Near-Infrared Dual-Emission Quantum DotsâGold Nanoclusters

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Near-Infrared Dual-Emission Quantum Dots-Gold Nanoclusters Nanohybrid via Co-Template Synthesis for Ratiometric Fluorescent Detection and Bioimaging of Ascorbic Acid In Vitro and In Vivo Peng Zhao, Kaiyu He, Yitao Han, Zhen Zhang, Mengze Yu, Honghui Wang, Yan Huang, Zhou Nie, and Shouzhuo Yao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02614 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 12, 2015

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Analytical Chemistry

Near-Infrared Dual-Emission Quantum Dots-Gold Nanoclusters Nanohybrid via Co-Template Synthesis for Ratiometric Fluorescent Detection and Bioimaging of Ascorbic Acid In Vitro and In Vivo Peng Zhao,a Kaiyu He,a Yitao Han,a Zhen Zhang,a,b Mengze Yu,a Honghui Wang,b Yan Huang,a Zhou Nie, a,* and Shouzhuo Yaoa a

State Key Laboratory of Chemo/Biosensing & Chemometrics, College of Chemistry

& Chemical Engineering, Hunan University, Changsha, P. R. China b

College of Biology, Hunan University, Changsha, 410082, P. R. China

* Corresponding author. Tel.: +86-731-88821626; Fax: +86-731-88821848 E-mail address: [email protected]

ACS Paragon Plus Environment


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Abstract Near-infrared (NIR) quantum dots (QDs) have emerged as an attractive bioimaging toolkit for exploring biological events because they can provide deep imaging penetration and low fluorescence background. However, the quantitation process of such NIR QDs generally relies on single-emission intensity change, which is susceptible to a variety of environmental factors. Herein, for the first time, we proposed a protein-directed co-template strategy to synthesize NIR based dual-emission fluorescent nanohybrid (DEFN) constructed from far-red gold nanoclusters and NIR PbS QDs (AuNCs-PbS-QDs). The convenient protein-directed co-template synthesis avoids the tedious chemical coupling and modification required in conventional preparation approaches of DEFNs. Additionally, the dual-emission signals of AuNCs-PbS-QDs exhibit two well-resolved emission peaks (640 nm and 813 nm) separated by 173 nm which can eliminate environmental interferences by the built-in correction of ratiometric signal, resulting in a more favorable system for bioimaging and biosensing. Next, the target-responsive capability of this NIR-based DEFN to ascorbic acid (AA) was discovered, enabling the proposed DEFN to ratiometrically detect AA with a linear range of 3 - 40 µM and a detection limit of 1.5 µM. This DEFN sensor possesses high selectivity, rapid response, and excellent photostability. Moreover, the feasibility of this NIR nanosensor has been fully proved by the ratiometric detection of AA for fruit internal quality assessment, in vitro cellular imaging, and in vivo imaging in nude mice.


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Introduction The utilization of near-infrared (NIR) fluorescence for biomedical imaging and detection has attracted tremendous interests due to its multifaceted advantages, including minimum photo-damage, deep penetration into biomatrics, lower fluorescence

background,

and

weak

scattering

of

emission

light.1-2

NIR

semiconductor quantum dots (QDs) have emerged as the representative NIR material possessing excellent optical properties, such as broad excitation spectra, narrow emission bands, size-tunable photoluminescence, and robust photochemical stability.3-5 To date, a limited number of NIR QDs, such as Ag2Se,3 Ag2S,5 PbS,6 CdTeSe/CdS/ZnS7 etc., have been successfully synthesized and demonstrated as promising candidates for tissue imaging and biomarker detection. For instances, Wang’s group has reported the use of Ag2S QDs for imaging xenograft tumors and tracking of transplanted human mesenchymal stem cells.5 CdTeSe/ZnS NIR QDs were utilized for the insulin detection in complex human plasma based on the NIR fluorescence energy transfer (FRET).8 Consequently, the development of NIR QDs-based biosensors will make a significant impact in clinical diagnosis, treatment assessment, and drug discovery. However, most of the bioimaging and biosensing probes derived from NIR QDs were designed based upon single-emission intensity change of QDs, which may be susceptible

to

instrumental

efficiency,

environmental

conditions,

and

the

concentration of QDs. In recent years, a novel kind of nanomaterials named



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“dual-emission fluorescent nanohybrid” (DEFN) have emerged as ideal fluorescence imaging labels and assay platforms due to their single-excitation and dual-emission fluorescence,9-12 enabling a high accuracy in analytical applications because the ratio of the fluorescent intensities at two wavelengths is capable of built-in correction to avoid the environmental interference.10-12 Currently, most of DEFNs have the two emissions located in visible light range, which hampered their applications in deep tissue and whole animals. Accordingly, the construction of NIR QDs-based DEFN not only presents a potential solution to improve the bioimaging performance of NIR QDs with single emission, but also provides a new NIR-based DEFN material competent in in vivo ratiometric bioimaging. In order to build high performance DEFNs, the initial but essential step is choosing proper counterpart nanomaterials. Gold nanoclusters (AuNCs) consisting of several to tens of gold atoms shows intense fluorescence with far-red or NIR emission.13 The unique optical properties of AuNCs, such as large Stokes shifts, great biocompatibility, and facile synthesis, render them competent as the potent fluorescent probes in living tissues visualization.13 These facts implied that it is potential to integrate broad excitation spectra of NIR QDs and large Stokes shifts of AuNCs into a new type of NIR-based dual-emission fluorescent nanohybrid with only single excitation. The conventional synthetic methods of dual-emission nanohybrid are normally dependent on “three-step” approach, including two synthetic steps to respectively prepare two kinds of component nanomaterials with different emission, and, sequentially, the conjugation step of two nanomaterials via biological affinity


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interaction10 or chemical covalent coupling12. Nevertheless, tedious multi-step preparation and sophisticated modification processes are required in this approach. It has been noted that natural proteins have been employed as biotemplates for the bottom-up

preparation

biomineralization.6,

14-16

of

fluorescent

nanomaterials

via

protein-directed

Notably, some proteins serve as the potent and versatile

biomolecular templates for preparing various fluorescent nanoparticles, for instance, bovine serum albumin (BSA) has been widely exploited to synthesize a series of nanomaterials, including QDs (CdS and ZnxHg(1-x)Se) and AuNCs.13, 16-17 However, the strategy using one protein as the only template to construct binary nanoparticles was scarce. Inspired by the facts, we were curious about whether one protein could be employed as a “co-template” for the facile synthesis of dual-emission fluorescent nanohybrid. Ascorbic acid (AA) is an essential micronutrient in human diet, and functions as a key coenzyme in many metabolic pathways and a potent antioxidant against oxidative damages which have been implicated in many chronic diseases, such as cardiovascular disease, cancer, and cataracts. AA deficiency also causes fatigue, depression, connective tissue defects, and the supplement of AA is proved to be favorable for reducing the incidence of chronic disease and mortality.18 Therefore, the development of AA probe with high sensitivity and selectivity is of significance to the prevention of disease and the medical assay. There have been several strategies utilizing fluorescence, electrochemistry, chromatography and colorimetry for the determination of AA. Among them,



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fluorescence analysis offers many new advantages due to its simplicity and low detection limit for the assay and imaging in vitro and in vivo. Recent examples of AA nanoprobes include the CdTe QDs fluorescent probe for probing AA in biological fluids,19 the CoOOH-modified persistent luminescence nanoparticles (PLNPs) for imaging of AA in living cells and in vivo,18 and the 7-Hydroxycoumarin-modified MnO2 nanosheets20 or CoOOH-modified carbon nanodots for monitoring AA in the brain microdialysate.21 However, for most of the existing fluorescent nanoprobes, the quantitative process was based on the change of single fluorescent emission in visible light region. In this way, the high fluorescent background in the organisms and tissues may interference measurement results, and the limited penetration depth of visible light restricted its application in the bioimaging. Thus, it is still in high demand to design simple and efficient fluorescent nanoprobes for the biosensing and bioimaging of AA with low fouorescent background and deep penetration depth. Herein, we developed a novel NIR-based dual-emission fluorescent nanohybrid of PdS quantum dots and gold nanoclusters (termed as AuNCs-PbS-QDs) for ratiometric fluorescent biosensing and bioimaging of ascorbic acid in vitro and in vivo. This NIR-based DEFN was facilely synthesized with high-yield and high-quality at ambient conditions via a protein-directed co-templated strategy (Scheme 1). AuNCs-PbS-QDs shows the comparable and well-resolved dual-emission signals located in far-red to NIR region with a tremendous spectral shift of above 170 nm, allowing high-resolution and sensitive biosensing and imaging. It was found that AA can efficiently quench the NIR emission of PbS QDs component of DEFN and has no


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influence on far-red fluorescence of AuNCs component. The plausible quenching mechanism of PbS QDs component was explored and found to be the charge transfer between the PbS QDs and AA. Thus, the NIR-based DEFN AuNCs-PbS-QDs has been utilized as the target-responsive NIR fluorescent probe to ratiometrically detect AA with high sensitivity and selectivity. The feasibility of AuNCs-PbS-QDs as potent AA probe has been fully demonstrated by detection of fruit internal quality, ratiometric imaging in living cells, and whole-body in vivo imaging in mice. Experimental Section Materials All chemicals were purchased from Sigma Aldrich. The fruits were purchased from the local supermarket. Ascorbate oxidase was purchased from Sangon (Shanghai, China). Nude mice were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). All the chemicals were of analytical grade and used without further purification. Ultrapure water was obtained from a Millipore Milli-Q system (18.2 MΩ·cm) was used in all runs. Preparation of AuNCs-PbS-QDs The synthesis of AuNCs and PbS QDs is shown in the Supporting Information. The AuNCs-PbS-QDs were synthesized as follows. The synthesized AuNCs were first diluted 10 times by 50 mM Tris-HCl (pH 7.5), then 95 µL of diluted AuNCs was mixed with 12 µL of 10 mM Pb(NO3)2 and incubated at room temperature for 10 min. Next, 6 µL of 10 mM Na2S was quickly injected into the solution followed by intense agitation on a vortex mixer for 10 s.



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Detection of Ascorbic Acid: Aliquots of 100 µL of freshly prepared AuNCs-PbS-QDs were added to 1.5 mL microcentrifuge tubes, and then different concentrations ((0, 3, 5, 10, 15, 20, 25, 30, 35 and 40 µM) of AA solutions were added. The fluorescent spectra of the resulting solutions were measured immediately with excitation at 450 nm. Fluorescence Imaging of the Interaction of AuNCs-PbS-QDs and AA In Vitro: Hela cells were plated on a 35-mm Petridish with 10-mm bottom well in the culture medium for 24 h. Hela cells were then incubated with AuNCs-PbS-QDs at 37 °C for 1.5 h for cell uptake. In the assay of AA, the cells were washed three times, followed by the addition of 40 µM AA, and incubated for 10 min before fluorescence imaging. Fluorescence Imaging of the Interaction of AuNCs-PbS-QDs, AA and AOase In Vivo: To demonstrate the applicability of the AuNCs-PbS-QDs to monitor AA and AOase in vivo, animal imaging was conducted as follows. First, three nude mice were anesthetized using isoflurane and O2 was utilized as carrier gas. The living mice were injected with 0.1 mL of the freshly prepared AuNCs-PbS-QDs, the mixture of the AuNCs-PbS-QDs and 40 µM AA, the mixture of the AuNCs-PbS-QDs and the reaction product of 40 µM AA and 10 U/µL AOase for 0.5 h, respectively. Then fluorescence signals were collected by a Lumina XR imager under emission filter of ICG or DsRed and excitation filter of GFP. Results and Discussion


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Fabrication and Characterization of the AuNCs-PbS-QDs The co-template synthesis process of AuNCs-PbS-QDs is shown in the Scheme 1A. BSA-templated AuNCs was synthesized by a simple and environmentally friendly synthetic approach as reported previously (route 1 in Scheme 1A).13 Subsequently, the as-prepared BSA-functionalized AuNCs was further used as the template for PbS QDs synthesis. The Pb(NO3)2 was incubated with BSA-templated AuNCs for 10 min under ambient conditions, and then the Na2S solution was quickly injected followed by intense vortex for 10 seconds, resulting in the instant formation of AuNCs-PbS-QDs (route 2 in Scheme 1A). Compared with the conventional methods for DEFNs preparation, the proposed “co-template” strategy greatly simplified the operation via integration of the second nanomaterial synthesis and two nanomaterials coupling into one facile and quick step (in about 10 min), significantly shortening the preparation time relative to the several hours to a few days required in the conventional methods. The optical properties and the structure of the nanohybrid were studied in detail. At first, we investigated the optical properties of AuNCs, PbS QDs, and AuNCs-PbS-QDs, respectively. Figure 1A shows that far-red emission maximum at 640 nm was acquired from BSA-AuNCs by excitation at 450 nm, presenting a large Stokes shift of 190 nm. As QDs possess a wide excitation range, PbS QDs gave rise to NIR fluorescence maximum at 813 nm under the same excitation wavelength at 450 nm (Figure 1A). These facts ensure the AuNCs-PbS-QDs to be capable of simultaneous excitating at 450 nm. As expected, the nanohybrid displayed a typical



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spectrum with two well-resolved fluorescence peaks at 640 nm and 813 nm (Figure 1A) with a great emission separation of 173 nm which is wider than that of previously reported dual-emission nanohybrids.9-12 The UV-vis absorption spectra of BSA, AuNCs, PbS QDs and AuNCs-PbS-QDs are shown in Figure S1 (Supporting Information, (SI)), from which we can see that all these BSA-templated nanomaterials possess an apparent absorption peak at 274 nm which is the characteristic peak of protein BSA. As for the synthesized AuNCs-PbS-QDs, its spectrum showed a gradual rise in absorbance from 700 nm which is similar to that of PbS QDs, and a slightly higher absorbance increase started from around 450 nm which is derived from the absorption characteristics of AuNCs. These results demonstrate the successful formation of the DEFN using the co-template strategy. The quantum yield of AuNCs and PbS QDs was found to be 5.5% and 2.4% respectively, which was calculated by using rhodamine B in H2O as the reference. These results were similar to the quantum yield of AuNCs and PbS QDs reported by existing reference,6, 13 instructing that the co-template strategy could guarantee the simplified operation, shortened preparation time and satisfactory quantum yield at the same time. In order to exhibit the merits of this new DEFN in fluorescent imaging, we further analyzed the fluorescence of AuNCs-PbS-QDs in a 96-well plate with an IVIS Lumina XR imaging system. A GFP filter (445-490 nm) was used for excitation, and two different band-pass emission filters were used: DsRed (575-650 nm) and ICG (810-875 nm). The far-red fluorescence of AuNCs is detectable only under the DsRed filter, and NIR fluorescence from PbS can only be observed via an ICG filter (Figure


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1B). More importantly, no matter which filter (DsRed or ICG) was used for imaging, there was no interference between the emission light of AuNCs and PbS QDs (Figure 1B), demonstrating that there was no cross talk between the two filter channels. Therefore, the unique optical features, including far-red-NIR dual emission, a large Stokes shift, and considerable peak separation of two emissions, allow AuNCs-PbS-QDs to achieve high-resolution ratiometric bioimaging and biosensing with deep tissue penetration and low background noise. TEM images (Figure 1C) shows that the obtained BSA-functionalized AuNCs-PbS-QDs nanohybrids were monodispersed with an average size of 17.8 nm ± 5.3 nm and the size of 85.7% of the observed nanohybrids distributed from 10 nm to 25 nm (Figure 1D). The hydrodynamic diameter of AuNCs-PbS-QDs measured by dynamic light scattering (DLS) was 68.1 ± 8.2 nm (Figure S2, SI), resulting from that the protein template of AuNCs-PbS-QDs nanohybrid was charged in water, generating an electrical double layer on the nanohybrid surface which increased the colloidal hydrodynamic diameter.22 High resolution TEM images revealed that numerous AuNCs and PbS QDs were embedded together (Figure 1E). The lattice spacing of 0.21 nm agreed with that of PbS (220)23 (Figure S3A, SI) and the 0.24 nm spacing was ascribed to the Au (111)11 (Figure S3B, SI). The phase structures of the obtained AuNCs-PbS-QDs were characterized by XRD (Figure 1F). The results of the XRD indicate that the sample crystallized well, apart from the diffraction of BSA phase,24 and the pattern of five major peaks was attributed to (111), (200), (220), (311), and (222) planes of PbS cubic phase, which were in good agreement with the



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bulk PbS crystal phase (JCPDS 05-0592).25 And the other two peaks could also be indexed as Au(111) and Au(220) planes which were aligned with the bulk Au crystal phase.25 The component of AuNCs-PbS-QDs was further analyzed using X-ray photoelectron spectroscopy (XPS) (Figure S4, SI). Apart from the common peaks of carbon, nitrogen, sulfur and oxygen in BSA protein, AuNCs-PbS-QDs exhibited additional peaks of gold and lead. The data showed that the as-prepared AuNCs-PbS-QDs preserved the same composition and structure of individual AuNCs and PbS QDs. Next, in order to demonstrate that the AuNCs-PbS-QDs was the discrete BSA-decorated nanohybrid consisting of AuNCs and PbS QDs as the components rather than a simple mixture of BSA-AuNCs and BSA-PbS QDs, we examined the different nanomaterials utilizing 6% native polyacrylamide gel electrophoresis (PAGE) (Figure 2). Different examples, including BSA, BSA-AuNCs, BSA-PbS QDs, AuNCs-PbS-QDs, and the mixture of BSA-AuNCs and BSA-PbS QDs, were loaded on the gel and analyzed using coomassie blue staining to visualize the protein component, and fluorescent imaging with Lumina XR system under DsRed filter to illustrate the AuNCs component, or ICG filter to display the PbS QDs component. Figure 2A shows the results after staining with coomassie blue. It can be seen that the pure BSA only (lane 1) exhibited a single band with high mobility, while the BSA-AuNCs (lane 2) presented smear bands with remarkably slower mobility relative to BSA, which also exhibited fluorescence under DsRed filter (Figure 2B, lane 2), suggesting the encapsulation of fluorogenic AuNCs in BSA enabled the BSA


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scaffolds to form large complex.26 The BSA-PbS QDs (lane 3) showed a single band with retarded mobility relative to BSA and being emissive under ICG filer (Figure 2C, lane 3), suggesting the increasing overall size of BSA after in situ generation of PbS QDs. Additionally, the relative sharp bands in lane 3 indicated the quite narrow size distribution of BSA-PbS QDs. It was noted that the PbS QDs has a relatively higher mobility in comparison with AuNCs, probably due to the less number of BSA ligands in BSA-PbS QDs relative to BSA-AuNCs. In lane 4, although the bands pattern of AuNCs-PbS-QDs sample after coomassie blue staining was similar to that of BSA-AuNCs, both the fluorescent images under DsRed and ICG filters showed a single smear emissive band at the same position near to the gel loading well (Figure 2B and 2C, lane 4), whose mobility was significantly slower than that of BSA-PbS QDs under ICG filter, evidencing the AuNCs and PbS QDs migrated as the assembly in the gel electrophoresis. In lane 5, a simple mixture of BSA-AuNCs and BSA-PbS QDs, as the negative control, showed two distinguishable bands, which belonged to BSA-AuNCs and BSA-PbS QDs, respectively. Correspondingly, their fluorescent bands under DsRed and ICG filters, respectively, were separated (Figure 2B and 2C, lane 5). Hence, the co-localization of AuNCs and PbS QDs during PAGE analysis of AuNCs-PbS-QDs has strongly demonstrated that the formed nanocomplex was composed of AuNCs and PbS QDs. Target Responsive Capability of AuNCs-PbS-QDs to AA The excellent photoluminescence properties of AuNCs-PbS-QDs prompted us to apply it to target-responsive analysis and bioimaging. Interestingly, we found that the



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unique fluorescence signal of AuNCs-PbS-QDs is selectively responsive to AA and its two emission peaks have significantly different responses. As shown in Figure 3A and Figure S5 in the SI, 40 µM AA eliminates about 70% of the fluorescence intensity at 813 nm but has negligible effect on the fluorescence at 640 nm. The respective investigation of the PbS QDs and AuNCs (Figure S6, SI) upon AA treatment indicated that PbS QDs emission is selectively quenched by AA while AuNCs remain impervious, which were in accordance with the results shown in Figure 3A. To investigate possible mechanism of the fluorescence quenching, we have examined the absorption spectroscopy for the AA and PbS QDs. First, FRET was ruled out as no absorption around 813 nm was observed for the AA (Figure S7A, SI). Next, about 25% decrease in the absorption of PbS QDs with 40 µM AA (Figure S7B, SI) implied that the fluorescence quenching may be caused by the charge transfer in which an electron is transferred from AA to the conduction band of PbS QDs and a hole moves in the opposite direction.27 To further prove this hypothesis, by using differential pulse voltammetry (DPV), we obtained that the oxidation potential of PbS QDs is 0.52 V (vs. SCE) in Figure S7C in the SI which was equal to 0.76 V (vs. NHE), thus, the EHOMO was calculated to be -5.26 eV. The calculated results are in accordance with the value of -5.1 eV in previous study.28 On the other hand, the redox couple of AA is 0.082 V (vs. NHE).29 Therefore, the quenching mechanism is plausibly due to the charge transfer between the PbS QDs and AA (Figure S7D, SI). We then investigated the capability of the AuNCs-PbS-QDs as a nanosensor for


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ratiometric AA assays (Figure 3B). When incubated with AA, the fluorescence at 813 nm decreased with the increase of AA concentration, while there was little change in fluorescence intensities of the reference band at 640 nm. The ratios of fluorescence intensities at 640 and 813 nm (I640/I813) showed a linear relationship with increasing AA concentrations (Figure 3C), and the resulting linear equation was I640/I813 = 0.04368c + 1.24974, where c represents the concentration of AA (µM). The detection limit of this ratiometric sensor was 1.5 µM (S/N = 3) with a linear range of 3-40 µM, demonstrating the potential of AuNCs-PbS-QDs for sensitive detection of AA. For comparison, the AA-induced quenching of individual PbS QDs was also quantitatively studied (Figure S8, SI), and the corresponding calibration curve shows that the decreasing fluorescence of PbS QDs was correlated linearly with AA concentration in the range of 5-20 µM and the detection limit was 2.5 µM (S/N = 3). Notably, compared with the “single-emission” based PbS QDs sensor, the ratiometric detection of AA using “dual-emission” AuNCs-PbS-QDs has a twice wider linear range and the detection limit was decreased about by 40%, probably due to its internally calibrated ratiometric signal. Further comparison of our AA sensor with other previously reported methods (Table S1, SI) implied that the proposed DEFN sensor possesses the comparable detection limit and possesses unique intrinsic features, such as the ratiometric detection in NIR emission region, which shows great potential for bioimaging of AA in vitro and in vivo. Kinetic studies (Figure S9, SI) showed that the nanohybrid responded rapidly to 40 µM AA and the fluorescence signal has reached a minimum value within 2 min. In addition, the resistance to photo



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bleaching of AuNCs-PbS-QDs was characterized by continuous irradiation by an Hg lamp with a power of 18 W (Figure S10, SI). The fluorescence intensity of two emission peaks at 640 nm and 813 nm could retain more than 80% after 300 min of continuous irradiation, while indocyanine green (ICG) completely photobleached after 180 min of continuous irradiation. These results indicate that the as-prepared AuNCs-PbS-QDs nanohybrid has a good photostability. Then, the selectivity experiments were carried out by challenging the AuNCs-PbS-QDs ratiometric sensor by potential interferents such as abundant cellular cations (Ca2+, K+, Na+, etc.), saccharides (glucose, fructose, sucrose), organic acidic molecules (citric acid, tartaric acid), biological reductant substrates (reduced cysteine, glutathione, dopamine, and uric acid) and other species structurally-related to AA including dehydroascorbic acid, L-gluconic acid-γ-lactone, xylose, catechol, resorcin, and quinol. The experimental results (Figure S11, SI) were suggestive of that either the fluorescence of AuNCs-PbS-QDs or its signal response to AA (40 µM) is insusceptible to these coexisting interferents (50 µM), indicating that the AuNCs-PbS-QDs sensor has a good selectivity for the ratiometric detection of AA. The good selectivity of this sensor is probably due to the strong electron-donating ability and coordination property with Pb2+ of AA, and the detailed explanation are given in SI. The ability of AuNCs-PbS-QDs to detect AA by the fluorescence imaging system was also investigated. As shown in Figure 3D, the luminescence of AuNCs-PbS-QDs under the emission filter of ICG gradually decreased as the concentration of AA increased while the intensity remained stable under the filter of


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DsRed. This has provided a foundation for the quantitative assay of AA through the strategy of fluorescence imaging. Thus, we further constructed three aspects of bioimaging applications to demonstrate the great potentials of the AuNCs-PbS-QDs as a target-responsive probe in different complex matrics (Scheme 1B). Detection of Fruit Internal Quality using AA as a Model Target Fruit is one of the most important sources of AA in human diet. Since AA content is key component of fruit physical quality, AA analysis is the essential part of the internal quality control of fruits.30 In this work, a rapid and simple quantitative model was established based on AuNCs-PbS-QDs for the determination of fruit AA content. The above-mentioned selectivity experiments indicated that excessive other substances such as saccharides and ions hardly affected the dual-emission property of AuNCs-PbS-QDs (Figure S11, SI), therefore, we first in situ directly performed imaging of AA content on the surface of fruit slice (Figure 4A). After AuNCs-PbS-QDs spread on the slice of banana, the fluorescence imaging through the DsRed filter reflected the fluorescence intensity distribution, however, no fluorescence was detected under the ICG filter (Figure 4A, 1, 4, 7). This verified that banana is abundant in AA, and thus the NIR fluorescence of PbS QDs was completely quenched. The similar results were acquired from the slices of other fruits, including apples, pitayas, peaches, and cantaloupes (Figure S12, SI). We further attempted to utilize AuNCs-PbS-QDs probe to estimate inner content of AA inside intact fruit. Taking tangerine as the example, upon direct injection of AuNCs-PbS-QDs into the pulp (injected from the side of tangerine, about 5 mm under peel), the fluorescence



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remains under the DsRed filter but no detectable signal was observed via ICG filter because of the AA content in tangerine juice (Figure 4A, 2, 5, 8). However, the AuNCs-PbS-QDs injected into pith (injected from the top, about 5 mm under peel) couldn’t contact the juice and the dual emission was unaffected (Figure 4A, 3, 6, 9). The AuNCs-PbS-QDs also opened the possibilities to quantitatively detect AA content in the fruit juice. The juices freshly squeezed from 4 kinds of oranges, involving ponkan, orange, Gannan orange, and navel orange were examined (Table S2, SI). Fluorescence spectra and two-channel imaging were utilized respectively to construct “dual-mode” detection. Figure 4B showed that I640/I813 increased with the increase of orange juice volumes and reached a platform when 5 µL extracts was added. According to the as-built calibration equation of AA, the AA concentration in four kinds of oranges was calculated (Table S1, SI) and the results reflected that AA content per 100 g ponkan, orange, Gannan orange, and navel orange are 28 mg, 46 mg, 50 mg, and 36 mg, respectively, which were comparable to the previously reported data.31 Next, the two-channel imaging of corresponding juice samples with the nanohybrid probe was adopted for region of interest intensity (ROIs) measurements, and the ROIs ratio of two channels (ROIsDsRed/ROIsICG) increased monotonically with increasing volumes of orange juice (Figure S13A), presenting a convenient alternative approach for quick semiquantitative detection of AA (Figure S13B). Taken together, all of the above results revealed that AuNCs-PbS-QDs is a promising tool for fruit internal quality assessment targeting AA content. In Vitro Cellular Imaging


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Because of the uses of AA as both a preventive agent and a cure in modern cancer therapy,32 the investigation on cellular uptake of AA is of significance. Thus, we then explored the ability of AuNCs-PbS-QDs for living cell imaging of AA. The cytotoxicity of AuNCs-PbS-QDs in the complex matrices was firstly investigated. We performed an MTT assay in Hela cells with varying AuNCs-PbS-QDs concentrations (0, 0.2, 0.5, 1.0, 1.5, and 2.0 mg/mL BSA component). Only marginal toxicity could be observed for the AuNCs-PbS-QDs up to 2.0 mg/mL with the cell viability decreased by ~5% after 12 h incubation (Figure S14, SI). This data demonstrated the high biocompatibility of the AuNCs-PbS-QDs. We next applied the AuNCs-PbS-QDs for imaging of AA in living cells using confocal microscopy. After the nanohybrid (1.5 mg/mL) was incubated with Hela cells for 1.5 h at 37 °C, we observed that the cells at channel (550-650 nm, green) (Figure 5B) and channel (700-800 nm, red) (Figure 5C) displayed a bright fluorescence signal, which gave immediate evidence for efficient uptake of AuNCs-PbS-QDs into living cells. However, after the addition of 40 µM AA in the same sample, almost no fluorescence could be observed from the 700-800 nm channel as shown in pseudocolored red images (Figure 5G,H), demonstrating that the nanohybrid could image AA in living cells. The overlay of fluorescence images and bright-field image confirmed that the cells were viable throughout the imaging experiments (Figure 5D,I). The fluorescence ratio (F550-650/F700-800) images clearly showed a significant increase of the fluorescence ratio after the addition of 40 µM AA (Figure 5E,J). The above experiments confirmed that the AuNCs-PbS-QDs could be used for ratiometric imaging in Hela cells.



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In Vivo Imaging on Nude Mice Model Within the “optical window” in biological tissue, the hemoglobin, oxygenated hemoglobin, fat or water has its minimum absorption in the NIR range.33 Therefore, the AuNCs-PbS-QDs is appropriate for in vivo imaging with a relatively low interference. As a proof-of-concept experiment, we injected AuNCs-PbS-QDs into nude mice muscle tissues at varied depths (1-5 mm) and visualized them using a Lumina XR imaging system under different emission filters. As shown in Figure 6A, the AuNCs-PbS-QDs can be visualized at a depth of no more than 3 mm under an emission filter of DsRed. Under the emission filter of ICG, however, the AuNCs-PbS-QDs can even be detected at 5 mm beneath the tissue surface. These results implied that both the far-red and NIR fluorescence of the proposed DEFN shows satisfactory tissue penetration abilities. We next assessed the ability of the nanohybrid to image AA in a nude mouse model (~16 g) (Figure 6B). As shown in Figure 6B, the 1st mouse treated with AuNCs-PbS-QDs showed strong fluorescence under both DsRed and ICG emission filters, while the 2nd mouse treated with AA (40 µM) and the AuNCs-PbS-QDs exhibited unaffected fluorescence via DsRed filter, but the fluorescence under ICG filter was totally quenched. These phenomena were in good agreement with those of in vitro detection of AA by the AuNCs-PbS-QDs DEFN probe. To verify that the quenching effect was resulted from the AA rather than the other substances in the mouse body, AA (40 µM) was treated with AOase (10 U/µL) for 0.5 h to suppress the reductive properties,34 and the resulting product was mixed with AuNCs-PbS-QDs


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and injected into the 3rd mouse. The recovery of fluorescence under the ICG filter indicated that the AuNCs-PbS-QDs has a high selectivity to AA for the imaging in vivo. The suppression of quenching effect of AA by AOase enzymatic catalysis was further demonstrated by in vitro fluorescence spectra (Figure S15, SI) and the oxidation product of AA by AOase treatment, the dehydroascorbic acid (DHA), hardly has any effect to the fluorescence of AuNCs-PbS-QDs. It has been proved that the dual-emission AuNCs-PbS-QDs is highly efficient for avoiding interferences from autofluorescence and light scattering of biological samples under in situ excitation and allows sensitive imaging of the AA in mice with high contrast.

Conclusion In summary, we have successfully synthesized a novel NIR-far red dual emission fluorescence nanohybrid of AuNCs-PbS-QDs for the first time based on a co-template method. The convenient co-template strategy avoids the tedious chemical coupling and modification, and greatly reduced the synthetic time. This novel nanomaterial is highly competent for high resolution bio-imaging because of its two dual-emission falling in the “optical window” of in vivo imaging with large Stokes shift and great separation of two emission. The intriguing target-responsive capability of this NIR-based DEFN to AA was discovered, which functions as the novel ratiometric nanosensor for AA with high sensitivity and selectivity, rapid response, and excellent biocompatibility. The potency of this nanosensor has been demonstrated by its applications in AA detection for internal quality estimation of intact fruits, the ratiometric imaging of AA uptake in the living cells, the in vivo imaging of AA in



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nude mice with high contrast. We expect that the NIR-based DEFNs and its protein co-template synthetic approach could pave the way for developing novel bio-imaging and diagnosis nano-reagents for future therapeutic and medical applications.

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21222507, 21175036, 21235002, and 21475038), the National Basic Research Program of China (973 Program, No. 2011CB911002), the Foundation for Innovative

Research

Groups

of

NSFC

(Grant

21221003),

the

Hunan

Provincial Innovation Foundation for Postgraduate (521293011), and the Presidential Scholarship for Doctoral Students, Hunan University (531107016011). Supporting Information Available Additional information including the description of extensive methods and figures as noted in text is available free of charge via the Internet at http://pubs.acs.org.

References (1)Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science, 2005, 307, 538-544. (2)Weissleder, R.; Tung, C. H.; Mahmood U.; Bogdanov, A. Nat. Biotechnol., 1999, 17, 375-378.


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(3)Gu, Y. P.; Cui, R.; Zhang, Z. L.; Xie, Z. X.; Pang, D. W. J. Am. Chem. Soc., 2011, 134, 79-82. (4)Cui, R.; Gu, Y. P.; Lei, B.; Zhao, J. Y.; Qi, B. P.; Zhang, Z. L.; Xie, Z. X.; Pang, D. W. Anal. Chem., 2012, 84, 8932-8935. (5)Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. Angew. Chem. Int. Ed., 2012, 124, 9956-9959. (6)Ma, N.; Marshall, A. F.; Rao, J. J. Am. Chem. Soc., 2010, 132, 6884-6885. (7)Li, L.; Chen, Y.; Lu, Q.; Ji, J.; Shen, Y.; Xu, M.; Fei, R.; Yang, G.; Zhang, K.; Zhang, J. R.; Zhu, J. J. Sci. Rep., 2013, 3, 1529. (8)Wang, Y.; Gao, D.; Zhang, P.; Gong, P.; Chen, C.; Gao, G.; Cai, L. Chem. Commun., 2014, 50, 811-813. (9)Wu, L.; Guo, Q. S.; Liu, Y. Q.; Sun, Q. J. Anal. Chem., 2015, 87, 5318-5323. (10) Wu, P.; Xu, C. Y.; Hou, X. D.; Xu, J. J.; Chen, H. Y. Chem. Sci., 2015, 6, 4445-4450. (11) Ju, E.; Liu, Z.; Du, Y.; Tao, Y.; Ren, J.; Qu, X. ACS Nano, 2014, 8, 6014-6023. (12) Chen, T.; Hu, Y.; Cen, Y.; Chu, X.; Lu, Y. J. Am. Chem. Soc., 2013, 135, 11595-11602. (13) Xie, J.; Zheng, Y.; Ying, J. Y. J. Am. Chem. Soc., 2009, 131, 888-889. (14) Wu, P.; Zhao, T.; Tian, Y. F.; Wu, L.; Hou, X. D. Chem. Eur. J., 2013, 19, 7473-7479.



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(15) Zhao, L.; Asfura, K. M. G.; Xu, J.; Patel, R. A.; Dadlani, A.; Mahinay, M. S.; Cushmore, M.; Rastoqi, V. K.; Shah, S. S.; Leblanc, R. M. Chem. Commun., 2011, 47, 7242-7244. (16) He, X.; Gao, L.; Ma, N. Sci. Rep., 2013, 3, 2825. (17) Ghosh, D.; Mondal, S.; Ghosh, S.; Saha, A. J. Mater. Chem., 2012, 22, 699-706. (18) Li, N.; Li, Y.; Han, Y.; Pan, W.; Zhang, T.; Tang, B. Anal. Chem., 2014, 86, 3924-3930. (19) Chen, Y. J.; Yan, X. P. Small 2009, 5, 2012-2018. (20) Zhai, W.; Wang, C.; Yu, P.; Wang, Y.; Mao, L. Anal. Chem. 2014, 86, 12206-12213. (21) Li, L.; Wang, C.; Liu, K.; Wang, Y.; Liu, K.; Lin, Y. Anal. Chem. 2015, 87, 3404-3411. (22) He, Y.; Zhong, Y.; Su, Y.; Lu, Y.; Jiang, Z.; Peng, F.; Xu, T.; Su, S.; Huang, Q.; Fan, C.; Lee, S. T. Angew. Chem. Int. Ed., 2011, 50, 5695-5698. (23) Lee, H.; Leventis, H. C.; Moon, S. J.; Chen, P.; Ito, S.; Haque, S. A.; Torres, T.; Nüesch, F.; Geiger, T.; Zakeeruddin, S. M. Adv. Funct. Mater., 2009, 19, 2735-2742. (24) Zhang, J.; Han, B.; Chen, J.; Li, Z.; Liu, Z.; Wu, W. Biotechnol. Bioeng., 2005, 89, 274-279. (25) Yang, J.; Elim, H. I.; Zhang, Q.; Lee, J. Y.; Ji, W. J. Am. Chem. Soc., 2006, 128, 11921-11926.


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(26) Lin, H.; Li, L.; Lei, C.; Xu, X.; Nie, Z.; Guo, M.; Huang Y.; Yao, S. Biosens. Bioelectron., 2013, 41, 256-261. (27) Choi, J. H.; Chen, K. H.; Strano, M. S. J. Am. Chem. Soc., 2006, 128, 15584-15585. (28) Pal, B. N.; Robel, I.; Mohite, A.; Laocharoensuk, R.; Werder, D. J.; Klimov, V. I. Adv. Funct. Mater., 2012, 22, 1741-1748. (29) Wang, G. L.; Xu, P. P.; Xu, J. J.; Chen, H. Y. J. Phys. Chem. C, 2009, 113, 11142-11148. (30) Scherer, R. A.; Rybka, C. P.; Ballus, C. A.; Meinhart, A. D.; Filho, J. T.; Godoy, H. T. Food Chem., 2012, 135, 150-154. (31) Silva, F. O. Food Control, 2005, 16, 55-58. (32) Chen, Q.; Espey, M. G.; Krishna, M. C.; Mitchell, J. B.; Corpe, C. P.; Buettner, G. R.; Shacter, E.; Levine, M. Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 13604-13609. (33) Wang, Y.; Chen, J. T.; Yan, X. P. Anal. Chem., 2013, 85, 2529-2535. (34) Smirnoff, N. Curr. Opin. Plant Biol., 2000, 3, 229-235.



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Figure captions Scheme

1.

Schematic

illustration

for

(A)

the

synthesis

of

NIR-based

AuNCs-PbS-QDs exploiting co-template strategy, (B) the detection of AA and three aspects of bio-imaging applications utilizing AuNCs-PbS-QDs. Figure 1. (A) Different fluorescent characteristics of AuNCs, PbS QDs and AuNCs-PbS-QDs. (B) Fluorescent imaging under the emission filters of DsRed (575-650 nm) and ICG (810-875 nm), respectively. (C) TEM image and (D) corresponding size distribution of AuNCs-PbS-QDs nanohybrids. (E) High resolution TEM images (Blue circles represent PbS and red circles represent AuNCs) and (F) XRD spectrum of AuNCs-PbS-QDs. Figure 2. Polyacrylamide gel electrophoresis (PAGE) analysis of BSA (I), AuNCs (II), PbS QDs (III), and AuNCs-PbS-QDs (IV) by visualization of (A) protein using coomassie blue staining, fluorescent imaging under (B) DsRed filter or (C) ICG filter using Lumina XR imaging system. Lane 1 to lane 5 represent BSA, AuNCs, PbS QDs, AuNCs-PbS-QDs, and the mixture of AuNCs and PbS, respectively. The gray boxes represent position of PAGE well. Figure 3. (A) Fluorescent spectra of AuNCs-PbS-QDs before and after the addition of 40 µM AA. (B) Fluorescent spectra of AuNCs-PbS-QDs with the addition of different concentrations (from a to j: 0, 3, 5, 10, 15, 20, 25, 30, 35, and 40 µM) of AA. (C) The fluorescence ratio (I640/I813) of the proposed sensor as a function of the different AA concentrations. (D) Fluorescent imaging of AuNCs-PbS-QDs in the absence and


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presence of various concentration of AA under filters of DsRed and ICG, respectively. Figure 4. (A) Digital photos and corresponding in situ fluorescence imaging of banana (1, 4, 7) and side view (2, 5, 8) and stop view (3, 6, 9) of tangerine with different treatments. The red cross marked the different injection position of about 5 mm under peel. (B) Ratio of fluorescent intensity at 640 nm and 813 nm with the addition of different volume of fruit juice from 4 kinds of oranges. Figure 5. Confocal fluorescence images of Hela cells before (upper panel) and after (lower panel) addition of 40 µM AA. (A, F) Bright field, pseudo-color fluorescence images from (B, G) 550-650 nm filter and (C, H) 700-800 nm filter, (D, I) overlay of fluorescence images and bright field images, and (E, J) fluorescence ratio (F550-650/F700-800) images. The scale bar was 30 µm.

Figure 6. Fluorescence images of (A) the nude mice muscle tissues injected with AuNCs-PbS-QDs in different depth and (B) the nude mice injected with AuNCs-PbS-QDs,

AuNCs-PbS-QDs/AA,

and

AuNCs-PbS-QDs/AA/AOase,

respectively. All the images were taken under two filters of DsRed (575-650 nm) and ICG (810-875 nm), respectively. The GFP (445-490 nm) filter was used for excitation.



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Scheme 1


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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