Two-Photon Active Boron Nitride Quantum Dots for Multiplexed

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Two-photon active boron nitride quantum dots for multiplexed imaging, intracellular ferric ion biosensing and pH tracking in living cells Alireza Dehghani, Sara Madadi Ardekani, Pooria Lesani, Mahbub Hassan, and Vincent G. Gomes ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00145 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Two-photon active boron nitride quantum dots for multiplexed imaging, intracellular ferric ion biosensing and pH tracking in living cells Alireza Dehghani,a Sara Madadi Ardekani,a Pooria Lesani, a Mahbub Hassana, Vincent G. Gomes∗a aThe

University of Sydney, School of Chemical and Biomolecular Engineering, Sydney, NSW 2006, Australia

*Corresponding author. E-mail: [email protected]; Fax: +61293512854; Tel: +61293514868

Keywords: boron nitride; quantum dots; fluorescence lifetime; multiplexed imaging; intracellular sensing; two-photon excitation

Abstract Nanoparticles are key vehicles for targeted therapies because they can pass through biological barriers, enter into cells and distribute within cell structures. We investigated the synthesis of blue/green emissive hexagonal boron nitride quantum dots (hBNQDs) using a liquid exfoliation technique followed by hydrothermal treatment. A distinct shift from blue to bright green emission was observed on surface passivating the dots using poly (ethylene glycol) or PEG200 under the same UV-irradiation. The quantum yield of the hBNQDs increased with the surface passivation. Multiplexed imaging was accomplished using the hBNQDs in conjunction with organic dyes. The hBNQDs provided images with distinctive emission wavelengths and fluorescence lifetimes. Though the fluorescence signals of blue/green-emissive hBNQDs overlap spectrally with those of the emission wavelengths of the organic dyes, the fluorescence lifetime data was resolved temporally using software-based time gates. The blue emissive hBNQD-b quantum dots were validated as sensitive platforms for detecting intracellular ferric ions with a low limit of detection (20.6 nM). The green emissive hBNQD-g quantum dots successfully identified intracellular variations in pH, and localization of human breast cancer cells as a function of their life cycles via fluorescence lifetime imaging.


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Abbreviations AFM CLSM

Atomic force microscopy Confocal laser scanning microscopy

FFT FLIM

Fast Fourier transform Fluorescence lifetime imaging microscopy

FTIR

Fourier transform infrared

hBN

Hexagonal boron nitride

HEPES

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

HRTEM

High-resolution transmission electron microscopy

HOMO

Highest occupied molecular orbital

LUMO NIR

Lowest unoccupied molecular orbital Near infrared

PEG

Poly (ethylene glycol)

QD

Quantum dot

QY

Quantum yield

SNR

Signal to noise ratio

TCSPC TEM

Time correlated single photon counting Transmission electron microscopy

TPE XPS

Two-photon excitation X-ray photoelectron spectroscopy

1. Introduction Graphene and carbon quantum dots have been instrumental in inspiring research into metal-free low dimensional nanomaterials suitable for bio-applications1,2. Hexagonal boron nitride (hBN) has attracted a great deal of interest recently because of its unique physicochemical and structural properties. In contrast to graphene, hBN monolayer shows localized electronic states, which lead to its superior chemical and thermal stabilities. This is due to its surface ionicity compared to graphene covalent C–C bonds3. Excellent electrical insulation, thermal stability and high mechanical strength and inertness of hBN have led to a variety of applications in optoelectronics 4 energy conversion 5, catalysts 6 and nanomedicine 7. Compared to the already investigated 2D BN nanosheets, hBN

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quantum dots (hBNQDs) have been characterized to a much less extent. Initial attempts at the synthesis of hBNQDs focused on improving the quantum yield (QY) of the nanoparticles using various synthesis methods. Lin et al.8 successfully fabricated monolayer hBN-QDs with 2.5% QY using a three step synthesis process. Further studies by Lei et al 9, Xue et al 10, and Thangasamy et al. 11, reported QYs of 8.6, 1.8 and 2.5%, respectively. A study by Li et al 12 on the effect of reaction parameters on the final properties of hBNQDs found that increasing the reaction time and temperature resulted in smaller QDs with narrower size distributions. Interestingly, smaller QDs were obtained on lowering the hydrothermal reactor filling capacity from 66.7% to 40%. The authors reported a relatively high QY of 19.5% at optimal conditions. Subsequently, Liu et. al 13 studied the effect of solvent on the final properties of hBNQDs. The emission was red-shifted by changing the polarity of the solvent with the highest QY of 21% for the hBNQDs synthesized in N-methyl-2 pyrrolidone (NMP) medium. Despite the high QY, the use of the organic solvent pose challenges for bioimaging and drug delivery applications where an aqueous medium is desired. Multiplexed imaging – an extension of spectral imaging provides unique opportunities for simultaneously colocalizing multiple components within cells. The widely used dye based bioimaging suffers from major limitations of photobleaching and instability. Therefore, capturing high-resolution images using a photostable, non-blinking and non-bleaching system would be highly desirable. Most importantly, probes with overlapping emission wavelengths but different fluorescent lifetimes can be resolved via temporally multiplexed imaging. The combination of spectral and temporal imaging increases the number of resolvable channels and opens new avenues for investigating cell/nanoparticle interactions. This method has the potential of precise drug delivery and biosensing using biocompatible fluorophores, such as, hBNQDs in conjunction with commercially available organic dyes, where the user can resolve


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the fluorescence signals spectrally and temporally based on emission wavelength and fluorescence lifetime values, respectively. Image quality at visible wavelengths is negatively affected, especially if low concentrations of fluorophores are used because of light scattering and interference from cell auto-fluorescence, requiring new approaches to enhance the signal-to-noise ratio (SNR)14. Low concentrations of fluorophores necessitate the use of strategies to reduce auto-fluorescence and improve SNR. The FLIM modalities at NIR wavelengths are able to successfully distinguish lifetime values and provide methods to sort cell populations. Furthermore, when co-incubated with dyes, lifetime distributions of cells allow estimate of the relative concentrations of nanoparticles and dyes in complex cellular micro-environments. Thus, real-time fluorescence imaging requires advances in new fluorescent probes in conjunction with analytical tools. The inertness and stability of hBNQDs provide unique opportunities for 3D fluorescence imaging of biological samples at NIR wavelengths using multi-photon fluorescence microscopy and offers the advantages of less photodamage15. In multi-photon microscopy, photodamage to biological specimens is reduced to sub-femtoliter volume which can result in increased viability for the samples, especially during extended periods of imaging 16,17. Typically, a fluorophore that can be excited at a particular wavelength can potentially be excited by absorbing two photons of nearly twice the wavelength at the same time. However, these two excitation processes are fundamentally different with very dissimilar selection rules. A fluorophore that can be excited by one-photon at a particular wavelength might not necessarily show good two-photon absorption/emission. The existing gaps in knowledge in this field motivated us to explore an efficient and green synthesis method, gain an understanding of the unique PL properties and investigate the two-photon excitation (TPE) features of hBNQDs for application in multiplexed cell imaging and intracellular ferric ion sensing. The relatively longer fluorescence lifetime values of hBNQDs enables the use of fluorescence lifetime imaging microscopy (FLIM) to accurately distinguish the emission signals of nanoparticles from relatively shorter cell autofluorescence and those from organic dyes. This

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important property, when combined with imaging techniques enable applications where determining the precise position and translocation of nanoparticles is crucial. Here, we report green synthesis of hBNQDs and tuning of their emission from blue to green spectrum through hydrothermal treatment of bulk hBN sheets followed by surface passivation. The morphological and optical characteristics of the synthesized hBNQDs were investigated along with their two-photon excitation (TPE) capabilities. The non-passivated hBNQDs were first tested for detection of ferric ions in mixtures. Subsequently, the application of hBNQDs via spectral and temporal imaging were explored. In particular, the fluorescence lifetime decay was used to track the intracellular distribution and endocytic pathways of nanoparticles as the cells advanced through the cell cycles over a 4 hr period.

3. Method 3.1 Materials Dulbecco's modified Eagle's medium (DMEM, high glucose), fetal bovine serum (FBS) and Trypsin were acquired from Gibco BRL (San Francisco, CA, USA). The RL-14 human fetal ventricular cardiomyocytes and MCF-7 breast cancer cell lines were obtained from Lonza (Allendale, NJ, USA). Boron nitride (98%, AR), potassium hydroxide (90%, AR), quinine sulfate, PEG200, Aluminium, ferric, copper, ferrous, magnesium, manganese and zinc chlorides, lead acetate, nickel nitrate, solvents and other media were procured from Sigma-Aldrich (NSW, Australia). 3.2 Characterization TEM and HRTEM images were acquired on a JEOL 2200 transmission electron microscope (200 kV). A Thermo Scientific Nicolet 6700 spectrometer was used for the Fourier transform infrared (FTIR) spectra of samples. The X-ray photoelectron spectroscopy (XPS) measurements were carried out with a Thermo Scientific ESCALAB 250Xi X-ray Photoelectron Spectrometer microprobe using a monochromatic Al-K X-ray source (h = 1486.68 eV). Fluorescence ACS Paragon Plus Environment

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spectroscopy was performed using a Fluoromax-4 spectrofluorometer (Horiba Scientific, USA). UV-Vis absorption spectra were obtained on a Cary 60 UV-Vis Spectrophotometer (Agilent Technologies). The PL lifetime decay profile and mathematical fitting was done on a Nikon A1 inverted confocal microscope (412 nm excitation), with a 550 nm long-pass filter. The CLSM imaging was carried out using a confocal microscope equipped with a multi-photon laser at 780 nm (Leica TCS SP II). 3.3 Synthesis of hBNQDs The bulk hBN flakes were exfoliated to hBN sheets by solvent exfoliation. Briefly, 1.0 g hBN was added to a 3M solution of KOH in EtOH and stirred until the hBN sheets were dispersed in the solution and sonicated for one hr using a tip sonicator. After EtOH evaporation, the solid was transferred into a furnace at 400 ºC for four hr (N2 atmosphere, ramp rate 10 ºC/min) and cooled to room temperature (same cooling rate). The solid was then crushed into a powdery form and washed several times to remove the KOH and dispersed in 20 mL DI water (pH 7.0) followed by sonication for 1 hr. The mixture was transferred to a stainless-steel autoclave with a Teflon liner (100 mL capacity with 40% filling) and kept at 240 ºC for 8 hr. The autoclave was cooled to room temperature and the final product was collected after vacuum filtration and centrifugation (twice at 5000g; 30 min). The aqueous media containing hBNQDs was dialyzed using a 30 kDa membrane. Surface passivated hBNQDs were prepared by adding 2 g PEG200 to the synthesized hBNQDs as described above on a hotplate for five minutes (180 ºC). Unreacted PEG was removed by dialysis using a 3000 kDa centrifuge column. 3.4 Quantum yield measurements The quantum yield (QY, ϕ) of the synthesized hBNQDs was calculated following our previously published procedure 2. In brief, the absorbance peak and the integrated fluorescence intensity (excitation at 320 nm) of the samples at five different concentrations were recorded. The QY values

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were calculated by comparing the gradient of the plotted graph (absorption vs integrated emission intensity) to a reference sample (quinine sulfate). ீ௥௔ௗ

ೣ ߶௫ = ߶௦௧ ∗ ቄ ቅ ∗ 100 ீ௥௔ௗ ೞ೟

(1)

3.5 Cell culture and cell viability The RL-14 cells were cultured in full media (DMEM with 10% fetal bovine serum or FBS). Cell suspensions were incubated (Thermo Electron Corporation, USA) at 37 ºC with 95% humidity 5% CO2. A TC10 cell counter was used to measure the cell viability. Briefly, cells were seeded in a six-well plate in 1 mL full media (75k cells/well). After 12 hr of incubation, the media was removed and hBNQDs were suspended in fresh media to obtain final concentrations of 20 to 700 μg/mL (1 mL final volume) and incubated for 24 hr. The cells were washed with sterile PBS and 10 μL trypsin was added; the cells were then centrifuged and re-dispersed in fresh media. 10 µL of cell suspension was mixed with the trypan blue dye at 1:1 ratio and inserted into the counting slides. Untreated cells were used as the control. 3.6 Spectral and temporal multiplexed cell imaging Fluorescence cell images were taken on a Leica TCS SP5 II inverted confocal microscope. The RL-14 cells were cultured in 35 mm Mattek dishes (density of 105 cells/well) and incubated at 37 ºC in a 5% CO2 incubator for 24 h. After the removal of the media, hBNQDs suspended in full media (500 μg/mL) was added (1 mL final volume) for 24 hr incubation. The cells were washed with PBS (×2) to remove extracellular hBNQDs and fixed with ethanol. For spectral microscopy, images were acquired with a CLSM (Leica TCP SP5 II), 25× water immersion objective. Two detectors were used to collect two sets of images: (a) In the blue channel with hBNQD-b (430-470 nm); (b) in the green channel with hBNQD-g (500-550 nm). The images were taken using proprietary software (Leica LAS X) and analyzed using Image J (NIH, US).


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Fluorescence lifetime imaging microscopy (FLIM) in conjunction with time-correlated single photon counting (TCSPC) was applied for imaging the cells using the inverted microscope (Leica TCS SP5 II) equipped with a FLIM system (Becker and Hickl, SPC830, TCSPC) having 256 × 256 pixel resolution and 256-time channels with a 25× water immersion objective. A 780 nm picosecond pulsed laser with 40 MHz repetition rate was used as the excitation source. Bi-exponential decay fits using an incomplete decay model was used to calculate the fluorescence lifetime values in the FLIM image. 3.7 Fluorescence “turn-off” assay for sensing Fe3+ ions The detection of Fe3+ was performed in DI water at room temperature. In a typical procedure, 200 µL of the hBNQD-b containing aqueous solution (200 µg/mL) was added to 2.8 mL of DI water in a spectrophotometer quartz cuvette to form the probe solution. Then 10 µL of a known concentration of Fe3+ (10-600 µM) stock solution was added to the cuvette. The cuvette was vibrated for 5 minutes to form a homogenous mixture. The fluorescence spectra were recorded (345-600 nm) with excitation at 335 nm. For the intracellular Fe3+ sensing, MCF-7 cells were cultured in a T25 flask (90% confluency) and then lysed using iced water. The cells were then scraped from the flask and dispersed in HEPES solution. Then, 200 µL of the hBNQD-b containing aqueous solution (200 µg/mL) was added to 2.8 mL of the cells/HEPES solution in a spectrophotometer quartz cuvette to form the probe solution. Then 10 µL of a known concentration of Fe3+ (100-20 µM) stock solution was added to the cuvette. The cuvette was vibrated for 5 minutes to form a homogenous mixture. The fluorescence emission spectra were recorded (range: 345-600 nm) with excitation at 335 nm.

4. Results Several techniques were employed to characterize the physical and morphological properties of hBNQDs. Figure 1 shows the TEM, HRTEM, FFT, particle size distribution and AFM images of hBNQD-b. The as-prepared sample shows excellent dispersion in water. The measured particle

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sizes with the most probable size of 1.8 േ 0.3 nm follows a Gaussian distribution. The HRTEM images display crystalline structure with a lattice spacing of 0.21 nm, corresponding to (100) face of the BN crystal. The topographic height of hBNQD-b was 2.0 േ 0.5 nm (Figure 1C), suggesting that most of the hBNQDs had few layer boron nitride. These findings are in accord with the sharp decrease in peak intensity for the hBNQDs as compared to the BN raw materials at 1366-1370 cm-1 given by the Raman spectra (Figure S1, ESI). The decline in E2g phonon mode is consistent with previous studies and suggests the formation of a few layer structure of hBNQDs. Since the short (five minute) reaction to passivate the surface of the hBNQDs with PEG does not incur any significant changes in size and morphology, we conclude that the color shift is due to changes in functional groups that form on the surface of hBNQD-g after surface passivation.

Figure 1. (A) TEM image, (B) Particle size distribution and (C) AFM image of hBNQD-b. (Insets: (A) HRTEM image of hBNQD-b, FFT and reverse FFT images; (C) height profile at selected black line of AFM image) The surface functional groups of hBNQDs were analyzed by FTIR and XPS measurements. The similar FTIR spectra of the two hBNQDs samples (Figure S2 ESI) indicate their similar chemical compositions. Both hBNQDs showed absorption peaks at 1350 and 830 cm−1, which is attributed to the B-N stretching and bending modes, respectively 8. Characteristic peaks in the FTIR spectra of hBNQDs at 1060, 1350 and 1610 corresponding to B-O, B-N and C-N bonds, respectively showing the formation of oxygen-rich functional groups and the attachment of the solvent molecules on the


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surface of hBNQD 13. As expected, the two samples showed similar spectra indicating their similar chemical structure. Due to the high polarity of B(-)=N(+) in the hBN structure, the reaction between the PEG and hBNQDs resulted in the formation of nitrile bonds as shown in the FTIR spectrum at 1900-2100 cm-1. The CN bond is strongly polarized towards nitrogen, resulting in high molecular dipole moments. XPS analysis to characterize the chemical composition of hBNQDs in Figure 2 shows four dominant peaks at 535.2 eV (O1s), 401.2 eV (N1s) and 285.2 eV (C1s) and 193.1 eV (B1s) in the full XPS survey spectra. The presence of the O1s peak indicates that the EtOH/water molecules can attach to the surface of the hBNQDs. Figure 3D and H show the deconvoluted spectra of B1s. Peaks centered at 190.5 eV and 191.1 eV are attributed to (B-N) and (B-O) bonding, respectively. The peaks at 398.3 eV and 399.6 (399.8 or 399.4) eV in the high-resolution spectra of N are assigned to N-B and N-O/N-C, respectively. The relative intensity ratio of

(஻ିை)

(஻ିே)

and

(ேିை) (ேି஻)

increased after

surface passivation. The surface charge properties of hBNQDs, obtained by measuring their ߞpotentials (-21.1 and -35.1 mV for hBNQD-b and -g, respectively) also indicates the presence of additional negatively charged oxygen-rich functional groups on the surface of the hBNQD-g.

Figure 2. XPS full survey spectra (A, E) and high resolution XPS spectra of C1s (B, F), N1s (C, G) and B1s (D, H) of hBNQD-b and g, respectively

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Based on the characterization discussed earlier the following formation mechanism is proposed for the synthesis and color shift for hBNQDs. During the K-hBN exposure, highly oxidative KO2 forms, which is responsible for the presence of O-dopants bound to the B sites during the heat treatment process 8, while the C dopants arise mainly from the ultrasonication treatment in the presence of EtOH. On implementing sonication in the presence of 3M KOH in EtOH, potassium intercalated hBN sheets start to form because of the weak interlayer van der Waals forces 8. The heat treatment in the presence of K leads to the formation of surface defects on the edges and the basal planes of hBN sheets and partial formation of oxygen-containing groups which act as chemically reactive sites and break down the hBN sheets to subsequently form hBNQDs on hydrothermal reaction. UV-Vis absorption and PL spectroscopy of hBNQDs were explored to understand the correlation between the synthesis process and changes in the optical properties of hBNQDs (Figure 3). The absorption spectrum of the hBNQD-b shows a peak at 260 nm (4.77 eV). However, a second peak appears after the surface passivation reaction at 330 nm (3.76 eV). The two hBNQDs show blue and green luminescence under the same UV irradiation (360 nm) but show different PL responses when excited using variable wavelengths. After surface passivation, the maximum excitation intensity for hBNQD-g changed from 340 to 360 nm and the main peak emission from 442 (2.80 eV) to 464 nm (2.67 eV). A careful analysis of the two fluorescence spectra shows two emission centers. Emission center 1 is excitation independent from 280-320 nm for hBNQD-b and 280-340 nm for hBNQD-g. Emission center 2 is excitation dependent with excitation wavelength ranging from 330-420 nm and 350-500 nm for hBNQD-b and -g, respectively. One factor contributing towards the excitation dependent behavior PL is the presence of inhomogeneous functional groups on the surface of the hBNQDs. A closer inspection of center 2 emission spectra (Figure 3 b and F) for both hBNQDs show a small shoulder (more prominent in the hBNQD-b) and a main peak. Interestingly, the small peak’s relative intensity increases as the sample is excited at higher wavelengths. While this small peak in the emission center 2 also appears ACS Paragon Plus Environment

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in the hBNQD-g emission spectra, its relative intensity is not as strong. This behavior is expected since the surface passivation with PEG covers the surface of hBNQD-g. The sensitivity of the PL behavior of hBNQDs to their chemical environment was assessed by varying the pH values from 4 to 12 (Figure S3 ESI). A sharp drop in the emission intensity in a highly basic environment is due to the presence of large populations of OH− ions that induce changes to the electron density of the emission centres. Subsequently, the electronic transitions of the defects are disrupted or even prohibited. The luminescence decay profile of the hBNQDs was measured using the time-correlated single photon counting technique and the luminescence lifetimes, fitted satisfactorily with two-exponential functions for both hBNQD-b and -g (Figure 3 D and H). This also suggests that the decay process is dominated by contributions from different emissive centers. Compared to organic dyes, hBNQDs show long lifetime decays of 16.60 and 19.91 ns for hBNQD-b and g, respectively. Based on the foregoing results, we conclude that the emissions from hBNQD samples originate from two different luminescent centers: (a) carbon-replaced N vacancy point defects (3-B and 1-B centers) with singlet carbene structures formed at zigzag edges of the samples (both π→π∗ transitions), and (b) BO-X species (x=1 and 2) 18. The 1,3-B centers consist of unpaired electrons trapped in the vicinity of 11B nuclei. The luminescence is generated directly by the electron transition between 1,3-B centers and lower carbon levels (some N atoms were replaced by C to form 1,3-B centers) 8. The formation of carbene structures at the edges of the hBNQDs can act as a radiative transition pathway from LUMO to HOMO as has been observed previously with GQDs19. The BO-x moieties are closed-shell species with linear ground states. These emission centers are formed during ultrasonication and heat treatment in the presence of KOH, where surface oxidation caused the formation of BO-x species with non-bonding electron pairs (n→π∗ transitions). The carbon-replaced N vacancy point defects and carbene structures were formed during the hydrothermal treatment (both π→π∗ transitions).

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The two-photon excitation characteristics of the hBNQDs in Figure 4 show that while the maximum excitation wavelength for both samples is at 750 nm, the emission spectrum of hBNQD-b has a single emission peak at 510 nm, while the hBNQD-g shows two distinct peaks at 480 and 520 nm. This finding is consistent with the UV-Vis and PL spectra during single photon excitation. The two peaks in the UV-Vis and PL spectra, and the two lifetime values of the hBNQD-g are indicative of the presence of two distinct emission centers.

Figure 3. UV-Vis absorption, PL spectra and luminescent decay profile of the hBNQD-b (A, B, C and D) and hBNQD-g (E, F, G and H) in aqueous dispersion (insets from left to right: hBNQDs excited at 320 and 365 nm wavelengths, respectively)

Figure 4. Normalized two-photon excitation spectra of (A) hBNQD-b and (B) hBNQD-g


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5. Applications: Multiplexed imaging, intracellular ferric ion biosensing and detecting pH variations in live cells 5.1 Multiplexed cell imaging To evaluate the biocompatibility of hBNQDs, RL-14 cells were cultured with variable concentrations of hBNQDs from 20 to 700 mg/mL (Figure S4 ESI). After 24 hr cell culture, we observed that more than 92% of the cells were viable at concentrations as high as 500 µg/mL, indicating excellent biocompatibility of hBNQDs and their potential for in vivo cell imaging and biological labelling. The hBNQD-b was used for qualitative and quantitative sensing of ferric ions in cell suspensions and inside MCF-7 breast cancer cells. As shown in Figure 5, strong fluorescence from the MCF-7 cells can be seen after incubation with hBNQD-g for 24 h. The imaging was performed in multi-photon mode at 780 nm in order to minimize cell autofluorescence and photodamage. The results show that hBNQD-g penetrated into the cytoplasm of the MCF-7 cells and emitted bright fluorescence. Multiplexed imaging is a powerful technique for concurrently obtain multiple signals from biological targets and analyzing physiological processes. Because of the long FL lifetime of hBNQDs and tunable emission wavelength, when they are used in conjunction with organic dyes, their fluorescent signals can be resolved spectrally and temporally based on their emission wavelengths and fluorescent lifetimes, respectively, which make them suitable for multiplexed imaging using multiple channels to measure desired entities.

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Figure 5. Two photon imaging of MCF-7 breast cancer cells with 780 nm pulsed laser: (A) Brightfield; (B) Fluorescence signal from hBNQD-g; (C) Overlay; (Scale: 20 µm) The combination of spectral and temporal imaging is particularly useful when multiple probes with overlapping emission wavelengths but have different fluorescent lifetimes. Contrary to light microscopy, fluorescence lifetime is independent of concentration, illumination intensity, photobleaching or light path length – parameters that are difficult to control in a cellular system 20. Table 1 shows the fluorescent lifetimes of hBNQDs in water and MCF-7 cells after 24 h incubation. Biexponential decay analysis using an incomplete model in aqueous solution gave satisfactory fits (χ2 of 1.06 and 1.05) showing a short and a long lifetime component and comparable fractional amplitudes. Due to the sensitivity of FL lifetime to the pH and biochemical species in local cellular environment a sharp drop in the lifetime values of the hBNQDs inside MCF-7 cells is observed. While the fractional amplitudes remain unchanged both in water and in the intracellular environment, the longer lifetime component (τ2) dropped from 16.6 and 19.9 ns to 5.6 and 5.2 ns for the hBNQD-b and -g probes, respectively. Since the outer layer of the hBNQDs is covered with ionized carboxylic acid groups, the protonated -COOH groups can form hydrogen bonds between the nanoparticle and biomolecules, resulting in close interactions and reduced lifetime values. Previous studies also showed a reduction in lifetime for negatively charged QDs which interact with the positively charged intracellular proteins 21.

Table 1. Photoluminescence emission lifetimes (τ, ns) and fractional amplitudes (A) of hBNQDs for 24 h with 780 nm pulsed laser excitation hBNQD lifetimes and fractional amplitudes (A) Aqueous Solution

MCF-7 Cells

τ1 / (A1)

τ2 / (A2)

hBNQD-b

2.59 (0.61)

hBNQD-b

16.6 (0.39)

hBNQD-g

2.47 (0.63)

hBNQD-g

19.9 (00.36)

hBNQD-b

0.96 (0.61)

hBNQD-b

5.68 (0.36)

hBNQD-g

0.95 (0.66)

hBNQD-g

5.21 (0.34)


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To achieve the temporally and spectrally resolved fluorescent signals, two nuclear stains with blue and green fluorescence (DAPI and Sytox green) but with much shorter FL decay lifetimes (4 ns (red).

5.2 Intracellular ferric ion (Fe3+) detection and quantification Figure 7A shows the sensitivity and selectivity of hBNQD-b in the presence of various metal ions. No significant change in fluorescence intensity was observed in the presence of Al3+, Ca2+, Cu2+, K+, Mg2+, Ce3+ and Ni2+ at the excitation wavelength of 335 nm. Figure 7B shows a gradual decrease in the fluorescence intensity at 440 nm on adding Fe3+ at concentrations of 20-500 µM. The reduction in fluorescence intensity is related to the formation of complexes between hBNQD-b and Fe3+ ions due to the electron deficiency of Fe3+ and its interactions with the oxygen-rich groups on the surface of hBNQD-b. In particular, the excited electrons from hBNQD-b tend to transfer to the unfilled “d” orbital of Fe3+ 22. Apart from this, Huo et al. 23 proposed aggregation quenching mechanism for boron nitride quantum dots in the presence of Fe3+ and it was concluded that the aggregation of quantum dots gives rise to further fluorescence quenching.

Figure 7. (A) PL intensity of hBNQD-b in the presence of selected metal ions at 200 µM and excited at 335 nm; (B) PL intensity of hBNQD-b excited at 320 nm with variable concentrations (0 to 500 µM) of Fe3+ ions; (C) PL intensity of hBNQD-b as a function of Fe3+ concentration


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Figure 7C shows that the fluorescence quenching of hBNQD-b increases linearly with increase in Fe3+ concentration (0-500 µM) at the excitation wavelength of 335 nm (R2 = 0.994). These results show that the highly selective quenching effect with respect to PL intensity of hBNQD-b can be employed in applications involving the detection and concentration measurements of Fe3+. However, a direct correlation between the sensing data in DI water and the complex cellular microenvironment is not feasible. To measure the intracellular ion concentration, the interactions between hBNQD-b and different organelles inside the cell should be taken into account. In order to expose the hBNQD-b to the cell’s microenvironment, hBNQD-b was added to the lysed MCF-7 cells in HEPES. This strategy not only takes into account the interactions between different components in the system but also eliminates the pH variations, which can affect the FL intensity measurements as shown in Figure S3. Figures 8 shows that even when the surface of hBNQD-b is exposed to various organelles such as plasma membrane, DNA, proteins, lysosomes and endosomes, it can still detect Fe3+ at concentrations as low as 20 µM and the fluorescence quenching efficiency still maintains a linear correlation with the Fe3+ concentration. The intracellular detection limit of Fe3+ was calculated as 20.6 nM, which is among the highest sensitivity reported in the literature.

Figure 8. (A) PL intensity of hBNQD-b excited at 335 nm in the presence of Fe3+ ions at variable concentrations of 80 to 20 µM; (B) PL intensity of hBNQD-b versus Fe3+ concentration

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For intracellular sensing (Figure 9), the cells were incubated with hBNQD-b for 4 hours. After this incubation time, variable concentrations of ferric ion were added to the cells for 1 hr. The cells were washed 3 times with PBS before imaging. As shown below, the emission signal from the nanoparticles are quenched as the Fe3+ concentration is increased from 8 to 20 µM. Images were taken using identical settings on the microscope and the emission signal intensity of each image was measured. The signal intensity values started at 11 and decreased to 9.3, 7.4 and 6.5 (arbitrary units) with 8, 12, 16 and 20 µM of ferric ions, respectively.

Figure 9. PL intensity quenching of intracellular hBNQD-b excited by two-photon pulsed laser at 780 nm with variable Fe3+ concentrations of: (A) 8 µM; (B) 12 µM; (C) 16 µM and (D) 20 µM (Scale: 20 µm) 5.3 Tracking pH variations and visualization of endocytic pathway of hBNQD-g Fluorescence lifetime measurements provide absolute temporal and spectral values, and hence provide powerful means to determine the spatial location of nanoparticles for examining their microenvironment 24,25. Based on the sensitivity of an ensemble of nanoparticle FL lifetime, it has been reported 26 that the fluorescence lifetimes of CdSe-QDs varied in different cellular regions as a function of pH. In a similar study, CdSe-QDs were used for pH sensing in live cells owing to the sensitivity of their FL lifetime to pH, where increasing the solution pH from 5 to 9 resulted in longer FL lifetime values (8.7 to 15.4 ns) 27. In our work, the cells were incubated with hBNQD-g for 1-4 hr and the longer lifetime component (τ2) was monitored. After the initial 30 min incubation (when the hBNQD-g are either adherent to the membrane or in the cytosol), the average FL ACS Paragon Plus Environment

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lifetimes (τ2) were measured as 5.49 ns. Our measurements of the FL lifetime of the hBNQD-g in water suspensions having variable pH demonstrated a reduction in lifetime values in more acidic environments (Table S1 ESI). As shown by CLSM images (Figure 10), after 1 hr of incubation, a punctate pattern starts to form, showing that the hBNQD-g probes are predominantly localized in extranuclear vesicles, confirming endosomal sequestration of the nanoparticles. The vesicular distribution of endocytosed green luminescing hBNQD-g in the cell cytoplasm correspond to early endosome localization (pH ≈ 6.4-6.5). Increasing the incubation time resulted in a higher concentration of hBNQD-g in late endosomes (pH ≈ 6.0-5.0) followed by fusing with lysosomes. By increasing the incubation time, the punctate pattern started to spread throughout the cell cytoplasm (Figure 10 C/I/O/U).

Figure 10. Confocal and FLIM images of MCF-7 cells incubated with hBNQD-g for: 1 hr (A), 2 hr (G), 3 hr (M), 4 hr (S). FL images excited at 780 nm with overlay of phase contrast at corresponding times: B, H, N, T. Black and white FLIM images: C, I, O, U. False color FLIM images of cells showing different lifetimes throughout the cells: D, J, P, V. Fitting data with biexponential model: E, K, Q, W. Lifetime histograms of hBNQD-g following incubation with cells as a function of time: F, L, R, X (Scale: τ2 lifetime in picoseconds). 20



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As shown in Figure 10, with longer incubation time and matching of CLSM and FLIM images, the second component of the lifetime decay profile becomes shorter as the microenvironment of hBNQD-g becomes more acidic (Figure 10 F/L/R/X). These data are consistent with the decrease in PL intensity of hBNQDs-g in a more acidic environment (Figure S3 ESI). Previous studies also indicated the time dependent endosomal accumulation of QDs in endosomes using endosomal markers 20,28. A closer look at the FLIM images (Figure 10 C/I/O/U) indicates that a punctate pattern starts to form after 1 hr of incubation. Upon extended incubation time, this punctate pattern starts to spread throughout the cell cytoplasm (compare Figure 10 C/I/O to U). The initial punctate pattern changes to a more diffused distribution after 4 hr of incubation (see white arrow in Figure 10U), indicating the fusion of early endosomes with late endosomes. This resulted in the appearance of a second peak in the lifetime decay histogram (Figure 10X). In all cases, the two-component fits were adequate (1.04

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