A Novel Two-Photon Fluorescent Nanoprobe for Glutathione Sensing

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A Novel Two-Photon Fluorescent Nanoprobe for Glutathione Sensing and Imaging in Living Cells and Zebrafishes Using Semiconducting Polymer Dots Hybrid with Dopamine and #-Cyclodextrin Junyong Sun, Ningning Chen, Xueli Chen, Qiang Zhang, and Feng Gao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03010 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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Analytical Chemistry

A Novel Two-Photon Fluorescent Nanoprobe for Glutathione Sensing and Imaging in Living Cells and Zebrafishes Using Semiconducting Polymer Dots Hybrid with Dopamine and β-Cyclodextrin Junyong Sun*, Ningning Chen, Xueli Chen, Qiang Zhang, and Feng Gao*

Laboratory of Functionalized Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chemo/Biosensing, Laboratory of Biosensing and Bioimaging (LOBAB), College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241002, P. R. China

*To

whom correspondence should be addressed.

E-mail: [email protected]; [email protected] 1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Fabrication of hybrid semiconducting polymer dots (Pdots) endowed with special applications in biosensing and bioimaging in living systems have recently received considerable attention. In this study, novel two-photon fluorescent hybrid Pdots, DA-CD@Pdots were firstly prepared by poly(styrene-co-maleic

anhydride)

(PSMA)

grafted

with

β-cyclodextrin

poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1’,3}-thiadiazole)]

(β-CD)

(PFBT)

and

through

nanoprecipitation method, following by covalently attaching with dopamine (DA) by using an EDC-catalyzed carboxyl-amine coupling reaction. The DA molecules anchored on the surface of Pdots were further oxidized to form their quinone-like structures (DQ) and act as good electron acceptors to magnifyingly quench the fluorescence of Pdots by intra-particle photo-induced electron transfer (PET) and “molecular-wire effect”. The finally achieved hybrids DQ-CD@Pdots display enhanced colloidal stability, higher resistibility to environment effect, and lower biological toxicity. In the presence of glutathione (GSH), DQ molecules on the surface of Pdots are reduced into catechol molecules and result in the inhibition of PET and restoration of fluorescence of Pdots. Based on the above demonstrations, the hybrids DQ-CD@Pdots are used as fluorescent probes for “turn-on” detection of GSH in the range from 0.01 to 3.0 μM with the detection limit of 2.7 nM. The prepared DQ-CD@Pdots probe is also applied to the GSH detection and imaging in living systems including human cervical carcinoma HeLa cells and living zebrafishes with satisfactory results.

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INTRODUCTION Glutathione (GSH) molecule is a low molecular weight sulfhydryl compound consisting of different structures including glutamic acid, cysteine and glycine. As the main antioxidant in the cell, GSH is almost exists in all cells and plays important roles in the process of controlling the redox state of cells.1 The abnormal changes of GSH content in cells are closely correlated with many diseases, such as heart disease, cancer and many neurological disorders.2 Therefore, it is of great significance to detect and monitor the concentration of GSH in biological systems. Up to now, different approaches, such as electrochemical, colorimetric method, and electroluminescence have been developed to detect GSH.3-5 Among them, fluorescent sensors have attracted significant attention due to the high sensitivity and non-invasive.6-9 Furthermore, with the progress of fluorescence microscope techniques, mapping the spatial and temporal distribution of the GSH in bio-organism can be obtained. 10 However, the majority of these fluorescent sensors are generally excited with short-wavelength light and usually interfered by background fluorescence, leading to reducing the precision and imaging resolution. 11, 12 Two-photon fluorescence microscopy (TPM) technique, which employs two near-IR photons as the excitation source, 13 is capable of tackling the aforementioned challenges with unique merits, such as deeper imaging depth, low phototoxicity and high imaging resolution. 14 Therefore, it is necessary to develop two-photon fluorescent probes with excellent performances for GSH sensing and imaging in various living systems. The analytical performances of fluorescence probes, such as the detection limit, sensitivity, spatiotemporal resolution and analytical reliability, strongly depend on the photophysical properties of fluorophores including fluorescence brightness, emission rates and photostability. 15,16 Compared with conventional fluorescent materials including small organic dyes, fluorescent proteins, and inorganic semiconductor quantum dots (QDs), semiconducting polymer dots (Pdots) made of conjugated polymers, have attracted a great deal of interest for biosensing and bioimaging with excellent comprehensive features. 17, 18 It is worth mentioning that Pdots-based fluorescence probes

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usually require to be functionalized with small molecules as recognition units to respond to various target analytes.

19-22

Among these functionalization methods of Pdots, covalent coupling and

electrostatic assembly have been intensively used for constructing fluorescent sensors owing to the fact that recognition units fixed on the particle surface achieve fast and adequate response. However, the functionalization process of Pdots with small recognition molecules may destroy the chemical balance of Pdots surface and reduce the stability of the colloid, which brings difficulties for subsequent purification and application. In addition, the small molecules fixed on the surface of particles are likely to decrease the biocompatibility. Therefore, it is very meaningful to maintain good colloidal stability and excellent biocompatibility of Pdots-based probes during functionalization process to expand their further biological applications. Herein, we develop a two-photon hybrid fluorescent probe with improved colloidal stability and better biocompatibility for GSH sensing and imaging via two-step method (Scheme 1). In this study, a commonly used stabilizer, amino-β-cyclodextrin (β-CD), was firstly grafted to the poly(styrene-co-maleic anhydride) (PSMA) to form an amphiphilic polymer (CD-PSMA). The obtained

CD-PSMA

was

then

blended

with

poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,10,3}-thiadiazole)] (PFBT) to form β-CD modified and carboxy-abundent semiconducting polymer dots (CD@Pdots) via conventional nanoprecipitation method. The obtained CD@Pdots were further covalently bonded with dopamine molecules (DA) via typical EDC-catalyzed carboxyl-amine coupling reaction, and then the DA molecules anchored on the surface of Pdots was oxidized to form quinone-like structures (DQ) via base-catalytic oxidation reaction. After the two-step preparation, the resulting hybrid fluorescent probe, DQ-CD@Pdots, was achieved. For the designed hybrid fluorescent probe, the β-CD molecules anchored on the surface of Pdots significantly improve the biocompatibility, the colloidal stability and the fluorescent brightness of the hybrid Pdots. In addition, quinone-like structures resulted from the oxidation of DA can be employed as excellent electron acceptors, 23

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and therefore turn off significantly the fluorescence of Pdots via amplified photo-induced electron transfer (PET) and “molecular-wire effect” of conjugate polymer. The presence of GSH can reduce DQ and subsequently inhibit the PET process, resulting in fluorescence recovery of Pdots. These merits of β-CD functionalized Pdots and excellent quenching ability of DQ endow hybrid fluorescent probe with satisfied analytical performances in biosensing and bioimaging of GSH in living systems.

Scheme1. Schematic illustration of the designed two-photon hybrid Pdots for GSH sensing.

EXPERIMENTAL SECTION Reagents and Apparatus. Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1’,3}-thiadiazole)] (PFBT, polydispersity 3.4, MW 164 000) was purchased from American Dye Source, Inc. Poly(styrene-co-maleic anhydride) (PSMA), tetrahydrofuran (THF, anhydrous, ≥ 99.9%, inhibitor-free), polyethylene glycol (PEG, MW 3350), 2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic

acid

buffer

(HEPES)

and

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich. L-Glutathione (reduced) and

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N-methylmaleimide (NEM) were obtained from Aladdin. Bovine serum albumin (BSA) and dopamine hydrochloride were purchased from Sinopharm Chemcial Reagent Co., Ltd. (Shanghai, China). Amino-β-cyclodextrin (NH2-β-CD) was acquired from Shandong Binzhou Zhiyuan BioTechnology Co., Ltd. (Shandong, China). The human serum samples were provided and pretreated by the Hospital attached to Anhui Normal University. Ultrapure water ( ≥18.25 MΩ ) was used throughout the experiments. The fluorescence and absorption spectra were obtained on PerkinElmer LS-55 fluorescence spectrophotometer and PerkinElmer Lambda 35 spectrophotometer, respectively. Transmission electron microscopic (TEM) images were recorded on Hitachi HT 7700 TEM instrument, operating at 100 kV. Dynamic light scattering (DLS) and zeta potential analysis was performed on a Malvern ZS90 Zetasizer Nano instrument. Representative fluorescence images were captured using a Fusion FX7 imaging system (Vilber Lourmat, Marne La Vallee, France). Absolute fluorescence quantum yields and fluorescence lifetimes were obtained with an FLS1000 fluorescence spectrophotometer (Edinburgh Instruments Ltd., United Kingdom). MTT assay was performed with a Multiskan Sky microplate reader (Thermo Scientific, Waltham, MA, USA). Two-photon fluorescence confocal fluorescence images were carried out on a confocal laser-scanning microscope (TCS SP8, Leica, Germany) equipped with a Ti:Sapphire laser (Chameleon Ultra II, Coherent). Covalent Linking NH2-β-CD to PSMA Polymer. The NH2-β-CD containing amino groups was grafted with PSMA polymer according to the previous reports with small modifications.

24

In a

typical procedure, 9 mg of PSMA polymer and 6 mg of NH2-β-CD were dissolved in 20.0 mL of anhydrous THF. The mixture was refluxed at 66°C for 48 h under nitrogen atmosphere. After the reaction was finished, the THF solvent was removed by rotary evaporation and weakly yellow powders were obtained. The resultant grafted polymer, termed as CD-PSMA, was used for the next step without further purification. Preparation of CD@Pdots and DQ-CD@Pdots. The β-CD modified semiconducting polymer

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dots (CD@Pdots) were prepared by a nanoprecipitation technique which has been described in detail previously.

25

Typically, PFBT and CD-PSMA were dissolved into anhydrous THF with a

concentration of 1 mg mL-1, respectively, following by filtering with 0.7 µm filter to remove aggregates. Then, 250 μL of PFBT solution and 100 μL of CD-PSMA were added into 5.0 mL of anhydrous THF and the mixed solution was sonicated for 2 minutes. The resulting homogeneous solution was quickly injected into 10 mL ultrapure water under high sonication power and sonicated for 5 minutes. The THF of the solution was removed by bubbling nitrogen on a hot plate at 95°C, and followed by filtration through 0.2 μm membrane filter to remove aggregates. Finally, CD@Pdots colloidal solution was obtained and then concentrated to 50 μg mL-1 via ultrafiltration centrifuge (Amicon®Ultra-4, MWCO:100 kDa). The DA-CD@Pdots was synthesized via the amidation reaction catalyzed with EDC according to previously reported procedure with small modifications. 26, 27 Typically, 80 μL of 1.0 M HEPES were added into 4 mL of CD@Pdots solution with 0.1% PEG. Then 80 µL of 1 mg mL-1 dopamine hydrochloride was added to the solution and mixed well on a vortex. After that, 80 µL of freshly prepared 10 mg mL-1 EDC was added to the above solution, and the mixture was mixed for 4 hours by magnetic stirring at room temperature under inert atmosphere. The resultant product was purified through a Bio-Rad Econo-Pac 10DG column to remove the free dopamine moleculars, and then the dopamine-functionalized Pdots, DA-CD@Pdots was obtained. The DA molecules anchored on the surface of Pdots was further oxidized into quinone-like structures (DQ-CD@Pdots) via catalytic oxidation reaction with base. Briefly, 40 μL of HEPES (1.0 M) buffer was added into 2.0 mL of the prepared DA-CD@Pdots solution and followed by adjusting the pH to 10.0 with 1 M NaOH solution, then magnetic stirring for 2 h. After the oxidation reaction was completed, the pH of the system was adjusted to 7.0 with 1.0 M HCl, and the salts were removed by ultrafiltration centrifuge (Amicon®Ultra-4, MWCO:100 kDa). Procedures for GSH Sensing. Typically, 20 μL of DQ-CD@Pdots solution (50 μg mL-1) and 40

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μL of HEPES buffer (1.0 M, pH 7.4) were added into a series of 2 mL colorimetric tubes, respectively. Then different amounts of GSH standard solution were added into the colorimetric tubes and diluted to 2 mL with water and mixed thoroughly, respectively. The mixture was incubated at 37°C for 30 min, and the fluorescence spectra were collected with an excitation of 450 nm. For the detection of the GSH level in human serum, 10 μL of serum sample was placed in colorimetric tubes and the experimental procedures were similar to those stated above for GSH detection. Cell Culture and Imaging. For the living imaging experiment of DQ-CD@Pdots nanoprobe, human cervical carcinoma HeLa cells were seeded into a 96-well plate at a concentration of 4 ×103 cells/well and grown in DMEM medium supplemented with 10% FBS, 100 U mL-1 penicillin, 100 mg mL-1 streptomycin at 37°C for 12 h under a humidified atmosphere containing 5% CO2. After the original medium was removed, HeLa cells were incubated with DQ-CD@Pdots at the desired concentration at 37°C for 2 h. Prior to fluorescence imaging, the treated cells were washed three times to remove any unbounded DQ-CD@Pdots. For the control experiments, HeLa cells were pre-incubated with GSH and NEM (a thiol-blocking reagent) for 10 min, respectively. The two-photon fluorescence images of DQ-CD@Pdots were collected in the range of 510-570 nm upon excitation with 810 nm. Zebrafish Maintenance and Imaging. The AB genotype transgeneic zebrafishes were acquired from Shanghai FishBio Co., Ltd and incubated with ultrapure water at 28.5°C. Those fish were fed twice a day to maintain higher nutritional status for spawn with a combination of Gemma fish food and freeze-dried bloodworms. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of NIAID, NIH. The zebrafish embryos were transferred into 90 mm petri dishes and maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 5-10% methylene blue, pH 7.5). The petri dishes were kept in the

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incubator at 28°C for seven days, refreshing the E3 embryo media every day. For the fluorescence experiment, the embryos were transferred into 35 mm glass bottom dish and incubated with DQ-CD@Pdots for 1 h, following by washing five times with embryo media to remove the remaining probes. In addition, for the control groups, the embryos were pre-incubated with GSH or NEM for 10 min, respectively, and then treated with DQ-CD@Pdots for 1 h. The two-photon fluorescence images of DQ-CD@Pdots were collected in the range of 510-570 nm upon excitation at 810 nm with 10x objective.

RESULTS AND DISCUSSION Design and Characterization of DQ-CD@Pdots. To achieve our design, we firstly prepared the amphiphilic grafted polymer, CD-PSMA, by linking NH2-β-CD to PSMA via the chemical reaction between anhydride and the amino compound according to the previous reports. 24 Subsequently, a green-emission

fluorescent

conjugated

polymer,

poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1’,3}-thiadiazole)] (PFBT), was employed as the luminescent component to prepare Pdots-based two-photon fluorescent probe due to its intrinsic superiorities including ultra-bright fluorescence, perfectly matching with the commercially available 488 nm laser equipped in confocal laser scanning microscopy (CLSM) and excellent two-photon excitation characteristics.

28

Using PFBT and CD-PSMA as the raw materials, the

CD@Pdots was prepared by the re-precipitation method. From the raw material ratio in the preparation of CD@Pdots and DLS result, the number of CD molecules on single particle was calculated to be 212 according to the published procedures.21, 29 During the formation of polymer dots, the hydrophobic polystyrene skeleton of CD-PSMA was enwound with polymer PFBT and embedded to form compact core, whilst the grafted β-CD units and carboxyl groups were hanged on the particle surface.

30

The resulting nanostructures are very stable and prevent the leakages of

PSMA-CD polymer chains from the Pdots although CD molecules are hydrophilic.

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In this study, CD molecules were selected to improve the colloidal stability, resistibility and biological compatibility of the prepared Pdots. It is well known that CD belongs to the family of cyclic oligosaccharides bonding with α-1,4-D-glucopyranose units and is widely used as drug compatibilizer and nanoparticle stabilizer due to the intrinsic merits such as good water solubility, good biocompatibility and low toxicity. In order to demonstrate the role of β-CD in the preparation of Pdots, the photophysical properties of Pdots without attaching β-CD (denoted as ppPdots), which were prepared using PFBT and PSMA as raw materials, were studied as control studies. As shown in Figure 1A, comparing with ppPdots, the emission wavelength of CD@Pdots displays slight red-shift from 537 nm to 541 nm. Meanwhile, its fluorescence intensity is 1.2-fold higher than that of ppPdots (Figure 1 A and B), which is consistent with the improved quantum yield from 0.252 to 0.306 for ppPdots and CD@Pdots, respectively. These results suggest that the β-CD molecules act as the stabilizer in the process of CD@Pdots formation and affect on the luminescent microenvironments of Pdots. As shown in Figure S1 (Supporting Information), the fluorescence lifetime of CD@Pdots and ppPdots were measured to be 1.31, and 1.14 ns, respectively. The elongated lifetime of CD@Pdots is resulted from the blocking of non-radiative decay pathways of PFBT through restraining their self-aggregation and the environment quenching influence. 28, 31 The zeta potential measurement shows that more charges are displayed on CD@Pdots (-34.7 eV) in comparison with those on ppPdots (-30.4 eV), indicating that the colloidal stability of CD@Pdots is improved through the linkage of β-CD in the preparation of Pdots. Dopamine molecules were coupled on the surface of CD@Pdots through the classical EDC coupling reaction. The morphologies of CD@Pdots and DA-CD@Pdots were assessed by TEM and DLS techniques. As can be seen in Figure 1C, the prepared CD@Pdots exhibit highly mono-disperse spherical shapes with an average diameter of 18.9 nm, which coincides well with the results from DLS measurement. After the conjugation of CD@Pdots with DA, the resultant Pdots slightly increased their micelle size to 21.5 nm (Figure 1D), suggesting the formation of

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DA-CD@Pdots. Base on the number of free carboxy groups on CD@Pdots nanoparticles, the average number of DA on a single CD@Pdots particle was calculated to be approximate 1060.21 The conjunction process was also confirmed by zeta potential studies, and the studies showed that less charges were observed on DA-CD@Pdots (−26.3 mV) in comparison to those on CD@Pdots (−34.7 mV).

Figure 1. (A) Fluorescence spectrum of CD@Pdots (curve a) and ppPdots (curve b). (B) Representative fluorescence images (pseudocolored) of ppPdots (left panel) and CD@Pdots (right panel) (Top), and representative fluorescence 3D images (pseudocolored) of ppPdots (left panel) and CD@Pdots (right panel) (Bottom) under UV light radiation. (C, D) Dynamic light scattering and TEM images of CD@Pdots (C) and DA-CD@Pdots (D).

It is known that dopamine can be oxidized under alkaline conditions to form oxidized DA-quinone structures (DQ), a good electron acceptor. 32, 33 In this study, to ensure that dopamine molecules on the surface of the particles were completely oxidized, the pH of DA-CD@Pdots solution was adjusted to 10.0 and then stirred in air for 2h. Compared with DA-CD@Pdots, the fluorescence brightness of DQ-CD@Pdots was significantly reduced under a 365 nm UV lamp 11



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(Figure S2A), indicating that dopamine oxidation products can effectively quench Pdots fluorescence. Glutathione Sensing and Its Sensing Mechanism. To investigate the feasibility of the prepared DQ-CD@Pdots probe to detect glutathione, a control experiment was first constructed. As indicated in Figure 2A, the prepared DA-CD@Pdots show strong fluorescence emission at 541 nm under the excitation of 450 nm (curve a), while the fluorescence of DQ-CD@Pdots was almost completely quenched (curve c in Figure 2A) under the same conditions, suggesting that the photoinduced electron transfer (PET) process from Pdots to oxidized dopamine-quinone (DQ), rather than dopamine occurs under excitation. To confirm the PET process between Pdots and DQ, the time resolved fluorescence decay of DA-CD@Pdots and DQ-CD@Pdots were measured. As show in Fig. 2B, the fluorescence lifetime of DA-CD@Pdots was 1.31 ns while the lifetime of DQ-CD@Pdots was shortened to 0.91 ns, suggesting the presence of PET from Pdots to oxidized DA-quinone. In addition, the density functional theory (DFT) calculations of the prepared probes were carried out to get further insight into the PET process. As represented in Figure 2C, the lowest unoccupied molecular orbital (LUMO) of DA is significantly reduced after oxidation (from -0.37 eV to -3.85 eV), which means that oxidized DA-quinone (DQ) can be used as a good electron acceptor.32 The HOMO of PFBT (-5.7 eV) is more closer to the LUMO of oxidized DQ (-3.85 eV) than that of DA, which favors the intra-particle PET between PFBT and DQ. The off-type fluorescent probe relied on intra-particle PET provides the precondition for turn-on measuring. Although many other semiconducting polymers such as PFO, PFPV, MEH-PPV, CN-PPV and PFDBT could fit the PET energy diagram with DQ as shown in Figure 2C. In this study, the conjugated polymer PFBT was selected to prepare two-photon fluorescent probe owing to its excellent photostability, attractive two-photon absorption properties and high brightness for two-photon laser-based fluorescence microscopy. In addition, comparing to those polymers excited with short wavelength (usually in the UV region) or emitted light in blue region, the PFBT polymer with maximum absorption at 450 nm

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and emission in green band can largely avoid interference derive from inner-filter effect and FRET of common substances.

Figure 2. (A) Fluorescence emission spectra of DA-CD@Pdots (curve a), DA-CD@Pdots with 3.0 μM GSH (curve b), DQ-CD@Pdots (curve c), DQ-CD@Pdots with 0.5 μM GSH (curve d), and DQ-CD@Pdots with 3.0 μM GSH (curve e) in pH 7.4, 20.00 mM HEPES buffer. The concentrations of DA-CD@Pdots and DQ-CD@Pdots are both 0.5 μg mL-1. (B) Time resolved fluorescence decay of DA-CD@Pdots (red circles) and DQ-CD@Pdots (black circles) in pure water. (C) Schematic energy level diagram of the electron transfer system calculated by density functional theory.

As shown in curve b in Figure 2A, the fluorescence spectrum of DA-CD@Pdots did not show obvious changes when GSH is added comparing to the fluorescence spectrum of bare DA-CD@Pdots (curve a in Figure 2A), indicating that the fluorescent DA-CD@Pdots does not response to GSH. However, when GSH was introduced into the DQ-CD@Pdots solution, the quenched fluorescence at 541 nm of the DQ-CD@Pdots probe was restored (curve d in Figure 2A) and the more fluorescence recovery was observed with increasing the concentrations of GSH (curve e in Figure 2A). According to previous report, GSH binds with DQ (dopaquinone) through a non‐enzymatic reaction to give rise to glutathionyldopas product.

34, 35

The fluorescence titration

study indicates that the degree of fluorescence recovery was close to its initial status when the

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concentration of GSH reaches at 3 μM (data nor shown). These results suggest that suppressed fluorescent probe resulted intra-particle PET can be used for turn-on measuring of GSH, which attributes to the fact that GSH can reduce DQ and subsequently inhibit the PET process. Furthermore, noticeable color changes of the probe solution from dark-yellow to light green were observed with a 365 nm UV lamp upon addition of GSH, as shown in Figure S2B (Supporting Information), which was consistent with the spectral results, indicating that DQ-CD@Pdots probe can be further used for GSH imaging in living systems. Analysis Performances of GSH Sensors. The experimental conditions including incubation time, incubation temperature and pH for GSH sensing with the DQ-CD@Pdots probe were optimized, as shown in Figure S3 (supporting Information). Under the optimum experiment conditions for GSH sensing, the standard fluorescence titrations were performed. As described in Figure 3A, with increasing GSH concentration, the fluorescence of the DQ-CD@Pdots at 541 nm was gradually restored. At the saturated concentration of 3 μM GSH, the recovery efficiency is calculated to be 334.93% with the formula (F-F0)/F0×100% (where F0 and F were the fluorescence intensity of the DQ-CD@Pdots at 541 nm in the absence and presence of GSH, respectively). As shown in Figure 3B, a good linearity was obtained in the range of 0.01 to 3 μM with a correlation coefficient of 0.9912 and a sensitivity of 1.0991 per μM. The limit of detection (LOD) for GSH detection was estimated to be 2.7 nM at S/N of 3. This LOD was lower than that of other nanofluorophore-based probes of GSH in previous reports, as shown in Table S1 (Supporting Information), exhibiting that the signal amplification deriven from Pdots can improve the sensitivity.

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Figure 3. (A) Fluorescence spectra of the sensing system with various GSH concentrations in pH 7.4, 20 mM HEPES buffer with 0.5 μg mL-1 DQ-CD@Pdots. (B) Plot of the recovery efficiencies as a function of GSH concentrations. The inset is the linear plot of recovery efficiency versus the concentration of GSH. F0 and F are the fluorescence intensity of the DQ-CD@Pdots in the absence and presence of GSH, respectively.

The selectivity of prepared DQ-CD@Pdots nanoprobe toward the potential interfering substances including various amino acids, common ions, uric acid, urea, glucose and ascorbic acid (AA) were also evaluated. As represented in Figure 4A and 4B, these substances except for AA with 500-fold of GSH concentration, show very little effects toward the DQ-CD@Pdots fluorescence in the presence of 2.5 μM GSH, indicating that DQ-CD@Pdots exhibits high selectivity for GSH detection. Furthermore, taking into account that other intracellular thiols may interact with DQ-CD@Pdots and interfere with GSH sensing, cysteine (Cys) and homocysteine (Hcy) were chosen to evaluate the specificity of the present sensor. Figure 4C shows the comparison of fluorescence signal response of the sensing system to 2.5 μM GSH with those of Hcy and Cys with different concentrations. We can see that Hcy with the concentration as higher as 50-fold do not result in a dramatic fluorescence change of DQ-CD@Pdots probe, comparing with the signal response of the present sensing system to 2.5 μM GSH. Although 50-fold of Cys exhibits significant turn-on effect on the DQ-CD@Pdots probe, intracellular content of Cys is much lower than that of GSH, 36, 37 suggesting that the present DQ-CD@Pdots possesses high selectivity and can be used for GSH analysis in real biological systems. In addition, to explore the potential applications of obtained biosensor as the imaging agent used in biological environments, the effect of ionic strength on the DQ-CD@Pdots probe was also investigated with increasing the concentration of KCl from 0 to 0.4 M. The green columns shown in Figure 4D displays the relative fluorescence ratio (F/F0) of the DQ-CD@Pdots biosensor towards

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various ionic strengths in the presence (F) or absence (F0) KCl, suggesting that it is practical for DQ-CD@Pdots to be used in physical salt concentrations. 38, 39 However, as a comparative study (shown in red columns in Figure 4D), the relative fluorescence ratios (F/F0) of DQ@Pdots are significantly lower than those of DQ-CD@Pdots at the same KCl concentration, especially at high ionic strength. The comparative study suggests that the linkage of β-CD to Pdots as a stabilizer could resist the effect of ionic strength on the fluorescence of Pdots.

Figure 4. (A) Fluorescence responses of the sensing system to 2.5 μM GSH in the presence of 18 essential amino acids with a concentration of 1.25 mM. (B) Fluorescence responses of the sensing system to 2.5 μM GSH in the presence of some possible coexisting substance with a concentration of 1.25 mM. (C) The comparison of fluorescence responses of the sensing system to 2.5 μM GSH with that of GSH, Hcy, and Cys with different concentration. (D) Ionic strength affects toward DQ-CD@Pdots (green column) and DQ@Pdots (red column). All the experiments are carried out in pH 7.4, 20 mM HEPES buffer with 0.5 μg mL-1 DQ-CD@Pdots or DQ@Pdots.

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Detection of GSH in Serum Samples. The feasibility for GSH detection with the prepared DQ-CD@Pdots in human blood samples was carried out. After the reasonable dilution of the serum samples, the sensing performance was evaluated by standard addition method, and the results were summarized in Table 1. The recoveries are varied from 97.19 to 106.64%, indicating that the proposed method possess the potential for monitoring GSH in real samples.

Table 1. GSH Detection in Serum Samples with the Proposed Probe. Sample

Without spiking

Spiked /1.0 μΜ GSH

Recovery / %

1

0.36±0.018

1.35±0.048

99.19±5.89

2

0.35±0.015

1.32±0.072

97.19±0.84

3

0.49±0.026

1.51±0.029

101.25±4.02

4

0.51±0.025

1.58±0.019

106.64±4.43

Cellular MTT Assays. The excellent performances of the proposed probe motivate us to further investigate the feasibility of GSH sensing and imaging applications in living systems. The biocompatibility of the probe was firstly evaluated by the standard MTT assays. In addition, The Pdots probe without β-CD modification, DQ@Pdots, was also used as a comparative study to demonstrate the effect of β-CD on improving biocompatibility of the probe. As shown in Figure 5A, the relative cell viability after incubated with DQ-CD@Pdots for 24 h is up to 81.96% even at 25 μg mL-1, which far higher than the normal concentration of probe used for imaging. Furthermore, the DQ-CD@Pdots show significant less cell toxicity compared with DQ@Pdots at all concentration levels, meaning that the functionalization of Pdots with β-CD is beneficial to improve the biocompatibility of probe. In all, the prepared DQ-CD@Pdots possesses favorable biocompatibility as expected and hold great promise for biological imaging.

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Figure 5. Cell viability treated with DQ-CD@Pdots (green pillars) and DQ@Pdots (red pillars) on HeLa cells at various concentrations for 24 h.

Imaging in Living Systems. Recently, multi-photon microscopy has emerged as a powerful technique for deep tissue imaging in biological systems.

40, 41

In this study, The prepared

DQ-CD@Pdots show typical advantages including fascinating analytical performances, improving biocompatibility and colloidal stability, inherent two-photon activity, and therefore are explored as two-photon imaging reagent. To investigate the capability of the DQ-CD@Pdots for intracellular GSH imaging, human cervical carcinoma HeLa cells were incubated with DQ-CD@Pdots in DMEM Medium and subsequently captured the image by using laser scanning confocal microscope with an 810 nm fs laser excitation. As can be seen from the control group of Figure 6, there is no apparent fluorescence in the HeLa cells without the incubation of probe (Figure 6A), implying negligible background fluorescence. As shown in Figure 6B, HeLa cells stained with the nanoprobe DQ-CD@Pdots demonstrated visible fluorescence in green channel (510 nm-570 nm), suggesting that the DQ-CD@Pdots hold good membrane permeability, which was likely attributed to the merits of Pdots such as soft nanoparticle, excellent biocompatibility, etc. In order to verify the dependence of green fluorescence on the amount of GSH, a control experiment was performed. After pretreated with N-methylmaleimide (NEM, a commonly thiols scavenger), HeLa cells were 18


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then incubated with the nanoprobe. As shown in Figure 6C, in this case, the fluorescence of green channel significantly decreased, suggesting DQ-CD@Pdots can efficiently activated by GSH. Moreover, another control experiment was carried out by first incubating the cells with GSH and then adding the DQ-CD@Pdots. In Figure 6D, as expected, significantly enhanced fluorescence in green channel was observed compared with that shown in Figure 6B, which is consistent with the above spectral results. The bright field photograph indicated that the cells were alive during the experiments, further confirming the good biocompatibility of the prepared DQ-CD@Pdots probe. Obviously, the present probe is promising nanoplatform for bioimaging and biosensing of GSH in cells.

Figure 6. Bright field and fluorescence images of untreated control HeLa cells (A), HeLa cells labelled with DQ-CD@Pdots (B), HeLa cells pretreated with N-ethylmaleimide (50 μM) for 10 min (C), and HeLa cells pretreated with GSH (50 μM) for 10 min (D). 2.5 μg mL-1 DQ-CD@Pdots was used throughout the imaging experiments. 19



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The DQ-CD@Pdots probe was further used to image the GSH level in living zebrafish upon excitation at 810 nm. As depicted in Figure 7A, the zebrafish shows nearly no background fluorescence (Figure 7A). However after incubating zebrafish with DQ-CD@Pdots for 3h, distinct green luminescence signals were observed in fish bodies (Figure 7B), suggesting that the probe is tissue-permeable and can be used to image in vivo. As shown in Figure 7D, the zebrafish pretreated with 100 μM GSH displays an enhanced fluorescence in green channel, revealing that the elevated GSH inside the zebrafish triggered the fluorescence of DQ-CD@Pdots. To confirm this point, an inhibition experiment was carried out by adding NEM to remove GSH. As shown in Figure 7C, the fluorescence resulted from zebrafish was significantly diminished. Based these observations, we believe that the proposed DQ-CD@Pdots probe is capable of monitoring changes of GSH levels in living bodies.

Figure 7. Fluorescence images of 7-day old zebrafish. (A) Untreated zebrafish (the control), (B) zebrafish incubated with DQ-CD@Pdots for 3h, (C) zebrafish pretreated with NEM (100 μM), and (D) GSH (100 μM) for 10 min and then incubated with DQ-CD@Pdots for 3h. The columns from 20


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left to right represent the fluorescence, bright field, merged and pseudocolored images of zebrafish, respectively. Scale bar = 250 mm. 2.5 μg mL-1 DQ-CD@Pdots was used throughout the imaging experiments.

CONCLUSIONS In summary, we have designed a two-photon Pdots-based GSH-sensing nanoprobe with improved fluorescence brightness, colloidal stability and biocompantibility via co-precipitating two polymers. Benefiting from the inherent merits of Pdots and rational functionalization, the applications of the functionalized Pdots in imaging of GSH in living systems with two-photon excitation have also been demonstrated in this study. We believe that this study provides a new method for preparation of Pdots-based probes with improved performances for bioapplications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Fluorescence decays of CD@Pdots and ppPdots, the corresponding fluorescence photographs taken under UV illumination, experimental condition optimization of GSH sensing using DQ-CD@Pdots, and comparing the biosensor performances of GSH detection with various schemes.

ACKNOWLEDGEMENTS The funds including the Natural Science Foundation of China (Grant No. 21874001, 21575004, 21605001), the Foundation for Innovation Team of Bioanalytical Chemistry of Anhui Province and PhD Research Startup Funds of Anhui Normal University (2018XJJ59) are acknowledged for supporting this work.

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For TOC only

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A Novel Two-Photon Fluorescent Nanoprobe for Glutathione Sensing

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