral stage at Clarkson University (NY, USA) in bioconjugates and nanohybrid systems. Since 2007, she joined to the Materials and Metallurgical Department at Federal University of Minas Gerais (Brazil) in the position of postdoctoral research fellow. She has over 70 peer-reviewed papers published in high quality indexed journals and over 150 presented in Conferences and Proceedings (H index = 17 and over 1200 citations ISI/JCR). Sandhra Maria de Carvalho: Degree Dentistry (1986), master in Biomaterials (2010) and PhD in Materials Science and Biomaterials (2014) from Federal University of Minas Gerais (Brazil). Research visitor at Institute of Biomedical Engineering (Portugal, 2013). Since 2014 she joined to the Materials and Metallurgical Department at Federal University of Minas Gerais (Brazil) in the position of Postdoctoral research, in the field of advanced biological assays for materials science and engineering applications. She has approximately 30 peer-reviewed papers published in high quality indexed journals and 30 presented in Conferences and Proceedings. Herman Mansur: Metallurgical and Materials Engineer (1985) and master in Chemistry of Semiconductors (1992) from Federal University of Minas Gerais (Brazil). PhD in Chemistry of Nanostructures from Melbourne University (Australia)/UFMG (1996). Post doctoral stage at Clarkson University (NY,USA) in bioconjugates and nanohybrid systems. In 1998, he joined to the Materials and Metallurgical Dept. at Federal University of Minas Gerais (Brazil) where he is currently Full Professor. He has over 150 peer-reviewed papers published in high quality indexed journals and over 400 presented in Conferences and Proceedings (H index = 29 and over 2500 citations ISI/JCR) and 6 patents deposited. He has attended over 20 international conferences as invited speaker and chaired 15 international scientific sections. He is a member of the Royal Society of Chemistry (RSC). Marcelo Navarro: Bachelor (1988), master (1991) and PhD degree in chemistry (1994) from University of São Paulo, Brazil (1994). Post doctoral stage (1996) at Joseph Fourier University, France, in electrochemical polymerization. Presently, he works as professor at the Federal University of Pernambuco since 1999. He has over 70 peer-reviewed papers published in high quality indexed journals and over 120 presented in Conferences and Proceedings. His research scope covers electrochemistry, electrosynthesis, conductor polymers and quantum dots. He is member of the Brazilian Society of Chemistry (SBQ), American Chemical Society (ACS) and International Society of Electrochemistry (ISE).

22

Figure Captions Fig. 1 Electrochemical cavity cell used for CdX-MPA (X = Te, Se, S) preparation. Fig. 2 UV-Vis absorption and emission spectra after heat treatment at 95°C for electrochemically synthesized CdX-MPA solutions (X = Te, Se, S): CdTe (A), CdSe (D) and CdS (G) at pH 9, and CdTe (B), CdSe (E) and CdS (H) at pH 11. Particle size vs. heating time (95 °C) for CdTe (C), CdSe (F) and CdS (I) QDs at pHs 9 and 11. Fig. 3 Typical bright-field HRTEM images for CdTe (A), CdSe (B) and CdS (C) QDMPA systems. EDS analysis of the CdX-MPA nanoparticles (X = Te (C), Se (D), and S (F); and corresponding crystallite size distribution histograms (CdTe (G), CdSe (H) and CdS (I)). Fig. 4 DRIFTS spectra for MPA (a) and CdTe-MPA (A), CdS-MPA (B) and CdSe-MPA (C), at pH 9 (b) and 11 (c). Fig. 5 XRD patterns of samples of CdX-MPA (X = Te, Se, S) QDs prepared after 6 hours of heat treatment (95°C). Fig. 6 (A) HeLa cell viability response by MTT assay towards CdS, CdSe and CdTe QD conjugates (positive control: Triton x-100; negative control: chips of sterile polypropylene Eppendorf tubes). (B) Microscopy images of HeLa cells incubated for 24 h with control culture (a), CdS (b), CdSe (c) and CdTe (d) QD conjugates with typical morphological features and approximately 100% confluence (scale bar = 100 μm, 100X). Fig. 7 Plot of fluorescent signal of the CdX-MPA nanoconjugates internalized via endocytic pathways by HeLa cancer cells versus the incubation time: (A) CdS; (B) CdSe; (C) CdTe; Inset: Schematic representation of the gradual endocytosis of CdX quantum dots with time-dependency characteristics.

23

Figure 1

24

(C)

(A)

(B)

CdTe

CdTe

pH 9

pH 11

CdTe

(D)

(E)

(F)

CdSe

CdSe

pH 9

pH 11

CdSe

(G)

(H)

(I)

CdS

CdS

pH 9

pH 11

CdS

Figure 2

25

(C)

(B)

(A)

0.36 nm

0.36 nm

(D)

(G)

(E)

0.35 nm

(F)

(H)

(I)

Figure 3

26

Figure 4

27

Figure 5

28

A

Cell viability (% of control)

100

80

60

40

20

Control

(a) a

CdS

100µm

(b) a

100µm

Cd Te

Cd Se

Cd S

Co nt ro l-

Co nt ro l+

B

Co nt ro l

0

CdSe

(c)

100µm

CdTe

(d)

100µm

Figure 6

29

14

CdS

“ON” “ON”

12

Cytosol CTFC CdS (r.u.)

10

QD

8

“OFF”

6

Cancer cell

4

Endosome 2

A

0

Cytosol 0

10

20

30

40

50

60

Time (min)

QD

CdSe

14 12

Cancer cell CTFC CdSe (r.u.)

10

Endosome

“ON”

8

Cytosol

6

“OFF”

4

Endosome

QD

2

B 0 0

10

20

30

40

50

Cancer cell

60

Time (min)

CdTe

14

Cytosol 12

QD

CTFC CdTe (r.u.)

10

“ON”

8 6

Endosome

“OFF”

Cancer cell

4 2

C

0 0

10

20

30

40

50

60

Time (min)

Figure.7

30

Table 1. pKa of the electrochemicallly generated species [17,18]. Entry

H2Te

H2Se

H2S

1

pKa1

2.64

3.73

6.93

2

PKa2

10.79

-

-

31

Table 2. Hydrodynamic radius (HD), zeta potential (ζ) and quantum yield (QY) for CdXMPA QDs (X = Te, Se or S). CdX-

HD (nm)

ζ (mV)

QY (%)

Diameter

Entry MPA/pH

HRTEM (nm)

1

CdTe/9

10.2 ± 0.4

-18.7 ± 2.9

9.6 ± 1.2

3.04 ± 0.60

2

CdTe/11

12.1 ± 0.3

-25.1 ± 3.0

3.9 ± 0.2

3.88 ± 0.82

3

CdSe/9

22.8 ± 0.6

-36.2 ± 2.3

1.9 ± 0.1

3.58 ± 0.58

4

CdSe/11

28.0 ± 0.4

-34.0 ± 3.2

1.0 ± 0.2

4.44 ± 1.01

5

CdS/9

30.9 ± 5.3

-30.6 ± 3.1

2.6 ± 0.7

3.44 ± 0.66

6

CdS/11

44.4 ± 6.8

-35.5 ± 2.5

1.5 ± 0.1

3.59 ± 0.63

32