Amino Nitrogen Quantum Dots-Based Nanoprobe for Fluorescence

Mar 7, 2017 - Zhijiao Tang, Zhenhua Lin, Gongke Li , and Yuling Hu. School of Chemistry, Sun Yat-sen University, Guangzhou 510275, China. Anal. Chem...
0 downloads 0 Views 1MB Size
Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

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

Analytical Chemistry

1 2

Amino Nitrogen Quantum Dots Based Nanoprobe for

3

Fluorescence Detection and Imaging for Cysteine in

4

Biological Samples

5 6

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

7 8

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

9 10 11

* Corresponding author: Gongke Li, Yuling Hu

12

Tel.: +86-20-84110922

13

Fax.: +86-20-84115107

14

E-mail: [email protected]

15

[email protected]

16 17

18

1

ACS Paragon Plus Environment

Analytical Chemistry

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

19

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

20

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

21

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

22

quantum yield, good photostability and excellent biocompatibility. We further

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

Especially, the nanoprobe exhibits good cell membrane permeability and excellent

32

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

33

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

34

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

35

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

36

to broaden applications in biological systems.

37 38

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

39

2

ACS Paragon Plus Environment

Page 2 of 30

Page 3 of 30

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

Analytical Chemistry

40 41

INTRODUCTION Fluorescent

quantum

dots,

such

as

semiconductor

quantum

dots,1

42

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

43

tremendous research attention owing to their outstanding electronic and photonic

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

element nitrogen is still imminently desired.

57

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

58

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

59

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

60

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

61

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

ACS Paragon Plus Environment

Analytical Chemistry

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

62

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

63

electronic characteristics and exhibit distinct luminescence properties, thus having a

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

biomarker in complex systems.

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

ACS Paragon Plus Environment

Page 4 of 30

Page 5 of 30

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

Analytical Chemistry

84

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

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

adenocarcinoma (A549) cells with high discrimination.

97

EXPERIMENTAL SECTION

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

ACS Paragon Plus Environment

Analytical Chemistry

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

Page 6 of 30

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

Reagent Factory (Shenyang, China).

116

Instruments. Microwave synthesis experiments were performed on a

117

MWave-5000 microwave apparatus (Sineo Microwave Chemistry Technology

118

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

119

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

120

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

121

instrument

122

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

123

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

124

absorption spectra were acquired by a NICOLET AVATAR 330 Fourier transform

125

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

126

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

127

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

(Philips,

Netherlands).

Raman

spectra

were

6

ACS Paragon Plus Environment

performed

on

a

Page 7 of 30

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

Analytical Chemistry

128

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

129

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

130

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

131

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

132

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

133

recorded at room temperature (25 °C).

134

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

135

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

136

2-azidoimidazole

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

as

a

starting

material

has

been

synthesized.

Briefly,

148

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

149

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

ACS Paragon Plus Environment

Analytical Chemistry

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

150

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

151

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

152

nanoprobe and appropriate aliquot of Cys solution were transferred into centrifuge

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

three times.

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

untreated cells.

170

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

171

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

ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30

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

Analytical Chemistry

172

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

173

plates containing sterile petridish and were cultured in DMEM supplemented with

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

after washing the cells with PBS buffer.

184

RESULTS AND DISCUSSION

185

Synthesis and characterizations of aN-dots.

186

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

187

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

188

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

189

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

190

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

191

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

192

of

193

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

amino-functionalized

quantum

dots,

the

present

9

ACS Paragon Plus Environment

“synthesis-modification

Analytical Chemistry

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

nuclear burst at supersaturation point.15

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30

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

Analytical Chemistry

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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

ACS Paragon Plus Environment

Analytical Chemistry

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

Page 12 of 30

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

multi-fluorescence

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

colors

under

different

excitation

12

ACS Paragon Plus Environment

wavelengths,

and the

Page 13 of 30

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

Analytical Chemistry

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

proposed detection principle. 13

ACS Paragon Plus Environment

Analytical Chemistry

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30

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

Analytical Chemistry

305

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

306

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+

309

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

311

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

313

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

318

analytical performance of the proposed nanoprobe in complicated biological samples,

319

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

326

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

327

indicating the practicability of the proposed sensing platform. 15

ACS Paragon Plus Environment

Analytical Chemistry

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

328

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

329

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

330

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

333

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

334

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

335

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

336

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,

338

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

339

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

340

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

341

living cells owing to their good cell membrane permeability and excellent

342

biocompatibility.

343

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

ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30

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

Analytical Chemistry

350

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

351

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

353

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

355

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

367

novel probe with membrane permeability and flexibility was constructed based on

368

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

369

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

ACS Paragon Plus Environment

Analytical Chemistry

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

372

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

373

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

374

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

375

preparation of high nitrogen component nanomaterials and broadening application of

376

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

18

ACS Paragon Plus Environment

Page 18 of 30

Page 19 of 30

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

Analytical Chemistry

389 390 391

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.

392

(2) Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. Nature Nanotech. 2012, 7, 11-23.

393

(3) Lei, W.; Portehault, D.; Dimova, R.; Antonietti, M. J. Am. Chem. Soc. 2011, 133, 7121-7127.

394

(4) Sun, Y.-P; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. A. S.; Pathak, P.; Meziani, M. J.; Harruff,

395

B. A.; Wang, X.; Wang, H.; Luo, P. G.; Yang, H.; Kose, M. E.; Chen, B.; Veca, L. M.; Xie, S.-Y. J.

396

Am. Chem. Soc. 2006, 128, 7756-7757.

397

(5) Du Y; Guo, S. Nanoscale 2016, 8, 2532-2543.

398

(6) Wang, X.; Sun, G.; Routh, P.; Kim, D. H.; Huang, W.; Chen, P. Chem. Soc. Rev. 2014, 43,

399

7067-7098.

400

(7) Li, Y.; Zhao, Y.; Cheng, H.; Hu, Y.; Shi, G.; Dai, L.; Qu, L. J. Am. Chem. Soc. 2012, 134, 15-18.

401

(8) Arcudi, F.; Đorđević, L.; Prato, M. Angew. Chem. Int. Ed. 2016, 55, 2107-2112.

402

(9) Park, Y.; Yoo, J; Lim, B.; Kwon, W.; Rhee, S. –W. J. Mater. Chem. A 2016, 4, 11582–11603.

403

(10) Tang, L.; Ji, R.; Li, X.; Bai, G.; Liu, C. P.; Hao, J.; Lin, J.; Jiang, H.; Teng, K. S.; Yang, Z.; Lau,

404

S. P. ACS Nano 2014, 8, 6312-6320.

405

(11) Lin, L.; Rong, M.; Lu, S.; Song, X.; Zhong, Y.; Yan, J.; Wang, Y.; Chen, X. Nanoscale 2015, 7,

406

1872-1878.

407

(12) Benítez-Martínez, S.; Valcárcel, M. Trends Anal. Chem. 2015, 72, 93-113.

408

(13) Hu, C.; Liu, Y.; Yang, Y.; Cui, J.; Huang, Z.; Wang, Y.; Yang, L.; Wang, H.; Xiao, Y.; Rong, J. J.

409

Mater. Chem. B 2013, 1, 39-42.

410

(14) Xu, H.; Zhou, S.; Xiao, L.; Wang, H.; Li, S.; Yuan, Q. J. Mater. Chem. C 2015, 3, 291-297. 19

ACS Paragon Plus Environment

Analytical Chemistry

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

411

(15) Chen, X.; Jin, Q.; Wu, L.; Tung, C.; Tang, X. Angew. Chem. Int. Ed. 2014, 53, 12542-12547.

412

(16) Wu, Z.; Feng, M.; Chen, X.; Tang, X. J. Mater. Chem. B 2016, 4, 2086-2089.

413

(17) Fu, Z.; Li, G.; Hu, Y. Anal. Bioanal. Chem. 2016, 408, 8813-8820.

414

(18) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D.

415

A.; Mowen, K.; Baker, D.; Cravatt, B. F. Nature 2010, 468, 790-795.

416

(19) Shahrokhian, S. Anal. Chem. 2001, 73, 5972-5978.

417

(20) Niu, W.; Guo, L.; Li, Y.; Shuang, S.; Dong, C.; Wong, M. S. Anal. Chem. 2016, 88, 1908-1914.

418

(21) Stachniuk, J.; Kubalczyk, P.; Furmaniak, P.; Głowacki, R. Talanta 2016, 155, 70-77.

419

(22) Rani, B. K.; John, S. A. Biosens. Bioelectron. 2016, 83, 237-242.

420

(23) Han, B.; Yuan, J.; Wang, E. Anal. Chem. 2009, 81, 5569-5573.

421

(24) Quach, A. D.; Crivat, G.; Tarr, M. A.; Rosenzweig, Z. J. Am. Chem. Soc. 2011, 133, 2028-2030.

422

(25) Deng, J.; Lu, Q.; Hou, Y.; Liu, M.; Li, H.; Zhang, Y.; Yao, S. Anal. Chem. 2015, 87, 2195-2203.

423

(26) Hou, K.; Ma, C.; Liu, Z. Chinese Chem. Lett. 2014, 25, 438-440.

424

(27) Zhu, H.; Wang, X.; Li, Y.; Wang, Z.; Yang, F.; Yang, X. Chem. Commun. 2009, 34, 5118-5120.

425

(28) Wang, Q.; Zheng, H.; Long, Y.; Zhang, L.; Gao, M.; Bai, W. Carbon 2011, 49, 3134-3140.

426

(29) Qu, S.; Wang, X.; Lu, Q.; Liu, X.; Wang, L. Angew. Chem. Int. Ed. 2012, 51, 12215-12218.

427

(30) Bhatnagar, D.; Kumar, V.; Kumar, A.; Kaur, I. Biosens. Bioelectron. 2016, 79, 495-499.

428

(31) Shen, P.; Xia, Y. Anal. Chem. 2014, 86, 5323-5329.

429

(32) Tetsuka, H.; Asahi, R.; Nagoya, A.; Okamoto, K.; Tajima, I.; Ohta, R.; Okamoto, A. Adv. Mater.

430

2012, 24, 5333-5338.

431

(33) Pan, L.; Sun, S.; Zhang, A.; Jiang, K.; Zhang, L.; Dong, C.; Huang, Q.; Wu, A.; Lin, H. Adv.

432

Mater. 2015, 27, 7782-7787. 20

ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30

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

Analytical Chemistry

433

(34) Shi, B.; Su, Y.; Zhang, L.; Liu, R.; Huang, M.; Zhao, S. Biosens. Bioelectron. 2016, 82, 233-239.

434

(35) Ma, S.; Chen, Y.; Feng, J.; Liu, J.; Zuo, X.; Chen, X. Anal. Chem. 2016, 88, 10474-10481.

435

(36) Li, Z.; Zheng, X.; Zhang, L.; Liang, R.; Li, Z.; Qiu, J. Biosens. Bioelectron. 2015, 68, 668-674.

436

(37) Xue, S.; Ding, S.; Zhai, Q.; Zhang, H.; Feng, G. Biosens. Bioelectron. 2015, 68, 316-321.

437

(38) Ye, H.; Cai, S.; Li, S.; He, X.; Li, W.; Li, Y.; Zhang, Y. Anal. Chem. 2016, 88, 11631-11638.

438

(39) Liu, Y.; Tian, Y.; Tian, Y.; Wang, Y.; Yang, W. Adv. Mater. 2015, 27, 7156-7160.

439

(40) Xiao, Y.; Zeng, L.; Xia, T.; Wu, Z.; Liu, Z. Angew. Chem. Int. Ed. 2015, 54, 5323-5327.

440 441

21

ACS Paragon Plus Environment

Analytical Chemistry

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

442

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.

449

22

ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30

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

Analytical Chemistry

450 451

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.

457

458

23

ACS Paragon Plus Environment

Analytical Chemistry

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

459

460 461 462

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

463

464

465

466

467 24

ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30

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

Analytical Chemistry

468

469

470

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

25

ACS Paragon Plus Environment

Analytical Chemistry

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

479

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

26

ACS Paragon Plus Environment

Page 26 of 30

Page 27 of 30

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

Analytical Chemistry

490

491 492 493

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

494

27

ACS Paragon Plus Environment

Analytical Chemistry

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

495 496

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.

503

28

ACS Paragon Plus Environment

Page 28 of 30

Page 29 of 30

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

Analytical Chemistry

504 505

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

507 508 509

29

ACS Paragon Plus Environment

Analytical Chemistry

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

510

Table of contents (TOC) image

511

512 513

30

ACS Paragon Plus Environment

Page 30 of 30