Amino Nitrogen Quantum Dots-Based Nanoprobe for Fluorescence

Mar 7, 2017 - Thereinto, the functionalization strategy of surface modification with time-saving for N-dots remains difficult but particularly importa...
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Amino Nitrogen Quantum Dots Based Nanoprobe for Fluorescence Detection and Imaging for Cysteine in Biological Samples Zhijiao Tang, Zhenhua Lin, Gongke Li, and Yuling Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00284 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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Analytical Chemistry

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Amino Nitrogen Quantum Dots Based Nanoprobe for

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Fluorescence Detection and Imaging for Cysteine in

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Biological Samples

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Zhijiao Tang, Zhenhua Lin, Gongke Li *, Yuling Hu*

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School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China

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* Corresponding author: Gongke Li, Yuling Hu

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Tel.: +86-20-84110922

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Fax.: +86-20-84115107

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E-mail: [email protected]

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[email protected]

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ABSTRACT: Fluorescent amino nitrogen quantum dots (aN-dots) were synthesized

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by microwave-assisted method using 2-azidoimidazole and aqueous ammonia. The

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aN-dots have nitrogen component up to 40%, which exhibit high fluorescence

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quantum yield, good photostability and excellent biocompatibility. We further

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explored the use of the aN-dots combined with AuNPs as a nanoprobe for detecting

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fluorescently and imaging for cysteine (Cys) in complex biological samples. In this

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sensing system, the fluorescence of aN-dots was quenched significantly by gold

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nanoparticles (AuNPs), while the addition of Cys can lead to the fluorescence signal

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recovery. Furthermore, we have demonstrated that this strategy can offer a rapid and

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selective detection of Cys with a good linear relationship in the range of 0.3–3.0

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µmol/L. As expected,this assay was successfully applied to the detection of Cys in

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human serum and plasma samples with recoveries ranging from 90.0% to 106.7%.

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Especially, the nanoprobe exhibits good cell membrane permeability and excellent

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biocompatibility by CCK-8 assay, which is favorable for bioimaging applications.

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Therefore, this fluorescent probe ensemble was further used for imaging of Cys in

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living cells, which suggests our proposed method has strong potential for clinical

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diagnosis. As a novel member of quantum-dot family, the aN-dots hold great promise

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to broaden applications in biological systems.

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KEYWORDS: Nitrogen quantum dots; microwave; cysteine; detection; bioimaging.

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INTRODUCTION Fluorescent

quantum

dots,

such

as

semiconductor

quantum

dots,1

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nanodiamonds,2 BCNO nanoparticles,3 and carbon dots (CDs)4 have garnered

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tremendous research attention owing to their outstanding electronic and photonic

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properties. However, their more widely used applications are greatly limited by some

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of their disadvantages such as potential biological hazards, low quantum yield and

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less active sites. Doping with heteroatoms as a principle way to obtain the remarkable

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properties of nanomaterials has been considered as an effective strategy to tune their

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intrinsic properties.5,6 For instance, doping with electron-rich N atoms is the most

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widely used method to obtain the exceptional properties of CDs.7,8 In the past few

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years, several methods have been developed for the advanced synthesis of N doped

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CDs.9 And it has been shown that, N-doping on CDs could drastically increase

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quantum yield and offer more active sites due to the electron-withdrawing ability of

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nitrogen atoms.10-12 Up to now, most of the nitrogen atoms were induced on particle

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surface under harsh conditions with relatively less content of element nitrogen.13,14

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Thus, a facile approach to synthesize new nano-structured materials based on the

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element nitrogen is still imminently desired.

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Recently, nitrogen-rich quantum dots (N-dots), as a new member of quantum-dot

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family were synthesized in methanol under mild conditions, which were demonstrated

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distinct and unique optical properties.15 These N-dots contain a higher percentage of

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nitrogen content compared to the neighboring carbon dots, which can effectively tune

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their photoluminescence properties. In view of the remarkable quantum-confinement 3

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and edge effects, these N-dots with electron-rich N atoms could drastically alter their

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electronic characteristics and exhibit distinct luminescence properties, thus having a

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wide range of applications. They also were further investigated in many other possible

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applications, such as biocompatible staining, determination of natural drug and

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detection of toxic cation.16,17 However, there are no attempts to explore the

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functionalization of N-dots; and their researches in the application of imaging are still

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inchoate, especially, as nanoprobes for imaging of clinical biomarker in living cells.

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Thereinto, the functionalization strategy of surface modification with time-saving for

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N-dots remains difficult but particularly important. In addition, it is in high demand to

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design novel probes based functionalized N-Dots for detection and imaging of clinical

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biomarker in complex systems.

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Cysteine (Cys) serves as a significant regulator of cell microenvironmental

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reactions, and abnormal levels of Cys in biological systems have been associated with

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many human diseases.18-20 Therefore, rapid and selective sensing of Cys is significant

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for early diagnosis and treatment of these diseases. Despite considerable efforts have

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been devoted to the development of Cys detection and imaging, some of these

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methods are complex, time consuming, and insensitive for rapid diagnosis.21,22

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Recently AuNPs and quantum dots-based fluorescent probes for Cys have been

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reported,23-25 which holds a great promise for developing facile nanosensors. However,

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only few reported nanoprobes with selectivity of intracellular Cys have been used for

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biological imaging so far. In fact, the discriminative imaging of Cys over

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homocysteine (Hcy) and glutathione (GSH) in living cells has been a focus and 4

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challenge. Therefore, it is highly pivotal to develop a facile and selective sensor with

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good biocompatibility for detection and imaging of Cys in biological samples.

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Herein, we present a facile and quick strategy of synthesizing fluorescent

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aN-dots using 2-azidoimidazole and aqueous ammonia by microwave-assisted

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approach. The as-prepared aN-dots with abundant amino groups, which exhibit good

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biocompatibility and high fluorescence quantum yield (34%). Moreover, we

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constructed a nanoprobe based on aN-dots and AuNPs, leading to the fluorescence

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(FL) quench of aN-dots, while the addition of Cys can lead to the FL signal recovery.

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On the basis of the FL change, we developed a facile nanoprobe for detection of Cys

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in human serum and plasma with flexibility. Especially, owing to the unique

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properties of the aN-dots/AuNPs nanoprobe with good membrane permeability and

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excellent biocompatibility, it was further used for imaging of Cys in human lung

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adenocarcinoma (A549) cells with high discrimination.

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EXPERIMENTAL SECTION

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Materials and reagents. L-cysteine (Cys, 98.0%), uric acid (UA, 99.5%),

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glutathione (GSH, 98.0%), ascorbic acid (AA, 99.7%), homocysteine (Hcy, 98.0%),

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L-histidine (His, 99.9%), L-Alanine (Ala, 99.8%), glucose( Glu, 98.5%), L-aspartic

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acid (Asp, 99.5%), L-isoleucine (Ile, 97.5%), potassium chloride (99.5%), ferric

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chloride (99.0%), quinine sulfate (98%, suitable for fluorescence) were purchased

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from Aladdin Chemistry Co. Ltd. (Shanghai, China). 2-aminoimidazole sulfuric acid

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salt (96.5%) was obtained from Shanghai Shaoyuan Co. Ltd. (Shanghai, China).

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Sodium chloride (99.5%), zinc nitrate (98.0%), magnesium sulfate (99%), and 5

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aluminium chloride (99.99%) were purchased from Guangzhou Chemical Reagent

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Factory (Guangzhou, China). N-methylmaleimide (NMM, 99.8%), sodium azide

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(NaN3, 98.5%) and sodium nitrite (NaNO2, 99.8%) were obtained from J&K (Beijing,

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China). Human serum and plasma samples were provided by Sun Yat-sen University

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Cancer Center (Guangzhou, China). Phosphate buffered saline (PBS) and Bovine

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serum were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China).

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Dulbecco's modified Eagle's medium (DMEM), human lung adenocarcinoma A549

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cells and Penicillin-Streptomycin were from Sigma-Aldrich (Missouri, U.S.A.). All of

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the other chemical reagents were of analytical grade, from Shenyang Chemical

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Reagent Factory (Shenyang, China).

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Instruments. Microwave synthesis experiments were performed on a

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MWave-5000 microwave apparatus (Sineo Microwave Chemistry Technology

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Company, Shanghai, China). 1HNMR spectra were recorded using a Bruker AVB-400

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MHz NMR spectrometer (Bruker biospin, Switzerland). Transmission electron

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microscopic (TEM) images were recorded with a PHILIPS TECNAI 10 TEM

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instrument

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battery-powered Raman spectrometer (model Inspector Raman, diode laser, excitation

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wavelength λex = 785 nm) in the range of 200-2000 cm-1 (DeltaNu, USA). Infrared

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absorption spectra were acquired by a NICOLET AVATAR 330 Fourier transform

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infrared (FT-IR) spectrometer (Nicolet, USA). Fluorescence (FL) spectra were

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obtained on a RF-5301PC fluorescence spectrometer (SHIMADZU, Japan). UV-Vis

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absorption spectra were recorded with the UV-Vis 2600 spectrophotometer (Shimadzu,

(Philips,

Netherlands).

Raman

spectra

were

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on

a

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Japan). Fluorescence imaging of A549 cells was examined by confocal laser scanning

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microscopy (Zeiss LSM 710NLO, Germany). The pH measurements were performed

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with a pH-meter PB-10 (Sartorius, Germany). The absorbance of the cell viability by

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CCK-8 was measured using a microplate reader (Thermo Scientific Multiskan GO,

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Finland). The fluorescence spectra and images of the resulting solutions were

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recorded at room temperature (25 °C).

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Synthesis of aN-dots. Fluorescence aN-dots with high percentage of the element

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nitrogen have been synthesized by microwave-assisted process. At most,

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2-azidoimidazole

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2-azidoimidazole was prepared from 2-aminoimidazole sulfuric acid salt, NaN3, and

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NaNO2 in aqueous HCl solution.15,26 Then the raw aN-dots were synthesized by

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microwave-assisted method using 2-azidoimidazole (0.50 g) and aqueous ammonia

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(50 mL, 25% in water) transferred into the microwave reactor. As the reaction

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temperature increased from 100 °C to 160 °C and the irradiation time increased for 8

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min under 500 W, the stock solution finally turned greenish black and even to dark

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brown, which suggested the formation of aN-dots. The supernatant was collected by

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removing the large dots through centrifugation at 12000 rpm for 20 min and then

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dialyzing against ultrapure water through a dialysis membrane for 24 h. After

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vacuum-freeze drying, cinnamon-colored solid residue was obtained. The procedures

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for other N-dots through different nucleophilic reagents were similar to the aN-dots.

as

a

starting

material

has

been

synthesized.

Briefly,

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Constructing fluorescent nanoprobe combined aN-dots with AuNPs. 4.0 mL

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of aN-dots solution (100 µg/mL) and 1.0 mL of AuNPs (4.0 nmol/L) solution were 7

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added to a 10 mL centrifuge tube, then incubated for 5 min for preparation the

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aN-dots/AuNPs nanoprobe. For Cys detection, the as-prepared aN-dots/AuNPs

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nanoprobe and appropriate aliquot of Cys solution were transferred into centrifuge

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tube. The Cys standard solution with different concentrations were diluted in 10

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mmol/L PBS buffer containing 50 mmol/L NaCl, 5 mmol/L KCl, 4 mmol/L MgCl2

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(pH 7.4), and then added 100 µL Cys into the aN-dots/AuNPs solution (100 µL) after

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incubation for 5 min at room temperature. The fluorescence intensity was recorded at

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419 nm with an excitation wavelength of 320 nm, and the slit widths of emission and

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excitation were 5 nm. The detection procedures for other interferences and biothiols

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were similar to Cys. When real samples were determined, Cys standard solution was

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substituted by the prepared human serum and plasma. Each experiment was repeated

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three times.

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Cytotoxicity assays. The cell viability was measured using the CCK-8 assay

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according to the manufacture’s protocol. Briefly, 5 × 103 A549 cells were incubated

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with different concentrations of the aN-dots or aN-dots/AuNPs nanoprobe in triplicate

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in a 96-well plate for 24 h at 37 °C in a final volume of 100 µL. The CCK-8 solution

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(10 µL) was added to each well and incubated with the cells for another 1 h. After

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thoroughly mixing, the absorbance was measured at 450 nm using a microplate reader.

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Each result was the average of three wells, and 100% viability was determined from

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untreated cells.

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Intracellular Cys imaging in A549 cells. To further demonstrate the quantitative

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detection in living cells, we then performed the measurements of Cys in A549 cells 8

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treated by the nanoprobe. Two days before imaging, A549 cells were seeded in three

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plates containing sterile petridish and were cultured in DMEM supplemented with

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10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100

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µg/mL) at 37 °C in a 95% humidity atmosphere under 5% CO2 for two days. Prior to

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imaging experiments, the cells were washed with PBS buffer for three times and then

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incubated with the aN-dots/AuNPs probe (1080 µg/mL) at 37 °C for 30 min. Then,

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the samples were rinsed with PBS buffer three times to remove the remaining probe.

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For negative and positive control groups, before the incubation with the nanoprobe,

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the A549 cells were initially pretreated with NMM (1.0 mmol/L) or Cys (0.5 mmol/L)

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at 37 °C for 1 h. After washing with PBS buffer three times, the A549 cells were

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further incubated with probe at 37 °C for 30 min. The cell images were then acquired

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after washing the cells with PBS buffer.

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RESULTS AND DISCUSSION

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Synthesis and characterizations of aN-dots.

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Synthesis of aN-dots. To avoid tedious and uncontrollable surface modification,

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herein, we presented a simple strategy that fluorescent aN-dots with high percentage

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of the element nitrogen have been synthesized using 2-azidoimidazole and aqueous

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ammonia by microwave-assisted process. Microwave-assisted strategy significantly

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decreased the reaction time and enhanced the fluorescent properties of aN-dots

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because of microwave effects.27-29 Compared with conventional two-step fabrication

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of

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integration” strategy via microwave irradiation is simpler and more efficient. As

amino-functionalized

quantum

dots,

the

present

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depicted in Scheme 1a, the aN-dots were synthesized by a microwave method with

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2-azidoimidazole in ammonia, where the formation of nanoparticles and the surface

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passivation were accomplished simultaneously. This reaction took just 8 min and the

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obtained aN-dots exhibited high quantum yield (QY), up to 34%. Herein, the possible

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mechanism for the formation of aN-dots from 2-azidoimidozole is that ammonia

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water is the nucleophilic reagent in a ring-opening reaction, which also plays a key

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role on passivating the active surface to give amine-modified on the surface of

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aN-dots.15,30,31 After decomposition of azido moiety, the active intermediate, nitrene,

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reacted with double bond of 2-azidoimidozole to form tricycle of aziridine moiety and

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polymers were then formed through self-polymerization. Aqueous ammonia may then

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be involved in a ring-opening reaction and finally aN-dots are formed by probable

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nuclear burst at supersaturation point.15

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To get better optical properties for the synthetic aN-dots, we optimized the

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reaction conditions. As Table S1 results show that, under the same temperature, the

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QY reaches maximum when the microwave treating time is 8 min and then a

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downward trend appears with time extension, while the size of the aN-dots increases

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with increasing heating time (Figure S1). The results suggested that the reaction was

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accelerated with time extension, which maybe generated in part of the aggregation of

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the aN-dots. And Figure S2 shows the full scan XPS spectrum and N1s XPS spectra

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of aN-dots, and the nitrogen content on the surface of aN-dots is increased as the time

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increased to 8 min but then decreased as increasing heating time. Therefore, the

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performance of aN-dots was reflected not only effects from particles of different sizes 10

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but also a considerable distribution of emissive trap sites on aN-dots.27 Further

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element analysis of these aN-dots indicated that the nitrogen proportion, which are

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consistent with the results of the above (Table S2).. Moreover, N-dots were

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synthesized with 2-azidoimidazole in different solutions such as methanol, water and

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aqueous ammonia at 120 °C for 8 min. It is worth mentioning that the QY of N-dots

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synthesized from aqueous ammonia is higher than that from methanol or water.

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Nitrogen atoms serve as n-type impurities providing excess electrons, which results in

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an upward shift of the Fermi level and a change in optical properties.8,10 What is more,

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we also investigated the effect of different temperature on absorption spectra (Figure

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S3) and fluorescence quantum yield (Table S3) of these aN-dots. With increasing

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temperature from 100 °C to 160 °C, the QY of different aN-dots reaches a maximum

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when the microwave treating temperature is 120 °C, and the aN-dots made by

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different microwave treating times had similar UV-Vis absorbance spectra and the

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same absorbance peaks at 300 nm or so, which is similar to that of previously

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reports.10,30 In summary, the aN-dots were synthesized by 8 min of microwave

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irradiation at 120 °C with 2-azidoimidazole and ammonia.

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Characterization of aN-dots. The morphology and structure of the as-prepared

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aN-dots were shown in Figure 1, and the aN-dots were well monodispersed and

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uniform with an average size of 5.0 nm, as shown in TEM and AFM images. To

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confirm the presence of various functional groups in aN-dots, a FT-IR experiment was

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measured, suggesting characteristic absorption of O–H and N–H stretching vibration.

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Raman spectrum was also employed to further characterize the microstructure of 11

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aN-dots, related to the presence of sp3 defects and sp2 carbon.32 In Figure 2, the full

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scan XPS spectrum of the aN-dots showed that C1s, N1s, and O1s signals appeared at

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286.2, 399.6, and 532.1 eV, respectively, and the typical XPS surveys of the aN-dots

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indicate a high nitrogen content, which was consistent to the results of their element

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analysis. To determine the N configurations in the aN-dots, N1s spectra were analyzed.

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The deconvoluted N1s XPS spectrum of aN-dots around 398.39 eV, 399.25 eV,

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399.93 eV and 400.72 eV were pointed to the nitrogen atom of C=N, NH2, C-N-C and

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N-C3, respectively.8,15,33 The aN-dots prepared by 8 min heating time have nitrogen

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component up to 40%, which is remarkable higher than the N-CDs reported

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previously.13,14,34 This indicated that the as-prepared aN-dots were abundant in amino

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groups on the surfaces, which was consistent with the corresponding FT-IR

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spectrum.35 FL is one of the most fascinating features of aN-dots, which produce

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multi-fluorescence

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excitation-dependent fluorescence behavior is one of the most special properties of

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quantum dots (Figure 3). The photograph of the dispersion under UV light exhibits a

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blue color (inset), further revealing that the resultant aN-dots exhibit blue fluorescence.

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It is interesting that aN-dots exhibit upconversion fluorescence when excited at

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long-wavelength from 620 to 760 nm, which also show excellent photostability, as the

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photoluminescence intensity did not change even within 180 days (Figure S4). It is

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worthwhile mentioning that the presence of rich hydrophilic groups (NH2) imparts

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excellent solubility in water, which are very stable for several months without the

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observation of any floating or precipitated dots. Intriguingly, the aN-dots internalized

colors

under

different

excitation

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and the

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into cells by the toxicity studies display good excellent biocompatibility, suggesting

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the potential application of biosensing and bioimaging (Figure S5).

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Construction of the sensing system combined aN-dots with AuNPs.

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The principle of the sensing system. In this study, we constructed a nanoprobe

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using the aN-dots combined with AuNPs for sensitive and selective detection and

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imaging of Cys. The principle of this sensing system based on the FL-quenching and

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FL-recovery of aN-dots is illustrated in Scheme 1b. This nanosensor is composed of

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aN-dots and AuNPs, where aN-dots serve as fluorometric reporter and AuNPs

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functions as fluorescence quencher. In the sensing system, the aN-dots with abundant

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amino groups are adsorbed on the AuNPs through Au-N bond, resulting in effcient

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quenching of their fluorescence, that is, upon addition of aN-dots into AuNPs solution,

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the aN-dots are prone to get close to the surface of AuNPs through the Au−N

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interaction owing to the aN-dots specifically interact with AuNPs.25 Meanwhile, the

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aggregation of AuNPs occurred due to the disorder assembly of AuNPs and aN-dots,

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which resulted in a color change from red to purple. After adding Cys to the sensing

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system, aggregated aN-dots/AuNPs dispersed again and the aN-dots desorbed from

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the surface of AuNPs because of the specific affinity between −SH and Au, and

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thereby result in remarkable recovery of the fluorescence of aN-dots. As revealed by

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TEM (Figure S6), AuNPs turn to aggregation in the presence of aN-dots, while the

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addition of Cys could induce the re-dispersion of the aggregated aN-dots/AuNPs.

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These results are in accordance with the FL spectra, which clearly verified our

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proposed detection principle. 13

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Optimization of the Cys sensing. In order to have a better response to the

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fluorescence probe, it is necessary to optimize these conditions which have a

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significant impact on the detection of Cys, such as the volume ratio of aN-dots to

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AuNPs, quenching time, recovery time, pH value and NaCl concentration (Figure S7).

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For the sake of good photorestoration, an obvious fluorescence quenching was needed.

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The appropriate volume ratio of aN-dots (4.0 mL, 100 µg/mL) and AuNPs (1.0 mL,

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4.0 nmol/L) was 4:1 when the FL was quenched effectively. As shown in Figure S7a,

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it indicated that both fluorescence quenching and recovery were completed within 5

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min. The pH may have effect on the surface charge of AuNPs, aN-dots and also the

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ionization of Cys, therefore we investigated the effect of pH value from 3.0 to 11.0 on

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the FL.25 Upon endocytosis, the microenvironmental pH value may vary from 7.2 to

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approximately 4.5. The emission intensity of the nanoprobe showed only a slight

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decrease as the pH value changed (Figure S7c). Therefore, we chose 7.4 as the pH

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value to examine the response of the nanoprobe based on the comprehensive

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consideration. In addition, the FL intensity of the nanoprobe changed less than 5% in

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NaCl solution with high concentration (1.0 mol/L). All these features make the

298

nanoprobe as an excellent candidates designed for biological applications.

299

High selectivity. The coordination priority of Cys to the aN-dots/AuNPs

300

nanoprobe formed the basis of sensing strategy. It is well-known that detection of Cys

301

will suffer from interference of GSH and Hcy due to their similar structures and

302

reactivity.36 In addition, the much lower intracellular level of Cys (30-200 µmol/L)

303

than that of GSH (millimolar range) also makes it difficult to detect Cys selectively

304

over GSH.37 To evaluate the selectivity of this nanosensor toward Cys, interference 14

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assays were performed under identical conditions using other molecules and ions,

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including competitive biothiols such as GSH and Hcy. As shown in Figure 4a, under

307

the same conditions, in sharp contrast to Cys, other molecules such as UA, Ile, AA,

308

Ala, Asp, Glu, and His, as well as ions including Na+, Zn2+, Mg2+, Al3+, K+, Fe3+

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showed almost no influence on the spectra of the nanoprobe. This result indicates that

310

nanoprobe has high selectivity for Cys over Hcy and GSH. It is worth noting that

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nanoprobe showed almost no response to GSH even it was present at a millimolar

312

level, which could be the main interference during detection of Cys in living cells.38

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The interferences in the serum and plasma were below the value of the tolerable,

314

which indicate that this method has a high selectivity for determination of Cys in

315

complex biological samples such as human serum, plasma and living cells.

316

Applications in complex biological samples.

317

Detection of Cys in human serum and plasma. In order to demonstrate the

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analytical performance of the proposed nanoprobe in complicated biological samples,

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the Cys concentrations in human serum and plasma have been determined

320

fluorometrically with the nanoprobe. Under the optimized parameters, there is a good

321

linear relationship between the fluorescence intensity and the concentration of Cys. As

322

shown in Figure 4b, the linear range was obtained from 0.3 µmol/L to 3.0 µmol/L

323

(R2=0.999), with a 0.1 µmol/L detection limit (signal-to-noise ratio of 3), which was

324

lower than the proper therapeutic level of Cys.18 Moreover, the capability of the

325

nanoprobe was evaluated by quantitative detection of Cys in human serum and plasma

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(Table 1). The recovery of the spiked samples ranged between 90.0% and 106.7%,

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indicating the practicability of the proposed sensing platform. 15

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Confocal microscopic imaging of Cys in A549 cells. To examine the capacity of

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nanoprobe for cell imaging, in vitro cellular uptake experiments in A549 cells were

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then performed and the images were taken under a laser scanning confocal

331

microscope. Toxicity is a crucial factor to be taken into account in the design of an

332

intracellular nanoprobe.38 Cell cytotoxicity experiments of the aN-dots/AuNPs

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nanoprobe were evaluated using A549 cell lines through the CCK-8 assay (Figure 5).

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The aN-dots/AuNPs nanoprobe shows no apparent toxicity to the cells even though

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the nanoprobe concentration was increased to 200 µg/mL, thus the probe showed low

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toxicity toward cultured cell lines under the experimental conditions. And these

337

results also indicate that the nanoprobe can be rapidly delivered into the cytoplasm,

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implying the nanoprobe is membrane-permeable.39 Moreover, the nanoprobe exhibits

339

high stability against photobleaching, which makes it promising probes for imaging

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applications. Therefore, the fluorescent probe is also favorable for imaging of Cys in

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living cells owing to their good cell membrane permeability and excellent

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biocompatibility.

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Then, the practical application of the nanoprobe for bioimaging of intracellular Cys

344

in A549 cells was also investigated (Figure 6). The A549 cells were first incubated

345

with the aN-dots/AuNPs nanoprobe (80 µg/mL) in culture media for 2 h and then the

346

fluorescence images were recorded using scanning confocal microscopy. A moderate

347

fluorescence emission in the 420-600 nm channel under excitation at 408 nm could be

348

readily observed for A549 cells incubated directly with the nanoprobe, thus

349

suggesting the recognition of intracellular Cys and a high level of Cys is expressed in 16

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A549 cells.38 To validate that the nanoprobe is able to respond to changes of the

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intracellular Cys level in living cells, we pretreated the A549 cells with 1.0 mmol/L of

352

N methylmaleimide (NMM), which is a thiol-reactive reagent for reducing the Cys

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level.40 As shown in Figure 6a, there are no fluorescence signal observed in A549

354

cells upon treatment with NMM due to a decrease in the Cys concentration. Whereas

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those pretreated with external Cys (0.5 mmol/L) showed significantly increased

356

fluorescence (Figure 6c). Therefore, these results confirmed that the nanoprobe can

357

be applied to monitoring change of the intracellular Cys in living A549 cells,

358

suggesting our strategy has potential for clinical diagnosis.

359 360

CONCLUSION

361

In summary, we developed a facile and quick “synthesis-modification integration”

362

strategy for preparation of fluorescent aN-dots by microwave-assisted approach using

363

2-azidoimidazole and aqueous ammonia. The aN-dots could be obtained within 8

364

minutes without additional surface passivation, which have high percentage of the

365

element nitrogen up to 40%, endowed them with tunable fluorescent emission, bright

366

luminescence (QY as high as 34%) and excellent biocompatibility. Furthermore, a

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novel probe with membrane permeability and flexibility was constructed based on

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aN-dots combined with AuNPs. The nanoprobe enables facile and robust Cys assay in

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human serum and plasma with excellent sensitivity and selectivity, which allows for a

370

rapid mix-and-read assay protocol without dye-modified oligonucleotides or complex

371

chemical modification. It was also uploaded into living cells and used to detect 17

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intracellular Cys levels with high discrimination. Therefore the strategy provides a

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reliable method for detection and imaging of Cys in complex biological samples,

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which has great potential for diagnostic purposes. Our study may give a new sight for

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preparation of high nitrogen component nanomaterials and broadening application of

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N-dots in bioimaging. Moreover, their superior optical properties should enable their

377

use in other applications, which are currently underway in our laboratory.

378

Acknowledgments

379

Foundation of China (Nos.21475153, 21575167 and 21675178), the Guangdong

380

Provincial Natural Science Foundation of China (No. 2015A030311020), the Special

381

Funds for Public Welfare Research and Capacity Building in Guangdong Province of

382

China (No. 2015A030401036), and

383

Program of China (No. 201604020165), respectively.

The work were supported by the National Natural Science

the Guangzhou Science and Technology

384 385

Compliance with ethical standards

386

competing interest.

The author(s) declare that they have no

387 388

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Figure captions

443

444 445 446

Scheme 1. (a) Synthesis strategy of the aN-dots using 2-azidoimidazole and aqueous ammonia by

447

a microwave method; (b) Schematic illustration of Cys detection principle based on the nanoprobe

448

combined aN-dots with AuNPs.

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452 453 454

Figure 1. (a) TEM image and the size distributions, (b) AFM image and the inset showing the

455

height profile along the line, (c) FT-IR spectrum, and (d) Raman spectrum of the as-prepared

456

aN-dots.

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Figure 2. (a) XPS survey, deconvoluted spectra of aN-dots: C1s (b), N1s (c) and O1s (d).

463

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469

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Figure 3. Fluorescence emission spectra of aN-dots in an aqueous solution with progressively

471

longer excitation wavelengths from 360 nm to 500 nm in 20 nm increments (with the normalized

472

spectra in the right inset). The inset (left) are the photographs of the aN-dots solutions under

473

sunlight and UV light illumination.

474 475 476 477 478

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480 481 482

Figure 4. (a) Fluorescence recovery efficiency F/F0 response of the nanoprobe toward Cys and

483

other interferents (10-fold concentration of Cys, red bars) and the subsequent addition of Cys (2

484

µmol/L, blue bars). (b) Fluorescence spectra (λex = 320 nm) of the nanoprobe upon addition of

485

increasing concentrations of Cys (0.0-3.0 µmol/L). The inset: linear relationship between F/F0 and

486

the concentrations of Cys (0.3-3.0 µmol/L). Error bars were obtained from three parallel

487

experiments. The incubation time was 5 min before detection in 10 mmol/L PBS of pH 7.4. The

488

slit widths of emission and excitation were both 5 nm.

489

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491 492 493

Figure 5. Cell viability of A549 cells incubated with the nanoprobe at different concentrations.

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497 498 499

Figure 6. Confocal microscopic images of A549 cells incubated with the aN-dots/AuNPs

500

nanoprobe. A549 cells were incubated with 1.0 mmol/L NMM (a) or 0.5 mmol/L Cys (c) for 1 h

501

before incubation with the nanoprobe. (b) Cells were incubated with the nanoprobe without

502

pretreatment.

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Table 1. Determination of Cys in human serum and plasma using aN-dots/AuNPs nanoprobe

506

Original

Added

Found

Recovery

RSD%

(µmol/L)

(µmol/L)

(µmol/L)

/%

(n=3)

0.3

0.95

106.7

3.4

1.0

1.65

102.0

2.6

2.5

3.10

98.8

3.7

0.3

1.04

90.0

2.7

1.0

1.81

104.0

3.1

2.5

3.18

96.4

1.5

Samples

Serum

Plasma

0.63

0.77

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Table of contents (TOC) image

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